"Heyuan Liji" is a joint venture between Heyuan Biotechnology (stock code: 688238) and Liji Biotechnology, specializing in the "Liji Biotechnology" and "Life-ilab" reagent brands

Language:

中文 English

HOT PRODUCTS

New products Cell Biology Common medium EZ Transfection Western Blot Electrophoresis

TECHNICAL SERVICE

Services Vector for gene therapy Genetic manipulation Cell experiments Animal testing Other special services

transcriptomics

Original link:Metabolomics - TranscriptomicsTranscriptome sequencing (RNA Seq) refers to the use of second-generation high-throughput sequencing technology for cDNA sequencing, which comprehensively and rapidly obtains almost all transcripts of a specific organ or tissue of a species in a certain state. With the advent of the post genomic era, various omics technologies such as transcriptomics, proteomics, metabolomics, etc. have emerged one after another, among which transcriptomics is the first to develop and the most widely used technology.RNA Seq has the following advantages:(1) High throughput, using second-generation sequencing platforms can obtain several to tens of billions of base sequences, which can meet the requirements of covering the entire genome or transcriptome;(2) High sensitivity, capable of detecting rare transcripts with as few copies in cells;(3) High resolution, the resolution of RNA Seq can reach a single base, with good accuracy, and there is no cross reaction or background noise problem caused by traditional microarray hybridization fluorescence simulation signals;(4) Unrestricted, full transcriptome analysis can be performed on any species without the need for pre designed specific probes, allowing for direct transcriptome analysis of any species. At the same time, it can detect unknown genes, discover new transcripts, and accurately identify variable splicing sites, SNPs, and UTR regions.RNA seq technology can detect the overall transcriptional activity of a specific species at the single nucleic acid level, thereby comprehensively and rapidly obtaining almost all transcriptional information of the species in a certain state. Due to its ability to obtain abundance information of all RNA transcripts and high accuracy, transcriptome sequencing has a wide range of applications. Mainly used for:(1) Detecting new transcripts, including unknown transcripts and rare transcripts;(2) Research on gene transcription levels, such as gene expression levels and differential expression between different samples;(3) Functional research on non coding regions, such as microRNA, non coding long RNA (IncRNA), and RNA editing;(4) Research on transcriptional structural variations, such as alternative splicing and gene fusion;(5) Develop SNPs and SSRs, etc.

Details

MiRNA sequencing

Original link:Meta organisms - miRNA SequencingSmall RNA is a general term for a class of non coding short RNAs in organisms that have important regulatory functions. Numerous studies have confirmed that small RNAs are involved in regulating almost all life processes in animals and plants, including cell proliferation, differentiation, apoptosis, and are closely related to the occurrence and development of human diseases. Small RNA sequencing is precisely the analysis of this important type of regulatory small RNA, especially microRNAs (miRNAs), using high-throughput sequencing technology to identify and analyze small RNA sequences ranging from 18 to 30 nucleotides in samples.Technical advantagesLibrary optimization:Optimize the library preparation process based on the different miRNA sequences of animals and plants, and maximize the enrichment of sample small RNA sequencesExclusive analysis software:We have independently developed data analysis software ACGT101 miR, whose reliability has been verified by hundreds of experimental projects; The generated charts can be directly used for paper writing.Rich project experience:One of the earliest companies in China to provide small RNA sequencing services, users have published over a hundred papers related to small RNA sequencing.technology roadmap Total RNA3 'and 5' connector connectionsreverse transcriptionPCR amplificationPAGE purificationMachine sequencing (HiSeq 2500, SE50)Data analysisAnalysis content1. Data quality control: distribution statistics of sequencing quality values; Quality control of sequencing bases; Sequencing data output statistics.2. Obtaining comparable sequencing data3. miRNA database and genome alignment of sequencing species4. Comparison with other RNA databases5. Predicting novel miRNAs6. Correlation analysis of miRNA sequences themselves7. Quantitative analysis of miRNA and differential expression analysis of miRNA in multiple samples; Differential expression miRNA screening; Differential expression miRNA statistics; Cluster analysis of miRNA expression patterns (limited to multiple sample projects).8. Duplicate correlation testing (limited to biological duplicate samples only).9. Prediction of Target Genes for Differentially Expressed miRNAs (Target Finder Software for Plants, Target Scan and miRanda Software for Animals)10. Differential expression of miRNA target genes GO, KEGG annotation and GO, KEGG enrichment, pathway pathway pathway analysis, and pathway network analysis. (Multiple sample projects only)Differential expression of miRNA target genes GO, KEGG annotation and GO, KEGG enrichment, pathway pathway pathway analysis, and pathway network analysis. (Multiple sample projects only)Sample typeCells, tissues, body fluids, serum, plasma, whole blood, total RNA, etcSuggested total RNA starting amount: 5 μ g, minimum 2.5 μ g, concentration ≥ 120 ng/μ LSample type1.Wu S, Li Y, Chen S, Liang S, Ren X, Guo W, Sun Q, Yang X. (2017) Effect of dietary Astragalus Polysaccharide supplements on testicular piRNA expression profiles of breeding cocks. International Journal of Biological Macromolecules 103(1), 957-964.2.Tritten L, Tam M, Vargas M, Jardim A, Stevenson MM, Keiser J, Geary TG. (2017) Excretory/secretory products from the gastrointestinal nematode Trichuris muris. Experimental Parasitology 178(1), 30-36.3.Sun J, Yao L, Chen T, Xi Q, Zhang Y. (2017) The effect of dietary ginseng polysaccharide supplementation on the immune responses involved in porcine milk-derived esRNAs. bioRxiv [Epub ahead of print].4.Ghorecha V, Zheng Y, Liu L, Sunkar R, Krishnayya NSR. (2017) MicroRNA dynamics in a wild and cultivated species of Convolvulaceae exposed to drought stress. Physiology and Molecular Biology of Plants 23(2), 291-300.5.Li H, Peng T, Wang Q, Wu Y, Chang J, Zhang M, Tang G, Li C. (2017) Development of Incompletely Fused Carpels in Maize Ovary Revealed by miRNA, Target Gene and Phytohormone Analysis. Frontiers in Plant Science 8(1), 463.Case PresentationAnalysis and comparison of small RNAs during pollen development in homologous tetraploid and diploid rice1. Research backgroundMicroRNAs (miRNAs) regulate the expression of plant genes by inhibiting their target genes and play an important role in plant reproduction. However, current research on miRNA profiling of homologous tetraploid rice is quite limited.2. Research resultsIn this study, researchers used small RNA sequencing to analyze the miRNA profiles during pollen development in diploid and polyploid rice. The research results showed that compared with diploid rice, 172 differentially expressed miRNAs (DEMs) were detected in tetraploid rice, and 57 miRNAs were specifically expressed in homologous tetraploid rice. Among these 172 DEMs, 115 miRNAs were upregulated and 61 miRNAs were downregulated. GO analysis of target genes upregulated in DEM showed that their functions were enriched in membrane transport during the pre meiotic interphase, meiotic reproduction, and nucleotide binding during the single microspore stage. Osa-miR5788 and osa-miR1432-5pR+1 are upregulated during meiosis, and their target genes reveal the interaction of meiosis related genes and suggest that they may be involved in gene regulation related to chromosome behavior. In addition, during the pollen development of homologous tetraploid rice, abundant 24 nt siRNA related to transposable elements were discovered; However, their content significantly decreased in diploid rice, indicating that 24 nt siRNA may play a role in pollen development. Venn diagram analysis of miRNA expression at different stages during pollen developmentThe results of this study provide new insights into the role of miRNA in pollen development in homologous tetraploid rice and its relationship with pollen sterility, laying the foundation for understanding the impact of small RNA expression profiles on polyploidy.referenceLi, et al. (2016) Comparative Small RNA Analysis of Pollen Development in Autotetraploid and Diploid Rice. InternationalJournal of Molecular Sciences. 17, 499; doi:10.3390/ijms17040499Result Display1. Statistical analysis of differential miRNA upregulation and downregulationThe frequency statistics of upregulation and downregulation of differentially expressed genes are used to determine the number of differentially expressed miRNAs under different experimental conditions. The horizontal axis represents the comparison group information, the vertical axis represents the number of upregulated and downregulated miRNAs, red represents upregulated miRNAs, blue represents downregulated miRNAs, and the number represents the number of upregulated and downregulated miRNAs.2. Cluster analysis of differentially expressed miRNAsDifferential miRNA clustering analysis is used to determine the clustering patterns of miRNA regulation under different experimental conditions. Based on the similarity of miRNA expression profiles in the samples, cluster analysis is performed to visually display the expression of miRNAs in different samples (or treatments), thereby obtaining biologically relevant information. Different colors represent different levels of miRNA expression, with colors ranging from blue to white to red indicating low to high levels of expression. Red represents high expression genes, while dark blue represents low expression genes. 3. Differential miRNA Venn diagramThe differential gene Venn diagram between different comparison groups can intuitively display the number of common and unique differentially expressed miRNAs between different comparison groups. The differential gene Venn diagram has obvious biological significance, such as the experimental design of the same control but different treatments, which can compare the differential genes under different treatments.4. GO Enrichment Bar ChartGO enrichment bar chart of miRNA target genes: used to reflect the distribution of differentially expressed genes on GO terms enriched in biological processes, cellular components, and molecular functions.5. GO enrichment scatter plotPerform GO enrichment analysis on differentially expressed miRNAs and display them in a scatter plot. The Rich factor represents the number of differentially expressed genes located in the GO divided by the total number of genes located in the GO. The smaller the P-value, the higher the degree of GO enrichment.common problem 1.What is the naming convention for miRNA?Regarding miRNA naming, it is based on the naming rules of miRBase: the Latin name of the species is abbreviated with 3 letters - miR/MIR - number (plant), miR represents the mature microRNA, and MIR is used as the precursor for plants. Refer to the official website of miRBase: Currently, * is no longer used to label complementary sequences of microRNAs and their hairpin precursors. Instead, "-3p" and "-5p" are used as suffixes to distinguish these two sequences, replacing the old naming convention. Please refer to the blog article on the miRBase database when version 17.0 was released http://www.mirbase.org/blog/2011/04/mirbase-17-released/  2. How to screen for differentially expressed genes?When filtering, it may be necessary to refer to this parameter. Generally, if the focus is on discovery, all copies should be retained. If the focus is on differential expression, only high and middle copies can be retained. Log2 (Treat/Control)>0 indicates upregulation, 1 indicates 2-fold upregulation, 0 indicates upregulation, and 1 indicates 2-fold upregulation,3.What are the advantages of the ACGT101 program?The advantages are as follows:1) Analyzing the entire process while considering sequencing quality scoring (phred score);2) Before comparing the mature body with the reported precursor sequence, in addition to discovering a completely new miRNA sequence, the mature body sequence at the other end and the flanking miRNA sequence of the reported mature body can also be discovered;3) The comparison and filtering with other RNA databases is based on the miRBase database, which avoids filtering out a small number of reported miRNAs with coding regions on the comparison;4) Table 5 has rich and comprehensive data information, and all results are traceable;5) 100 customer references.

Details

LncRNA sequencing

Original link:Genomics - lncRNA sequencingbrief introduction Long non coding RNA (lncRNA) is a type of non coding RNA (ncRNA) with a length greater than 200 nt, which is widely present in various organisms and plays an important role in epigenetic regulation, cell cycle regulation, and cell differentiation regulation. It is closely related to the growth and development of animals and plants, as well as the occurrence of human diseases. It can also serve as a biomarker or important target for disease diagnosis.The use of high-throughput sequencing technology for lncRNA sequencing and bioinformatics analysis can quickly and accurately identify lncRNAs with important regulatory functions, analyze their relationship with specific biological processes, and deeply explore the functions and expression regulation mechanisms of lncRNAs. This includes the ceRNA regulatory mechanism of competitive binding to miRNA, as well as a series of controls on chromatin structure such as enhancers, promoters, insulators, etc. Sample starting quantity and sample delivery suggestionsSample typeinitiation massAnimal and clinical organ tissues/brain tissues, etc>20mgAnimal and clinical skin/bone/blood vessels/adipose tissue, etc>100mgPlant leaf tissue/flowers>200mgPlant roots/stems/fruits/seeds>500mgPrimary cells/cell lines>5×106个Neutrophils/Eosinophils/Basophils>5×107个Extracellular vesicle samples>1×108个Serum/plasma/cerebrospinal fluid/joint effusion/follicular fluid>2mLCell culture supernatant>20mLurine>30mLTotal RNA>1μgAndRIN>7.0matters needing attention:① It is recommended to store the tissue samples in RNAlater, RNAHold, RNAProtect and other relevant tissue preservation solutions, and then store them at -80 ℃ or send them on dry ice;② After the cell samples are thoroughly lysed using TRIzol or other lysis buffer, they should be stored at -80 ℃ or sent on dry ice③ For a more detailed sample preparation guide, please contact our online customer service Bioinformatics analysis process and analysis contentlncRNAAnalysis content remarks Sequencing data quality controlStatistics of sequencing raw data volume and clean data volumeSequencing quality Q20 Q30 GC content statistics, removing adapters, low-quality, and contaminated sequences from the original offline datagenomic alignment Reference genome alignment/region alignmentChromosome density distributionExpression analysisGene/transcript expression profileStatistical distribution of gene expression values in samplesStatistical analysis of interval distribution of gene expression values in different samplesDepth statistics of sample gene transcript coverageGene expression density mapGene expression level boxplotSample correlation analysisPearson/Spermancorrelation analysis PCA clustering analysisMRNA analysisGene difference analysisTranscriptome differential analysisDifferential gene volcano mapOverall statistical chart of differentially expressed genesDifferential gene clustering heatmapDifferential Gene GO Enrichment AnalysisKEGG enrichment analysis of differentially expressed genesGSEA analysisLncRNA analysisTranscript reconstruction and lncRNA predictionAnalysis of lncRNA expressionDifferential expression analysis of lncRNADifferential lncRNA volcano mapOverall statistics of differentially expressed lncRNAsDifferential lncRNA clustering heatmapComparison of structural characteristics between lncRNA and mRNAComparison of lncRNA and mRNA LengthORF distribution of lncRNA and mRNAStatistics of lncRNA and mRNA exon numbersStatistical analysis of lncRNA and mRNA expression levelsPrediction of lncRNA target genesGO enrichment analysis of lncRNA targeting differentially expressed genesKEGG enrichment analysis of lncRNA targeting differentially expressed genesSNP/INDELanalysisSNP/INDELNumber statisticsLocation of SNP/NDELSNP mutation pattern statisticsGene region SNP and INDEL annotationVariable Shear AnalysisVariable cutting type statisticsVariable cutting visualization Application scenarios and cases Application scenario 1: Promoter/enhancer regulationScope of application: Research on nonlinear transcriptional regulation, discovery and functional identification of novel cis acting elements, screening and determination of tissue-specific promoters, etcThe self transcription of lncRNA can interfere with the transcription of genes encoding proteins in its vicinity. When the upstream lncRNA is transcribed, it crosses the promoter region of the adjacent target gene, interfering with the binding of transcription factors to the promoter of the target gene, thereby inhibiting the transcription of the target gene. In addition, lncRNAs have the function of promoting enhancer circularization and activating gene expression. Without cyclization, enhancers are in an inactive state.Application scenario 2: Combined with protein modification/DNA methylation/m6AScope of application: Clinical medicine, basic medicine, plant genetics and breeding research, etcLncRNAs are involved in epigenetic regulation, possibly by recruiting chromatin remodeling complexes to mediate gene silencing, particularly in regulating histone methyltransferases associated with histone modifications. In addition, lncRNA can also bind to some DNA methylation and demethylase enzymes, causing methylation changes in the promoter region of genes and affecting gene expression.Application scenario 3: ceRNA regulatory mechanismScope of application: Research on environmental stress, clinical medicine, plant genetics and breeding, early diagnosis of diseases, etcThe full name of ceRNA is competitive endogenous RNA, which is a functional element that can compete to bind to RNA. Usually, lncRNA and circRNA compete for binding to miRNA, and we generally refer to lncRNA and circRNA as ceRNA. The full name of the ceRNA regulatory network is ceRNA regulatory network, which refers to the entire cascade of regulatory networks involving ceRNA. And ceRNA analysis refers to the analysis of the entire ceRNA regulatory network. Generally, there are circRNA miRNA mRNA analysis or lncRNA miRNA mRNA analysis. Project Process Eukaryotic transcriptome sequencing can provide researchers with a complete service process from sample extraction, library sequencing, data analysis, etc., providing high-quality data results and strong reference for subsequent research.

Details

Single cell sequencing

Original link:Metaorganisms - Single Cell SequencingSingle cell sequencing technology is a new technique for high-throughput sequencing analysis of the genome, transcriptome, and epigenome at the single-cell level. It can reveal the gene structure and expression status of individual cells, reflect the heterogeneity between cells, and play an important role in fields such as oncology, developmental biology, microbiology, neuroscience, etc. It is becoming a focus of life science research.Single cell sequencing workflowThe workflow of single-cell sequencing includes four key steps: 1) single-cell preparation, 2) single-cell isolation and library preparation, 3) sequencing and primary analysis, and 4) data visualization and interpretation. In the entire workflow, there are some experimental considerations and key steps that affect the results and determine whether the research can be successful. To obtain accurate data and draw meaningful conclusions, it is necessary to carefully design experiments and conduct them according to requirements.In the past decade, significant progress has been made in the field of single-cell identification and research with the development of new technologies for cell isolation, sequencing, and techniques, as well as single-cell and new applications. With the increasing number of options for single-cell isolation and detection, the diversity of experimental protocols has significantly increased, each with its inherent advantages and disadvantages. Therefore, researchers face many decision-making issues, such as cell flux, sequencing depth, required transcript length, and whether to include epigenetic or protein level measurements.It is crucial to carefully design experiments and optimize every step of the workflow in order to fully utilize single-cell sequencing to elucidate complex biological systems. Researchers must have clear biological goals and reasonable experimental designs in order to select the best methods for their research questions.

Details

Eukaryotic transcriptome sequencing

Original link:Metaorganisms - Eukaryotic Transcriptome Sequencingbrief introductionTranscriptome broadly refers to all transcripts of a specific cell in a certain functional state, including mRNA and non coding RNA (ncRNA). It is an inevitable link between genomic genetic information and biological functions. Transcriptome sequencing in eukaryotes is based on high-throughput sequencing, which can quickly obtain a collection of all transcripts of a specific cell or tissue in a certain state of a species, used for studying gene structure and function, variable splicing, and predicting new transcripts.ranscriptome research can study gene function and structure at the overall level, and has become a priority research method to reveal the mechanisms of biological growth and development regulation and adaptation to stress, biological evolution laws, important mechanisms of disease occurrence and development, and discover key targets of pathogenic gene regulation. Currently, it has been widely applied in various fields such as basic research, clinical diagnosis and drug development, animal and plant breeding, etc.characteristicAbility to handle complex samplesRich experience in library construction, incorporating library homogenization technologyAssist clients in quickly and accurately conducting bioinformatics analysisCan flexibly customize information analysis based on customer needsInitial sample size and sample delivery recommendationsSample typeinitiation massAnimal and clinical organ tissues/brain tissues, etc>20mgAnimal and clinical skin/bone/blood vessels/adipose tissue, etc>100mgPlant leaf tissue/flowers>200mgPlant roots/stems/fruits/seeds>500mgPrimary cells/cell lines>5 x 106 piecesNeutrophils/eosinophils/basophils>5 x 107 piecesTotal RNA>1 μ g and RIN>7.0matters needing attention:① It is recommended to store the tissue samples in relevant tissue preservation solutions such as RNAlate, RNAOld, RNAProtect, etc., and then store them at -80 ℃ or send them on dry ice;② After sufficient lysis using TRIzol or other lysis solutions, the cell samples are stored at -80 ℃ or sent on dry ice③ For more detailed sample preparation guidelines, please contact online customer serviceBioinformatics analysis process and contentReferenced transcriptomeAnalysis contentremarksSequencing data quality controlRemove connector sequences, contaminated sequences, and low-quality error sequences from the original offline dataData volume statistics and quality evaluationSequence alignment and transcript reconstructionReference genome alignmentCompare the proportion of offline data to the upper genomeDistribution of reference genome alignment regionsStatistical comparison of the proportion of exons and introns in the genome sequenceReference sequence chromosome density distributionStatistical distribution of alignment sequences on chromosomesTranscriptome reconstructionContains sequence merged. fa and. gtf filesGene/transcript overall expression analysisGene expression summary tableTranscript expression summary tableBox plot of gene and transcript expression distributionStatistical distribution of gene and transcript expression intervalsTranscript coverage depth statisticsDistribution density map of gene and transcript expression levelsDifferential expression gene/transcriptome analysis (sample size ≥ 2)Differential expression genes and transcripts statistical bar chartDifferential expression genes and transcriptome expression profilesDifferential expression genes and transcriptome volcano mapCluster heatmap of differentially expressed genes and transcriptsGO enrichment analysis of differentially expressed genesIncluding GO enrichment bar charts, scatter plots, radar charts, etcEnrichment analysis of differentially expressed genes KEGGIncluding KEGG pathway enrichment scatter plots, pathway maps, radar maps, etcEnrichment analysis of differentially expressed genes Reactome (including only 19 common species)Including Reactome enrichment scatter plots, bar charts, etcEnrichment analysis of differentially expressed genes DO (disease annotation database) (including only human species)Including scatter plots, bar charts, etc. for DO database enrichmentstructural analysisVariable splicing analysisBy default, ASprofile variable shear analysis results are provided, and rMATS differential variable shear analysis results can be provided for free after salesSNP/InDel analysisSample correlation (sample size ≥ 2)Correlation coefficient chart and PCA (principal component analysis) chartApplication scenarios and casesApplication Scenario 1:Differential gene screening and functional analysisApplicable scope:Any direction including clinical medicine, basic medicine, biochemistry, animal and plant and fungal researchBy using eukaryotic transcriptome sequencing, differentially expressed genes can be screened by comparing the gene expression levels between the experimental group and the control group. Then, further locking of differentially expressed genes is carried out, such as GO, KEGG enrichment analysis and GSEA analysis, combined with published literature in Pubmed and some star molecules accumulated in the research group to annotate the functions of differentially expressed genes, and further analyze the functional genes of interest. After entering the experimental validation stage, qPCR, Northern, Western Blot, FISH validation, gene knockout, and overexpression can be performed on the screened differentially expressed genes.Application Scenario 2:Time series analysis or concentration gradient analysisApplicable scope:Clinical samples, cell samples, animal and plant samples with multiple time periods, or samples treated with different drug concentrationsIn the process of transcriptome data analysis, there is a special type of experimental design. Collect experimental samples from different time periods or test samples with different concentration gradients of drugs, reagents, etc. Subsequently, studying the expression patterns of different genes at different time periods or concentration gradients is commonly referred to as "time series analysis"Application Scenario 3:Discovery of upstream regulatory genes such as transcription factors/regulatory factors/splicing factorsApplicable scope:Any research direction including clinical medicine, basic medicine, biochemistry, animal and plant research, etcConventional transcriptome differential analysis is highly likely to yield a large number of differentially expressed genes, which poses a challenge for target localization in later experimental validation. Transcription factors are a great entry point without specific pathways of interest or star molecules. Transcription factors can regulate genomic DNA openness, recruit RNA polymerase for transcription processes, recruit cofactors to regulate specific transcription stages, and regulate various life processes such as immune response and developmental patterns. Therefore, analyzing the expression and regulatory activity of transcription factors is of great significance for deciphering complex life activities. Other regulatory factors, including variable splicing and other regulatory genes, can also participate in upstream regulation.Application scenario 4: Large sample researchApplicable scope:Animal and plant breeding, genetic populations and species origins, population cohorts and biomarker miningWith the rapid development of sequencing technology, transcriptome sequencing studies with a small number of samples are no longer able to explain complex biological problems. Researchers have begun to use large sample sizes of transcriptome samples, combined with statistical and machine learning methods, to identify core genes that conform to specific patterns and research objectives. Using methods such as Mendelian randomization, correlation analysis, linear regression, LASSO regression, Cox regression, etc., to analyze gene or genomic diversity in different samples and explore deeper and more comprehensive biological significance.Project ProcessTranscriptome sequencing with eukaryotic involvement in metaorganisms can provide researchers with a complete range of service processes, including sample extraction, library sequencing, and data analysis, providing high-quality data results and providing strong reference for subsequent researchAt the same time, the cloud analysis of metabiotic transcriptome has been fully upgraded, providing customers with a variety of analysis content to meet the standard and personalized analysis needs of researchers.

Details

CircRNA sequencing

Original link:Metaorganisms - circRNA sequencingbrief introductionCovalent closed, single stranded circular RNA (circRNA) is a relatively special type of RNA that does not have a free 5 'cap structure or a 3' poly (A) structure, and is not sensitive to nucleases, making it more stable than regular linear RNA. With the rapid development of high-throughput sequencing and bioinformatics analysis technology, thousands of circRNAs have been identified in different species, most of which are conserved and stable between different species, and the expression of circRNAs is cell specific, tissue specific, and developmental stage specific. In addition to the conventional ceRNA regulatory mechanism that competes for miRNA binding, circRNA also has new functions such as translation peptides, which have great potential in medical and animal and plant basic research and later transformation.Initial sample size and sample delivery recommendationsSample typeinitiation massAnimal and clinical organ tissues/brain tissues, etc>20mgAnimal and clinical skin/bone/blood vessels/adipose tissue, etc>100mgPlant leaf tissue/flowers>200mgPlant roots/stems/fruits/seeds>500mgPrimary cells/cell lines>5 x 106 piecesNeutrophils/eosinophils/basophils>5 x 107 piecesExtracellular vesicle samples>1x108 piecesSerum/plasma/cerebrospinal fluid/joint effusion/follicular fluid>2mLcell culture supernatant >20mLurine>30mLTotal RNA>1 μ g and RIN>7.0matters needing attention:① It is recommended to store the tissue samples in relevant tissue preservation solutions such as RNAlate, RNAOld, RNAProtect, etc., and then store them at -80 ℃ or send them on dry ice;② After sufficient lysis using TRIzol or other lysis solutions, the cell samples are stored at -80 ℃ or sent on dry ice;③ For more detailed sample preparation guidelines, please contact online customer serviceBioinformatics analysis process and contentcircRNAAnalysis content remarks Sequencing data quality controlRaw data quality control statistical tableReference genome alignment analysisReference genome gene statistics tableReference genome alignment reads statistics tableStatistical Table of the Number of CircRNA Identification SamplesStatistical chart of reference genome alignment region distributionReference genome nonlinear alignment region distribution statistical chartQuantitative analysis of circRNA identificationCircRNA fitting sequenceCircRNA expression profileBSJ statistical table for circRNACircRNA statistical analysisCircRNA type statistical pie chartStatistical boxplot of circRNA expression distributionDistribution map of circRNA expression densityStatistical chart of the distribution of circRNA chromosome numbersStatistical chart of the number of circRNA exonsStatistical chart of the number of circRNAs in parental genesCircRNA differential analysisStatistical chart of the number of differential circRNAsAnalysis of differential expression of circRNA in CaseVSControl comparison groupCase VSControl comparison group circRNA volcano mapCase VSControl comparison group circRNA heatmapCaseVSControl comparison group circRNA scatter plotApplication scenarios and casesApplication scenario 1: Structural molecule recruitment of core proteins and nucleic acidsApplicable scope: Any research direction such as basic medicine, biochemistry, and molecular biologyLncRNA and circRNA sometimes act as special RNA molecules, forming protein protein complexes or backbone molecules of protein nucleic acids, exerting a series of regulatory effects on downstream molecules. Some circRNAs recruit ubiquitinases to degrade downstream proteins, or some lncRNAs recruit transcription factors to bind to the promoter region of the target gene to activate downstream gene expression.Application scenario 2: ExosomesScope of application: Any research direction in clinical and translational medicine, basic medicine, etcDue to the fragmented form of lncRNA in exosomes, this random fragment is not only difficult to detect, but also has lower concentrations and poor repeatability, making lncRNA not one of the hot molecules in exosome research. However, due to its stability, circRNA can not only be isolated from extracellular vesicles in body fluid samples as a disease marker, but also be used for later treatment of extracellular vesicles, making it one of the more popular sub research directions for circRNA in the post ceRNA era. One direction is disease biomarkers, which are generally studied in large sample cohorts. One is the research direction of late stage transformation and mechanism, including the molecular mechanism of extracellular vesicle receptor cells and disease treatment.Application scenario 3: Translating peptidesScope of application: Any research direction including clinical and translational medicine, basic medicine, biochemistry and molecular biology, etcThere is an untranslated RNA sequence on circRNA, called IRES, which can fold into a structure similar to the starting tRNA and recruit more ribosomes. Under normal circumstances, IRES does not bind to eIF, but rather to a class of proteins called ITAF. The function of ITAF is to recruit ribosomes into the internal structure of circRNA to initiate protein translation. In addition, there may also be IRES enhancers, similar to UTR elements of circ-ZNF609. Some studies have also found through transcriptome sequencing and transcriptome sequencing that non coding RNA LINC-PINT can be translated into functional peptides after forming circular RNA.Application scenario 4: ceRNA regulatory mechanismThe full name of ceRNA is competitive endogenous RNA, which is an element that can compete for binding to RNA. Usually, lncRNA and circRNA compete to bind to miRNA, and we generally refer to lncRNA and circRNA as ceRNA. The full name of ceRNA regulation network is ceRNA regulation network, which refers to the entire regulatory network cascade in which ceRNA participates. And ceRNA analysis refers to the analysis of the entire ceRNA regulatory network. Generally, there are circRNA miRNA mRNA analysis or lncRNA miRNA mRNA analysis.Project Process Project ProcessTranscriptome sequencing with eukaryotic involvement in metaorganisms can provide researchers with a complete range of service processes, including sample extraction, library sequencing, and data analysis, providing high-quality data results and providing strong reference for subsequent research.

Details

Lentiviral vector

Original link:Metaorganisms - lentiviral vectorsLentivirus is a type of viral vector modified from human immunodeficiency virus (HIV), which is a type of retrovirus. The genome is RNA, and its toxic genes have been removed and replaced by exogenous target genes. Lentivirus belongs to the pseudotype virus, which can integrate foreign genes into the genome to achieve stable expression and has the characteristics of infecting dividing and non dividing cells.After the lentivirus genome enters the cell, it is reverse transcribed into DNA in the cytoplasm, forming a DNA pre integration complex. After entering the nucleus, DNA is integrated into the cell genome. The integrated DNA is transcribed into mRNA and returned to the cytoplasm to express the target protein or produce small RNA. The gene expression or small RNA interference mediated by lentivirus is sustained and stable, and divides with the division of the cell genome (Figure 1). Figure 1 Process of Slow Virus Packaging and Infection of Cells  Slow virus vectors have the characteristics of wide host range, low immunogenicity, large gene capacity, and long-term expression. Capable of effectively infecting various types of cells in culture, including tumor cells, liver cells, myocardial cells, neurons, endothelial cells, stem cells, etc.  The infection of lentivirus has the characteristic of integration, which can effectively integrate foreign genes into the host chromosome, achieve persistent expression, and thus construct stable cell lines for the study of gene cellular function. The use of lentiviral vectors to modify T cells can be used for CAR-T cell therapy.Slow virus packaging and purification  Slow virus packaging uses a three plasmid or four plasmid system for packaging, collects cell culture supernatant, and concentrates, purifies, and collects viruses through high-speed centrifugation.Slow virus titer detection  Detection method: Quantitative PCR is used to detect the copy number of exogenous DNA in the cell genome after infection  Experimental principle: lentivirus mediates the integration of exogenous genes into the target cell genome through reverse transcriptionAdvantages of lentiviral vectors① Long expression time: Slow viruses can integrate exogenous genes into the host cell genome, achieving stable gene expression for a long time without loss during cell division and passage. It is the preferred choice for cell experiments and is commonly used to construct stable strains.② High safety: No pathogenicity was found, and lentiviral vectors were used to modify T cells for CAR-T cell therapy.③ Low immunogenicity: Direct injection into live tissue does not easily cause an immune response and is suitable for animal experiments.Comparison of Biological Characteristics of Different VirusesCarrier selection  HarmonyBio has a rich range of lentiviral products for manipulating both coding and non coding genes, such as lncRNA microRNA、circRNA, The following slow virus vectors are available for you to choose from (not limited to this list):Regulatory methodsCarrier numberCarrier name (order of carrier components)Eukaryotic resistancefluorescencePromoterDefault labelCarrier capacityOverexpressionGL127pSLenti-CMV-EGFP-3xFLAG-WPREN/AEGFPCMV3FLAG4.8kbGL121pSLenti-EF1-EGFP-CMV-MCS-WPREN/AEGFPCMVN/A4.4kbGL129pSLenti-CMV-mCherry-3xFLAG-WPREN/AmCherryCMV3FLAG4.8kbGL119pSLenti-CMV-MCS-3xFLAG-PGK-Puro-WPREPuroN/ACMV3FLAG4.4kbGL120pSLenti-SFH-EGFP-P2A-Puro-CMV-MCS-3xFLAG-WPREPuroEGFPCMV3FLAG3.5kbGL107pSLenti-EF1-EGFP-P2A-Puro-CMV-MCS-3xFLAG-WPREPuroEGFPCMV3FLAG3.7kbGL109pSLenti-EF1-EGFP-F2A-Puro-CMV-MCS-WPREPuroEGFPCMVN/A3.8kbGL122pSLenti-EF1-EGFP-F2A-BSR-CMV-MCS-WPREblasticidinEGFPCMVN/A3.9kbGL130pSLenti-CMV-EGFP-3xFLAG-PGK-Puro-WPREPuroEGFPCMV3FLAG3.7kbGL132pSLenti-EF1-EGFP-F2A-Puro-WPRE2-CMV-MCSPuroEGFPCMVN/A3.7kbGL123pSLenti-EF1-mCherry-P2A-Puro-CMV-MCS-3xFLAG-WPREPuromCherryCMV3FLAG3.7kbGL125pSLenti-CMV-mCherry-3xFLAG-PGK-Puro-WPREPuromCherryCMV3FLAG3.7kbGL133pSLenti-EF1-mCherry-F2A-Puro-WPRE2-CMV-MCSPuromCherryCMVN/A3.7kbH114pLenti-CMV-Luc2-IRES-Puro-WPREPuroN/ACMVN/A2.2kbGL124pSLenti-EF1-Luc2-F2A-Puro-CMV-MCS-WPREPuroN/ACMVN/A2.8kbExpressing creH126pLenti-CMV-NLS-Cre-3xFLAG-WPREN/AN/ACMV3FLAG3.9kbH15108pLenti-CMV-DIO-MCS-WPREN/AN/ACMV3FLAG5.3kbThree standardsH7656pLenti-CBh-3xFLAG-Luc2-tCMV-mNeonGreen-F2A-Puro-WPREPuromNeonGreenCBh3FLAG1.3kbH7657pLenti-CBh-3xFLAG-Luc2-tCMV-tdTomato-F2A-Puro-WPREPurotdTomatoCBh3FLAG0.6kbH9911pLenti-CBh-3xFLAG-Luc2-tCMV-mNeonGreen-F2A-BSR-WPREblasticidinmNeonGreenCBh3FLAG1.5kbH9912pLenti-CBh-3xFLAG-Luc2-tCMV-tdTomato-F2A-BSR-WPREblasticidintdTomatoCBh3FLAG0.8kbCircRNA overexpressionH8384pLenti-EF1-EGFP-F2A-Puro-CMV-S-circRNA-WPREN/AEGFPCMVN/A3.0kbH8807pLenti-CMV-S-circRNA-WPREN/AN/ACMVN/A4.8kbH8399pLenti-EF1-EGFP-F2A-Puro-CMV-L-circRNA-WPREPuroEGFPCMVN/A1.0kbH8810pLenti-CMV-L-circRNA-WPREN/AN/ACMVN/A2.8kbMiRNA overexpressionGL109pSLenti-EF1-EGFP-F2A-Puro-CMV-MCS-WPREPuroEGFPCMVN/AN/AH3919pCLenti-U6-miR30(miRNA)-CMV-EGFP-F2A-Puro-WPREPuroEGFPU6N/AN/AH119pLenti-CMV-TurboGFP-IRES-Puro-miR30(miRNA)-WPREPuroTurboGFPCMVN/AN/AH3928pCLenti-U6-miR30(miRNA)-CMV-mCherry-F2A-Puro-WPREPuromCherryU6N/AN/AH146pCLenti-EF1-Puro-CMV-EGFP-3xFLAG-Sponge(miRNA)-WPREPuroEGFPCMV3FLAGN/AH7505pCLenti-U6-TuD(miRNA)-CMV-EGFP-F2A-BSR-WPREblasticidinEGFPCMVN/AN/AH7506pCLenti-U6-TuD(miRNA)-CMV-EGFP-F2A-Puro-WPREPuroEGFPCMVN/AN/AOverexpression (Tet on)H125pLenti-TRE-EGFP-EF1-rtTA3-IRES-Puro-WPREPuroEGFPTREN/A2.0kbH121pLenti-TRE-EGFP-3xFLAG-PGK-Puro-WPREPuroEGFPTRE3FLAG3.3kbH2057pLenti-EF1-rtTA3-IRES-Puro-WPREPuroN/AEF1N/AN/AShRNA interferenceGL401pCLenti-U6-shRNA-CMV-Puro-WPREPuroN/AU6N/AN/AGL404pCLenti-U6-shRNA-CMV-EGFP-WPREN/AEGFPU6N/AN/AGL427pSLenti-U6-shRNA-CMV-EGFP-F2A-Puro-WPREPuroEGFPU6N/AN/AGL428pSLenti-U6-shRNA-CMV-mCherry-F2A-Puro-WPREPuromCherryU6N/AN/AH7615pCLenti-U6-shRNA-CMV-mCherry-F2A-BSR-WPREblasticidinmCherryU7N/AN/ACRISPR KnockoutH5070pLenti-U6-spgRNA v2.0-CMV-Puro-P2A-3xFLAG-spCas9-WPREPuroN/AU6N/AN/AH7072pLenti-U6-spgRNA v2.0-CMV-BSR-P2A-3xFLAG-spCas9-WPREblasticidinN/AU6N/AN/AH6825pLenti-U6-spgRNA v2.0-CMV-sfGFP-P2A-3xFLAG-spCas9-WPREN/AsfGFPU6N/AN/AH5068pLenti-U6-spgRNA v2.0-CMV-EGFP-WPREN/AEGFPU7N/AN/AH5450pLenti-CMV-Puro-P2A-3xFLAG-espCas9_1.1-WPREPuroN/ACMV3FLAGN/ACRISPRa transcriptional activationH9517pCLenti-U6-gRNA-MS2-EFS-dCas9-VP64-T2A-BSD-WPREblasticidinN/AU6N/AN/AE2577pCLenti-EF1a-MCP-P65-HSF1-T2A-Hygro-WPREhygromycinN/AEF1aN/AN/AH7281pLenti-CMV-dCas9-VP64-T2A-Puro-WPREPuroN/ACMVN/AN/AConventional applications  In vitro infected cells   Slow virus vectors can effectively infect various types of primary cells and most cell lines, including tumor cells, liver cells, myocardial cells, neurons, endothelial cells, stem cells, etc.   The infection of lentivirus has integration characteristics, which can effectively integrate foreign genes into the host chromosome, thus achieving persistent expression and constructing stable transgenic cell lines. In vitro cellular functional experiments, including cell proliferation, invasion, migration, apoptosis, etc., are conducted. Stable expression of target genes in tumor cell lines can further construct tumor animal models for in vivo gene function validation, pharmacokinetic analysis, in vivo imaging, and other detection to further study the occurrence, development, metastasis, and drug treatment effects of tumors in vivo. Common MOI ListCell nameChinese nameChronic viral infection (MOI)(Reference value)MGC80-3Human gastric cancer cells10HT-29human colon cancer cell20RKOHuman colon adenocarcinoma cells10Caki-1Human renal clear cell carcinoma skin metastatic cells105637Human bladder cancer cells10U-118 MGHuman astroblastoma10RWPE-1Normal human prostate epithelial cells20HeLaHuman cervical cancer cells10~20Ca SkiHuman cervical cancer intestinal metastatic cells10MCF7 [MCF-7]Human breast cancer cells10A549Human non-small cell lung cancer cells10NCI-H1299Human non-small cell lung cancer cells20U-87 MGHuman astroblastoma10U251Human glioma cells10A172Human glioblastoma cells10K-562Human chronic myeloid leukemia cells10HL-60Human myeloid leukemia cells10GES-1Human gastric epithelial cells20U266Human myeloma cells20CAL 27Human tongue squamous cell carcinoma cells20PC9Human lung cancer cells5PC14Human lung cancer cells10MADB-106Rat breast cancer cells20F98Rat glioma cells20MHCC-97HHuman liver cancer cells (high metastasis)10~20  MOI stands for multiplicity of infection, also known as the plural number of infections, which refers to the ratio of the number of viruses to cells during infection. Generally, in an experiment, when a certain cell is infected and reaches 80%, it is defined as the MOI value of that cell, that is, the ratio of the number of viruses to the number of cells; The amount of virus added (μ l)=number of cells x MOI/titer (.../ml) x 1000Application cases of lentivirus in tumor research1.Nature Communications. (IF=12.353). Shi Y,et.al. (2017). Tumour-associated macrophages secrete pleiotrophin to promote PTPRZ1 signalling in glioblastoma stem cells for tumour growth.[lentivirus, glioma]Slow virus interference: Lenti-shNT/shPTN/shPTPRZ12. Hepatology. (IF=14.079). Ma JZ,et.al. (2016). METTL14 suppresses the metastatic potential of HCC by modulating m6 A-dependent primary miRNA processing. [lentivirus, liver cancer]Slow virus overexpression/interference:pLV-CMV-PGK-EGFP-T2A-puro/ pLV-U6-shRNA-CMV-EGFP-T2A-puroThe application of lentivirus in the nervous system (in vivo injection)1.Nature Neuroscience. (IF=19.912). Ding XL,et.al. (2017). Activity-induced histone modifications govern Neurexin-1 mRNA splicing and memory preservation.[Slow virus, learning and memory]Virus type: lentivirusCarrier name:LV-Suv39h1-RNAi(Uncertain details)Injection site: DG area of hippocampusVirus injection volume:2 μL,9.98 × 108 TU/mLTesting time: 2 weeks2.Nature Communications. (IF=12.353). Yao XH,et.al. (2016). Electrical coupling regulates layer 1 interneuron microcircuit formation in the neocortex.[lentivirus, neural circuit]Virus type: lentivirusCarrier name:LV- CX36-shRNA-EGFP(Uncertain details)Injection site: P1 mouse neocortex L1Virus injection volume:1μLTesting time: 2 weeks3.Molecular Psychiatry. (IF=11.64). Guo DJ,et.al. (2019). Autism-like social deficit generated by Dock4 deficiency is rescued by restoration of Rac1 activity and NMDA receptor function. [Chronic virus, autism]Virus type: lentivirusCarrier name:Lenti-CMV-EGFP-P2A-3FLAG-Rac1Injection site: CA1 area of mouse hippocampusVirus injection volume: 1 μ LTesting time: 4 weeksSlow virus extension service The production and quality control of metabiotic lentiviruses have adopted internationally recognized standard processes, using a three plasmid or four plasmid system for packaging in 293T cells. The obtained virus particles were purified by ultracentrifugation and the viral gene copies were titrated by qPCR. In general, the titer of our lentivirus is between 108~109TU/ml, which can fully meet the requirements of various experiments.

Details

Adenovirus ADV

Original link:Harmony with Metaorganisms - Adenovirus ADVAdenovirus (ADV) is a linear double stranded DNA virus without an envelope, with a wide range of cell and tissue infectivity. The infection process is intense, making it suitable for experiments with high gene expression in a short period of time. Compared with other viruses, adenoviruses have the advantage of longer insertion fragments and stronger expression activity.  When used in vitro, adenovirus vectors have high transgenic efficiency (close to 100% transduction efficiency) and can transduce different types of cells, making them a good tool for delivering genes to a large number of difficult to transfect cells. However, when applied in vivo, due to the strong immunogenicity and severe infection of adenovirus, it often causes local tissue inflammation and immune response in animals, which affects the objectivity of animal signs and experimental results.ADV packaging and infection processPackaging and purification of adenovirus  The adenovirus packaging skeleton plasmid and shuttle plasmid were co transfected into HEK-293 cells. After the cells showed CPE state, the virus was purified by collecting cell supernatant and lysate, and then concentrated or centrifuged with cesium chloride density gradient.   CPE(Cytopathic Effect):The cellular degeneration caused by virus infection on tissue cultured cells can be quantified using this pathological effect.Adenovirus titer detection  Detection method: Enzyme linked immunosorbent assay (ELISA) for adenovirus titer measurement. The virus titer is calculated based on the number of positive cells stained brown after infection.       Experimental principle: The capsid protein expressed in positive cells infected with adenovirus is stained brown.       Detection method: Immunocolorimetric method       Experimental principle: The monoclonal antibody against the coat protein of type 5 adenovirus binds to the cells of the adenovirus sample to be infected, and then the HRP labeled secondary antibody binds to the primary antibody. The infected cells turn brown after staining with the colorimetric solution. The viral titer is calculated based on the number of positive cells.Advantages of adenovirus vectors      ① Express quickly, after adenovirus infection of cells, it can be expressed within 1-2 days       ② Large carrier capacity, high infection efficiency, commonly used to infect cells that are difficult to infect       ③ Not integrated into chromosomes, no insertion mutagenicity       ④ It has hepatotropism and is prone to infecting liver cells. The in vivo use of adenoviruses is for infecting the liver    However, due to the high immunogenicity of adenoviruses, they are prone to inflammation in certain animal models and tissues that are sensitive to external stimuli, and caution should be exercised when used in vivo.Comparison of Biological Characteristics of Different VirusesCarrier selection  Heyuan Biotechnology has a rich range of adenovirus products, which are used to manipulate coding and non coding genes, such as lncRNA microRNA、circRNA。       The following adenovirus vectors are available for you to choose from:Regulatory methodsCarrier numberCarrier name (order of carrier components)Eukaryotic resistancefluorescencePromoterDefault labelCarrier capacityOverexpressionH225pADV-mCMV-MCS-3xFLAGN/AN/AmCMV3FLAG>6kbH201pADV-mCMV-EGFP-3xFLAGN/AEGFPmCMV3FLAG>5kbH213pADV-mCMV-3xFLAG-EGFPN/AEGFPmCMV3FLAG>5kbH221pADV-mCMV-3xFLAG-P2A-EGFPN/AEGFPmCMV3FLAG>5kbH204pADV-mCMV-mCherry-HAN/AmCherrymCMVHA>5kbH222pADV-mCMV-3xFLAG-T2A-mCherryN/AmCherrymCMV3FLAG>5kbH12588pADV-EF1-mNeonGreen-CMV-MCS-3xFLAGN/AmNeonGreenCMV3FLAG>4kbH12589pADV-EF1-mScarlet-CMV-MCS-3xFLAGN/AmScarletCMV3FLAG>4kbCircRNA overexpressionH6946pADV-CMV-S-circRNAN/AN/ACMVN/A>5kbMiRNA overexpressionH216pADV-U6-miR30(miRNA)-CMV-EGFPN/AEGFPU6N/AN/AExpressing creK0024 pADV-CMV-CreN/AN/ACMVN/AN/AinterfereDKD001pADV-U6-shRNA-CMV-EGFPN/AEGFPU6N/AN/ADKD004pADV-U6-shRNA-CMV-mCherryN/AmCherryU6N/AN/ACRISPR KnockoutH5064pADV-U6-spgRNA v2.0-CMV-3xFLAG-spCas9N/AN/AU63FLAGN/AH9350pADV-U6-spgRNA v2.0-CMV-sfGFP-P2A-3xFLAG-spCas9N/AsfGFPU63FLAGN/AH5066pADV-U6-spgRNA v2.0-CMV-EGFPN/AEGFPU6N/AN/AH218pADV-CMV-mCherry-P2A-3xFLAG-spCas9N/AmCherryCMV3FLAGN/AH5449pADV-CMV-mCherry-P2A-3xFLAG-eSpCas9_1.1N/AmCherryCMV3FLAGN/AApplication instance  1.Hepatology. (IF=14.079). Zhou YF,et.al. (2018). Cystathionine β-synthase is required for body iron homeostasis. [腺病毒, 肝脏]  Infection site: liver  Carrier name:Ad-CBS  Injection method: tail vein  Injection volume:1 x1011 PFU/mL,10ul  Testing time: 7 days  2. Diabetes. (IF=8.095). Xiao Y Z,et.al. (2015). Activation of ERK1/2 ameliorates liver steatosis in leptin receptor deficient (db/db)mice via stimulating ATG7-dependent autophagy. [Adenovirus, Liver]  Infection site: liver  Carrier name:Ad-ATG7  Injection method: tail vein  Injection volume:1 x109 PFU/piece  Testing time: 10 days  3. Molecular Neurobiology. (IF=5.397). Guo J,et.al. (2015). Overexpression of Fibulin-5 Attenuates Ischemia/Reperfusion Injury After Middle Cerebral Artery Occlusion in Rats. [Adenovirus, Rat]  Injection site: rat cerebral cortex  Carrier name:Ad-FBLN  Injection method: Brain stereotactic injection  Injection volume: three different doses  Testing time: 7 days  The production and quality control of adenovirus from Heyuan Biotechnology adopts internationally recognized standard processes, using AdEasy and Admax adenovirus packaging systems for packaging in HEK-293 cells. The obtained virus particles were subjected to ultrafast density gradient centrifugation and filtration, and their titers were determined by chitosan immunoassay. In general, the titer of adenovirus is between 1010~1011pfu/ml, which can fully meet the requirements of various experiments.

Details

Retrovirus

Original link:Metaorganisms - RetrovirusesRetrovirus, also known as retrovirus, is an RNA virus with long terminal repeat sequences (LTRs) at both ends. The virus is spherical, has a capsule, and has spikes on the surface. The particle diameter is about 100nm. The retroviral genome consists of three genes: Gag - the core protein of the virus; Pol - encodes reverse transcriptase; Env - Encodes the envelope glycoprotein of the virus.Retroviral packaging and cell infection processRetrovirus packaging uses a three plasmid or four plasmid system for packaging, collects cell culture supernatant, and concentrates, purifies, and collects the virus through high-speed centrifugation.Titer detection method:Quantitative PCR detection of the copy number of exogenous DNA in the cell genome after interference.Principle of titer detection:Retroviruses mediate the integration of exogenous genes into the target cell genome through reverse transcription.When expressing retroviral genes, RNA needs to be first reverse transcribed into DNA under the action of reverse transcriptase, forming a DNA pre integration complex, which then enters the nucleus and randomly integrates into the host cell DNA. After transcription and translation, the gene expression is completed.Retroviral packaging and infection mechanismRetroviral characteristicsThe biggest characteristic of retroviruses is their specific infection of dividing cells, because only when the infected cells are in the process of cell division can the retroviral DNA genome come into contact with the genetic material of the host cell.Comparison of Biological Characteristics of Different VirusesBased on the above characteristics, retroviruses are suitable for a large number of studies related to stem cells, such as neural stem cell research and hematopoietic stem cell research. When used in vivo, retroviruses are suitable for studying the work of adult newborn neurons or glial precursor cells.Application Cases of Retroviruses1、Customers publish articles:Scientific Reports. (IF=4.122). Xiong Y S,et.al. (2015). Early treatment of minocycline alle-viates white matter and cognitive impairments after chronic cerebral hypoperfusion. [Retrovirus, Stroke]Injection site: SVZ area of the lateral ventricle in micecarrier:pROV-EF1a-GFPVirus vector: RetrovirusVirus titer:1E+8cfu/mLInjection volume:1ul2、应用案例:Nature.(IF=41.577).Yu YC,et.al. (2009).Specific synapses develop preferentially among sister excitatory neurons in the neocortex. [Retrovirus, Developmental]Injection site: E12-E13 mouse embryoVirus vector: RetrovirusInjection volume:1ul

Details

Modification of viral vectors

Original link:Transformation of viral vectors in metaorganisms      The main research directions of basic R&D currently include lentivirus (LV) vector modification, adenovirus (ADV) capacity modification, adenovirus (AAV) serotype screening, specific promoter optimization, comprehensive development of CRISPR/Cas9 technology with in vivo application as the core, and the development of other biological research tools.  The viral vector is a key link in achieving gene delivery and completing gene manipulation, therefore, the modification of viral vectors plays a special and important role in research and application. The modification of virus vectors by the R&D department of Heyuan mainly includes the in vivo application modification of adenovirus, including the modification of dozens of targeted specific organ serotypes, various types of AAV serotype libraries, lentivirus and adenovirus.  The commonly used rAAV in current research is a hybrid viral vector produced by combining AAV2 genome with different capsid proteins, generally labeled as rAAV2/N (where N represents different capsid serotypes). The recombinant virus has stable expression and gene integration ability of AAV2 type, while obtaining tissue infection affinity for different serotypes (specific structural sites on the surface of the capsid of different serotypes determine the specificity of their respective receptors), exhibiting certain organ targeting specificity.  Tissue hydrophilicity of AAV with different serotypes SerotypeOrganizational affinityrAAV2/1The nervous system (high titer forward synaptic),Muscle, skeletal muscle, myocardium, smooth musclerAAV2/2Nervous system, muscles, liver,Smooth muscle of blood vessels, eyerAAV2/3Muscles, liver, lungs, eyesrAAV2/4Nervous system, muscles, eyes, brainrAAV2/5The nervous system, lungs, retina,Liver, synovial jointrAAV2/6Nervous system, lungs, muscles, heartrAAV2/7Muscle, liverrAAV2/8Nervous system, liver, pancreas,Retina, adipose tissuerAAV2/9Nervous system, myocardium, lungs,Retina, skin, muscle, adipose tissuerAAV2-retroThe nervous system (reverse non synaptic)AAV-PHP.eBCross blood-brain barrier (intravenous injection)AAV-PHP.SWhole peripheral nerve (tail vein injection)AAV-PANPancreatic (intraperitoneal injection)AAV-LUNGLung (tail vein injection)AAV-DJRetina, lungs, kidneys,Infection of cells in vitroAAV-7m8retinaAAV-ShH10YMuller cells in the retinaAAV-Rh10Liver, blood, heart,Infection of cells in vitroAAV-Anc80L65Inner ear, retina、Skeletal muscle, liverAAV-SCH9SVZ neural stem cellsAAV-2MretinaAAV-BR1Cerebrovascular endothelial cellsAAV6-TM6Microglia  Targeted specific organ AAV serotype infection case  1. RAAV2 retro: nervous system (reverse non synaptic)  Injection site: Mouse BPNInjection method: Brain localization injectionCarrier: rAAV2-retro hSyn-EYFPObservation time: 4 weeks 2. AAV-PHP-eB: Whole brain expression of the nervous system (across the blood-brain barrier)   Injection method: Tail vein injection  Carrier: pAAV-CMV-mScarlet-3FLAG&pAAV-CMV-mNeonGreen-3FLAGSerotype: AAV-PHP-eBVirus titers: 1.10 × 1013VG/mL&2.35 × 1013VG/mLInjection volume: 200nl (total amount of injected virus: 1.5 × 1011 VG)Observation time: 3 weeks3. AAV-PAN: Specific infection of pancreas  4. AAV-LUNG: Specific infection of the lungsInjection method: Tail vein injection Vehicle: pAAV-CMV-mScarlet-3FLAGSerotype: AAV-LUNGInjection volume: 2x1011 VG/animal (titer: 1.49 x1013 VG/mL),Injection volume: 200 uLObservation time: 3 weeks  5. AAV-DJ infection of 293T cells in vitroAAV-DJ(MOI=105)                            AAV8(MOI=105 )

Details

Adeno associated virus AAV

Original link:Meta organism - Adeno associated virus AAVAdeno associated virus (AAV) is a member of the parvoviridae family, which is a type of icosahedral parvovirus that cannot replicate autonomously and has no membrane. It has a diameter of about 20-26nm and contains approximately 4.7kb of linear single stranded DNA as its genome. The recombinant adeno-associated virus (rAAV) vector used in the study is a gene vector modified from non pathogenic wild-type AAV. Due to its diverse types, extremely low immunogenicity, high safety, wide host cell range (infectivity to both dividing and non dividing cells), strong spreading ability, and long in vivo gene expression time, rAAV is considered one of the most promising gene research and gene therapy vectors.AAV genome structureRAAV packaging purification and titer detection      During the AAV packaging process, the packaging plasmid is responsible for encoding the target gene and two terminal reverse repeat sequences (ITR), which play a decisive role in virus replication and packaging. The auxiliary plasmid contains the cap (encoding viral capsid protein) and rep (involved in virus replication) genes required for AAV packaging, as well as the adenovirus helper plasmid. After co transfection of three plasmids into 293T cells, AAV virus begins to replicate and package. The production and quality control of adenovirus associated with metaorganisms follow internationally recognized standard procedures and are packaged in 293T cells using a three plasmid system. The obtained virus particles were purified by ultracentrifugation and the virus gene copies were titrated by qPCR. In addition, we can also provide protein staining to detect the integrity of capsid proteins and endotoxin content according to user requirements. In general, the titer of rAAV is between 1012 and 1013 VG/ml, which can fully meet the requirements of various overall experiments.AAV Packaging Process DiagramTiter detection method: Quantitative PCR is used to detect the copy number of exogenous DNA in the viral genome.Titer detection principle: The genome of adenovirus is single stranded DNA, and the copy number of exogenous DNA represents the copy number of the virus genome.Process of rAAV Infection into Cells    The purified AAV viral vector can be used to infect cells. When infecting cells, AAV binds to specific receptors on the cell surface, activates intracellular signaling pathways, and triggers AAV to enter the cell through receptor-mediated endocytosis. With the assistance of organelles such as the nucleus and Golgi apparatus, AAV enters the nucleus and then the virus lyses. Its single stranded DNA needs to replicate into double stranded DNA to express the target gene.Mechanism of rAAV actionAdvantages of rAAV Carrier1. High safety and low immunogenicity: AAV is a replication defective DNA virus with no autonomous replication ability. Wild type AAV relies on the rep gene for low-frequency site integration, while rAAV does not integrate. At present, there are no reports of human and mammalian diseases caused by AAV, and it is also one of the safest viral vectors approved by the FDA for gene therapy drugs.2. Wide host cell range: AAV has a wide host range and has the ability to infect both dividing and non dividing cells.3. Strong diffusion ability: AAV has a diameter of about 20-26nm, a small volume, high titer, and good diffusion ability. Among them, AAV-PHP.EB and AAV9 have the ability to cross the blood-brain barrier and are widely used in the field of neuroscience.4. Long duration of gene expression in vivo: AAV has the ability to maintain long-term gene transcription expression, with peak expression typically occurring within 3 weeks and sustained high expression for more than 5 months.5. Diverse types: AAV serotypes are numerous, and in addition, we are constantly mutating and screening for new serotypes (AAV1-13, AAV2/1, 2/2, 2/5, 2/6, 2/8, 2/9, DJ; retro, PHP.eB…)。Comparison of Biological Characteristics of Different VirusesBased on the above characteristics, AAV is considered an efficient and safe in vitro and in vivo gene transduction tool. Especially in overall level research, compared with other commonly used viral tool vectors, AAV has a mild and long-lasting expression ability during the infection process, making it a powerful tool for gene manipulation.RAAV serotype selection      At present, the total number of registered AAV species has exceeded 196, and there are 13 different serotypes of AAV (i.e. AAV1-AAV13) in primates. Among them, AAV2, AAV3, and AAV9 originate from humans themselves. It is worth noting that AAV2 is the earliest cloned virus and the most thoroughly studied and widely used viral vector to date. With the continuous deepening of research, researchers have found that AAV of different serotypes can hybridize, and the hybridized AAV will have the characteristics of both heterozygotes. Therefore, AAV subtypes are born naturally.     The commonly used rAAV in current research is a hybrid viral vector produced by combining AAV2 genome with different capsid proteins, generally labeled as rAAV2/N (where N represents different capsid serotypes). The recombinant virus has stable expression and gene integration ability of AAV2 type, while obtaining tissue infection affinity for different serotypes (specific structural sites on the surface of the capsid of different serotypes determine the specificity of their respective receptors), exhibiting certain organ targeting specificity.Tissue hydrophilicity of AAV with different serotypesSerotypeOrganizational affinityrAAV2/1The nervous system (high titer forward synaptic), muscles,Skeletal muscle, cardiac muscle, smooth musclerAAV2/2Retina, nervous system,Muscle, liver, vascular smooth musclerAAV2/3Muscles, liver, lungs, eyesrAAV2/4Nervous system, muscles, eyes, brainrAAV2/5The nervous system, lungs, retina,,Liver, synovial jointrAAV2/6The nervous system, lungs,Muscle, heartrAAV2/7Muscle, liverrAAV2/8The nervous system, liver, muscles,Adipose tissue, pancreas, retina,rAAV2/9Nervous system, myocardium, lungs,Retina, skinrAAV2-retroThe nervous system (reverse non synaptic)AAV-PHP.eBCross blood-brain barrier (intravenous injection)AAV-PHP.SWhole peripheral nerve (tail vein injection)AAV-PANPancreatic (intraperitoneal injection)AAV-LUNGLung (tail vein injection)AAV-DJRetina, lungs, kidneys,Infection of cells in vitroAAV-7m8retinaAAV-ShH10YMuller cells in the retinaAAV-Rh10Liver, blood, heart,Infection of cells in vitroAAV-Anc80L65Inner ear, retina、Skeletal muscle, liverAAV-SCH9SVZ neural stem cells      Small suggestion: If you are unsure about which serotype to choose, you can try conducting pre experiments with Pandora's Virus (AAV serotypes) and compare the infection effects of different serotypes on the target tissue to explore the best experimental conditions (injection method, injection site, virus dosage, etc.), in order to obtain more ideal experimental results.     Pandora's Virus Details: Application case of rAAV carrier1. Applications in the field of neuroscience1.1 Lateral habenular nucleus&optogeneticsCustomer published article: Nature (IF=41.577).  Yang Y,et.al. (2018).  Ketamine blocks burning in the lateral habitua to rapidly relieve depressionInjection site: Mouse LHbVector: AAV2/9-hSyn-oChIEF tdTomatoSerotype: AAV2/9Virus titer: 6.29E+12 VG/mLInjection volume: 100nlObservation time: 1 month1.2 Brachial Nucleus&Chemical GeneticsCustomer published article: Science (IF=41.058).  Mu D,et.al. (2017).  A central neural circuit for itch sensingInjection site: Mouse PBNVector: AAV hSyn HA hM4Di IRES mCitrineSerotype: AAV2/9Virus titer: 1E+13 VG/mLInjection volume: 150nlObservation time: 3 weeks1.3 Thalamus and Calcium Ion ImagingCustomer article: Customer article: Science (IF=41.058).  Ren S,et.al. (2018).  The paraventricular thalamus is a critical thalamic area for wakefulnessInjection site: Mouse PVTVector: AAV CaMKII α - GCaMP6fInjection volume: 100nlObservation time: 4 weeks1.4 Periventricular nucleus of thalamus&killing neuronsCustomer published article: Science (IF=41.058).  Ren S,et.al. (2018).  The paraventricular thalamus is a critical thalamic area for wakefulnessInjection site: Mouse PVTVector: AAV-CaMKII α - Cre GFP&AAV-DIO-caspase-3Injection volume: Mix two viruses 1:1 and inject 100nlObservation time: 4 weeks1.5 Hippocampus, cortex&retrograde non synaptic (rAAV2/retro)Customer published article: Biological Psychiatry (IF=11.984).  Bing Xing Pan,et.al. (2018).  Chronic stress causes projection specific adaptation of amygdala neurons via SK channel downregulationInjection site: mouse dmPFC, VHPCVector: AAV2/1-reco-Syn-eYFP Cre AAV2/8-CaMKIIα-DO-SK2&AAV2/8-CaMKIIα-DIO-SK2-mCherryInjection volume: 300nlObservation time: 4 weeks1.6 Primary motor cortex&anterograde synapse (rAAV2/1)Nature Neuroscience. (IF=19.912). Yao J,et.al. (2018). A corticopontine circuit for initiation of urination. [Adeno associated virus, neural circuits]Injection site: Mouse cortex M1, PMCCarrier: rAAV2/1-hSyn-Cre&rAAV2/9-DIO-hChR2 (H134R) - mCherry, rAAV2/9-DIO-GCaMP6sVirus titer: rAAV2/1: 5E+12 VG/mL; rAAV2/9-DIO-hChR2:1.2E+13 VG/mL;rAAV2/9-DIO-GCaMP6s:1E+12 VG/mLInjection volume: multi-point injection, 30-40 ml per pointObservation time: 4 weeks1.7 Edge prefrontal cortex&cross synaptic tracing (WGA Cre)Customer published article: Science Advances (IF=11.511).  Ping Zheng,et.al. (2019).  Critical role of feedback signals from prellimbic cortex to basolatoral amygdala in the retrieval of morphological withdrawal memory. [Adeno associated virus, addiction, AAV1, AAV-WGA Cre]Injection site: Mouse PrLVector: AAV hSyn mCherry IRES WGA CrVirus titer: 4.28E+12 VG/mLInjection volume: 300nlObservation time: 4 weeks1.8 Cortical and AAV Vector (AAV shRNA)Customer posts articles:Nature Medicine. (IF=22.864). Cao X,et.al. (2013). Astrocyte-derived ATP modulates depressive-like behaviors.[Adeno associated virus, depression]Injection site: Mouse mPFCVector: AAV-CMV-P2rx2 shRNA EGFPVirus titer: 3E+12 VG/mLInjection volume: 1.5ulObservation time: 2 weeks1.9 Whole Brain Expression&Cross Blood Brain Barrier (AAV-PHP. eb)An example of whole brain infection caused by tail vein injection of rAAV-PHP-eB in He Yuan BiotechTotal virus load: 1.5E+11VGInjection volume: 200ulVirus expression time: Four weeks1.10 Sparse MarkingApplication Literature:Luo WS, et al., (2016) Supernova: A Versatile Vector System for Single Cell Labeling and Gene Function Studies in vivo. Sci Rep.Carrier information:pAAV-PTRE-tight-NLS-Cre(108-109VG/mL)、pAAV-EF1a-DIO(tTA-P2A-mScarlet)-WPRE(1012VG/mL)1.11 Application in the retinaHemibiome - intravitreal injectionViral vector:AAV-syn-GcaMP6s2. Application in other organs2.1 rAAV infection of the liverAn example of liver infection caused by tail vein injection of rAAV2/8 in He Yuan BiotechnologyTotal virus load: 1E+11VGInjection volume: 200ulVirus expression time: Four weeks2.2 rAAV infection of the heartAn example of cardiac infection caused by tail vein injection of rAAV2/9 in He Yuan BiotechTotal virus load: 2E+11VGInjection volume: 200ulVirus expression time: Four weeks2.3 rAAV infection in the lungs An example of pulmonary infection caused by tail vein injection of rAAV Lung in He Yuan BiotechTotal virus load: 5E+11VGInjection volume: 200ulVirus expression time: Two weeksCustomer posts articles:Cell. (IF=36.216). Huijuan Wu,et.al. (2020). Progressive Pulmonary Fibrosis Is Caused by Elevated Mechanical Tension on Alveolar Stem Cells. [Adeno associated virus, interference]Injection method: Tracheal injectionInfection site: lungsVector: pAAV-Tgfb1 shRNASerotype: rAAV2/9Virus injection volume: 1E+11 VG (50ul)Observation time: 3 weeks2.4 rAAV infection of kidneysAn example of renal infection caused by tail vein injection of rAAV2/8 in He Yuan BiotechnologyTotal virus load: 2E+11VGInjection volume: 200ulVirus expression time: Four weeks2.5 rAAV infection of adipose tissueExample of rAAV2/8 inguinal fat multi-point injection infection with adipose tissue from He Yuan BiotechnologyTotal virus load: 4E+10VG/sideVirus expression time: Four weeks2.6 rAAV infection in muscle tissueExample of Multi point Injection Infection of Muscle in Rear Legs by rAAV2/8 in He Yuan BiotechTotal virus count: 4E+10/sideVirus expression time: Four weeks2.7 rAAV infection of pancreasExample of intraperitoneal injection of rAAV PAN into the pancreas by He Yuan BiotechTotal virus load: 4E+11VGExpression testing time: 4 weeks

Details

Plasmid construction

Original link:Metaorganisms - Plasmid Construction  The Heyuan Biological Research Service Platform can provide cutting-edge, comprehensive, and rigorous vector design and construction services for gene function researchers, covering ordinary eukaryotic expression vectors, viral vectors, and more.  The virus vector construction service includes lentivirus, adenovirus, adenovirus, and retrovirus vectors, etc. The construction content includes gene overexpression vector construction, gene directed mutation expression vector construction, shRNA design and construction for gene silencing, microRNA expression and blocking vector design and construction, luciferase reporter gene vector construction (UTR, promoter, and other functional sequences), as well as Cre LoxP system and CRISPR/Cas9 gene editing tool vector design and construction, aiming to solve the needs of gene upregulation and gene downregulation in gene function research in a one-stop manner.  Plasmid advantages:  ① The expression time is fast, and the target gene expression can be observed about 24 hours after being introduced into the cell;  ② The construction technology is simple and the cost is low.  ③ The overexpression vector library of Heyuan is abundant, and the overexpression markers are abundant  Fluorescent labeling:GFP、mCherry、CFP、BFP、EYFP、mNeonGreen、tdTomato、 mScarlet…  Label marking: FLAG、 HA 、Myc、His tag…  Resistance markers:Puro、Neo、Hygro、BSD…  limitations:  ① Transfection reagents have cytotoxicity and can affect cell status after use;  ② Most cells are difficult to transfect and cannot achieve genetic manipulation goals;  ③ Plasmids are not integrated into the cell genome and can only be expressed instantaneously for a short period of time, making it impossible to conduct experiments with longer observation cycles.  Due to the limitations of pure plasmids, they need to be packaged into viruses to further meet the needs of different experiments.  Differences in different expression vectorsexpression system eukaryotic expression vectorLentiviral vectorRecombinant adenovirus vectorRecombinant adenovirus vectorCarrier gene characteristicsDouble stranded DNA plasmidRNA retrovirusSingle stranded DNA virusDouble stranded DNA virusExogenous gene expression timeExpression starts at 24 hours and lasts for 3-7 daysExpression starts from 2-4 days and stabilizes over a long period of timeStarting expression from 7-14 daysStarting from 1-2 days to expressInsert fragment sizeAround 8kbAbout 4kbAbout 2.8kbAbout 6kbStable cell line screeningDifficult to operatesuremay notNo, it can't be expressed instantaneouslyCell experimentsCell lines with low transfection efficiencyFirst choice, broad-spectrum, high infection efficiencyunsuitedBroad spectrum, high infection efficiencyAnimal experimentsunsuitedSuitable, depending on observation time and injection siteFirst choice, based on observation time and injection siteHigh immunogenicity, careful selectionTiter rangenothing108~109TU/ml1012-1013vg/ml1010-1011pfu/ml

Details

Gene overexpression

Original link:Metaorganisms - Gene OverexpressionIn gene function research, it is usually necessary to upregulate the expression of the target gene to observe changes in phenotype. Overexpression is the process of extensively expressing target genes in host cells. Its basic principle is to construct target genes onto tool vectors, introduce them into cells, and achieve extensive transcription and translation of genes, thereby achieving overexpression of gene products. Tool vectors typically include prokaryotic expression vectors, common eukaryotic expression vectors, lentiviral vectors, adenovirus vectors, adenovirus vectors, and retroviral vectors.Introduction to Tool CarrierAccording to different experimental needs and the size of the target gene, different vectors, including eukaryotic expression vectors and viral vectors, can be selected to achieve overexpression of both coding and non coding genes.expression systemeukaryotic expression vectorLentiviral vectorRecombinant adenovirus vectorRecombinant adenovirus vectorVector gene characteristicsDouble stranded DNA plasmidRNA retrovirusSingle stranded DNA virusDouble stranded DNA virusExogenous gene expression timeExpression starts at 24 hours and lasts for 3-7 daysStart expressing 2-4 days and maintain stable expression over a long period of timeExpression starts from 7-14 daysStarting from 1-2 days to expressInsert fragment size8kbabout4kbabout2.8kbabout4kbaboutStable cell line screeningDifficult to operatesuremay notNo, express it instantlyCell experimentCell lines with low transfection efficiency in some casesPreferred, broad-spectrum, high infection efficiencyCell lines, some serotypes have low transfection efficiencyBroad spectrum, high infection efficiencyAnimal experimentsunsuitedSuitable, based on observation time and injection siteFirst choice, based on observation time and injection siteHighly immunogenic, choose carefullyTiter range无108~109TU/ml1012-1013VG/ml1010-1011PFU/mlIntroduction to common components in carrier diagramsComponent NametypepurposeInitiatorCMV、CAG、EF1a、U6etcExpression of broad-spectrum or specific target geneshSyn、CaMKIIαetcSpecific promoters initiate the expression of target genes in specific tissue cellsProtein label3xFLAG、HA 、6xHis、MycetcFusion expression with the target gene, commonly used to detect the expression level of exogenous proteinsFluorescent labelingEGFP、mCherry、mNeonGreenetcCan be fused or non fused with the target gene for detecting transfection and infection effectsResistance genePuro、Neo、Hygro、BSDetcEukaryotic resistance, commonly used for screening stable strainsAmp、KanProkaryotic resistance, commonly used in prokaryotic bacterial culture and plasmid amplificationConnecting componentsLinkerUsed to connect two genes and commonly used for fusion expression, it can reduce the impact of fusion expression on protein folding and functionIRES、2AUsed to connect two genes to achieve non fusion expressionOther componentsWPREIncrease the expression of target genesCarrier selection (partial)The selection of viral vectors should refer to lentiviruses, adeno-associated viruses, adenoviruses, retroviruses, etc. according to the experimental requirements.Carrier categorynumberCarrier elementOverexpression of lentivirusGL127pSLenti-CMV-EGFP-3xFLAG-WPREGL121pSLenti-EF1-EGFP-CMV-MCS-WPREGL107pSLenti-EF1-EGFP-P2A-Puro-CMV-MCS-3xFLAG-WPREGL122pSLenti-EF1-EGFP-F2A-BSR-CMV-MCS-WPREGL124pSLenti-EF1-Luc2-F2A-Puro-CMV-MCS-WPREAdenovirus overexpressionH225 pADV-mCMV-MCS-3xFLAGH201pADV-mCMV-EGFP-3xFLAGH213pADV-mCMV-3xFLAG-EGFPH204pADV-mCMV-mCherry-HAOverexpression of adenovirusAOV024pAAV-CMV-EGFP-P2A-3xFLAG-WPREAOV025pAAV-CMV-mCherry-P2A-3xFLAG-WPREAOV064pAAV-hSyn-EGFP-P2A-3xFLAG-WPREAOV065AAV-hSyn-mCherry-P2A-3xFLAG-WPREH9429pAAV-CAG-EGFP-3xFLAG-tWPAH9525pAAV-CMV-EGFP-3xFLAG-tWPAH10881pAAV-CAG-EGFP-3xFLAG-tWPAH10886pAAV-CMV-EGFP-3xFLAG-tWPAService IntroductionBased on the gene information provided by the customer, retrieve the cDNA sequence of the gene from a bioinformatics website, construct the cDNA sequence into a tool vector, and overexpress the coding or non coding gene according to experimental requirements. For viral vectors, virus particles produced through packaging can be directly used for infecting cells or animal experiments.Virus vector service process

Details

Genetic interference

Original link:Metaorganisms - Gene InterferenceRNA interference (RNAi) is a phenomenon in normal organisms that inhibits the expression of specific genes. When double stranded RNA (dsRNA) homologous to the coding region of endogenous mRNA is introduced into cells, the mRNA undergoes degradation, leading to gene expression silencing. The antisense strand of small interfering RNA (siRNA) produced by exogenous dsRNA entering cells forms an RNA induced silencing complex (RISC) with various nucleases. RISC mediates RNA interference by binding and cleaving mRNA.Carrier selection (partial)The selection of viral vectors should refer to lentiviruses, adenoviruses, adenoviruses, retroviruses, etc. according to experimental needs.Carrier categorynumberCarrier elementSlow virus interferenceGL401pCLenti-U6-shRNA-CMV-Puro-WPRE GL404pCLenti-U6-shRNA-CMV-EGFP-WPREGL407pCLenti-U6-shRNA-CMV-EGFP-F2A-Puro-WPREGL408GL408 pCLenti-U6-shRNA-CMV-mCherry-F2A-Puro-WPREH7615pCLenti-U6-shRNA-CMV-mCherry-F2A-BSR-WPREAdenovirus interferenceDKD001pADV-U6-shRNA-CMV-EGFPDKD004pADV-U6-shRNA-CMV-mCherryAdenovirus interferenceAKD001pAAV-CBG-EGFP-3xFLAG-WPRE-H1-shRNAAKD003pAAV-CBG-mCherry-3xFLAG-WPRE-H1-shRNAH12663pAAV-U6-shRNA/spgRNA v2.0-CMV-EGFP-WPRE Service Introduction  Based on the target gene provided by the customer, design three siRNA targets for this gene, and then construct these targets into relevant vectors. For virus vectors, the packaged virus particles can be directly used for infecting cells or animal experiments.Virus Carrier Service Process

Details

CRISPR/Cas9

Original link:Metabiology - CRISPR/Cas9CRISPR/Cas9 is a technique that enables precise editing of specific loci in the genome. The principle is that the endonuclease Cas9 protein recognizes specific genomic sites through guided RNA (gRNA) and cleaves double stranded DNA, causing DNA double strand breaks. Cells repair the cleavage sites using non homologous end joining (NHEJ) or homologous recombination (HR) methods, achieving DNA level gene knockout or precise editing.The CRISPR/Cas9 system mainly consists of two parts:1. Single stranded guide RNA (sgRNA)2. Cas9 protein with endonuclease activityCRISPR gene knockoutWhen targeting gene knockout, Cas9 sgRNA can be used to cleave DNA to produce double stranded breaks. Subsequently, cells repair DNA through non homologous end connections. During this process, random deletions or additions of bases are introduced, causing frameshift mutations in the gene and resulting in the knockout of the coding gene. CRISPR gene editing When targeting gene insertion or replacement, the principle of homologous recombination needs to be utilized. The Cas9 sgRNA in the CRISPR/Cas9 system cleaves at the target site to produce DNA double strand breaks. In the presence of template DNA, cells repair the DNA through homologous recombination, and template DNA can be artificially designed as a gene that needs to be inserted or repaired. At this time, cells encode the target gene.Selection of carriers (partial)Provide virus vectors for single and double loading systems containing Cas9 protein, which can design multiple gRNAs based on the target gene sequence. The vectors carry multiple fluorescent resistance markers for easy screening.Carrier categorynumberCarrier elementCRISPR lentivirus single vectorH5070pLenti-U6-spgRNA v2.0-CMV-Puro-P2A-3xFLAG-spCas9-WPREH7072pLenti-U6-spgRNA v2.0-CMV-BSR-P2A-3xFLAG-spCas9-WPREH6825pLenti-U6-spgRNA v2.0-CMV-sfGFP-P2A-3xFLAG-spCas9-WPRECRISPR lentivirus dual vectorH5068pLenti-U6-spgRNA v2.0-CMV-EGFP-WPREH5450pLenti-CMV-Puro-P2A-3xFLAG-espCas9_1.1-WPRECRISPR AAV Single Carrier SystemH6941pAAV-CMV-SaCas9-U6-sagRNA v2.0H5273pAAV-hSyn-SaCas9-U6-sagRNA v2.0CRISPR AAV Dual Carrier SystemH6291pAAV-CMV-EGFP-WPRE-U6-spgRNA v2.0H12663pAAV-U6-shRNA/spgRNA v2.0-CMV-EGFP-WPREH11012pAAV-CMV-Luc2-WPRE-U6-spgRNAVirus Carrier Service Process

Details

Loop tracing

Original link:Metabiotics - Loop Tracing  The circuit connections between neurons are very complex, and a complete and accurate display of the structure and changes of these circuits is of great significance for revealing the causes of many diseases and developing new treatment methods.  Traditional neural tracing methods such as electron microscopy, Golgi staining, dyes, peptide markers, etc. can display the morphology and projection of neurons. A few tracers such as WGA, TTC, etc. can also achieve cross synaptic labeling. However, these methods have disadvantages such as indirect signal, non-specific direction, and severe signal attenuation after crossing synapses. Most importantly, these dyes cannot carry genes, so they can only be colored. Since Kristensson first applied herpes simplex virus (HSV) to label neurons across synapses in the 1980s, the application of various viral vectors in loop tracing has received increasing attention.  However, it should be noted that although these neurophilic viruses have strong infectivity and specificity for labeling neural circuits, their immunogenicity is strong, and animals often die a few days after injection, making behavioral testing or physiological research impossible. Currently, they are more suitable for searching for unknown neural circuits. In addition, such viruses require laboratories with a PII or higher level. Therefore, an increasing number of studies are using AAV for loop tracing labeling. Due to the lack of replication ability and extremely low immunogenicity of AAV, it does not cause significant toxicity or affect the normal physiology of animals. Therefore, AAV has become the most commonly used loop tracing tool as a viral vector.  Neurons are polarized cells, and information is transmitted from the axons of the higher-level neurons to the dendrites of the lower level neurons through synapses. Afterwards, information integration occurs between dendrites and cell bodies, and is emitted again through axons. Therefore, the labeling of neural circuits involves two fundamental issues: directionality (clockwise: labeling from cell body to axon; counterclockwise: labeling from axon to cell body) and whether it crosses synapses (Figure 1). Figure 1: The target of neural loop tracing technology (modified from Peter L Strike), et al., eLS, 2011)  Application cases of loop tracing  1. Forward non transsynaptic labeling based on AAV  ① Customers publish articles:Science. (IF=41.058). Mu D,et.al. (2017). A central neural circuit for itch sensation.[Adenovirus, itching, photogenetics, chemical genetics]  Injection site: PBN in mice  carrier:AAV-hSyn-HA-hM4Di-IRES-mCitrine  Serotype:AAV2/9  Virus titer:1.0× 1013 VG/mL  Injection volume: 150nl  Observation time: 3 weeks  ② Customers publish articles:Neuron. (IF=14.319). Tian-Le Xu,et.al. (2019). Central Processing of Itch in the Midbrain Reward Center. [AAV, Itch, Photogenetics]  Injection site: Vgat Cre mouse VTA  carrier:AAV-EF1a-DIO-ChR2(H134R)-mCherry  Serotype:AAV2/8  Virus titer:7.39×1012 VG/mL  Injection volume:300-400nl  Observation time: 2-3 weeks  2. Reverse non synaptic labeling based on AAV - rAAV2 intro  ① Customer published articles:Biological Psychiatry. (IF=11.984). Bing Xing Pan,et.al. (2018). Chronic stress causes projection-specific adaptation of amygdala neurons via SK channel downregulation. [Adenovirus, Anxiety Disorder, Photogenetics]  Injection site: mouse dmPFC, VHPC  Carrier:AAV2/1-retro-Syn-eYFP-Cre、AAV2/8-CaMKIIα-DO-SK2&AAV2/8-CaMKIIα-DIO-SK2-mCherry  Injection volume:300nl  Observation time: 4 weeks  ② Internal test results  Injection site: Mouse BPN  carrier:rAAV2-retro-hSyn-EYFP  Observation time: 4 weeks   3. Cross synaptic labeling based on AAV  3.1 Forward Transsynaptic Labeling Based on AAV - rAAV2/1  ① Customers publish articles:Nature Neuroscience. (IF=19.912). Yao J,et.al. (2018). A corticopontine circuit for initiation of urination. [Adenovirus, Neural Circuit]  Injection site: M1 and PMC in mouse cortex  Carrier:rAAV2/1-hSyn-Cre&rAAV2/9-DIO-hChR2(H134R)-mCherry、rAAV2/9-DIO-GCaMP6s  Virus titer:rAAV2/1: 5 × 1012 VG/mL;rAAV2/9-DIO-hChR2:1.2 × 1013 VG/mL;rAAV2/9-DIO-GCaMP6s:0.5 × 1012 VG/mL  Injection volume: Multi point injection, 30-40nl per point  Observation time: 4 weeks  ② Customer published articles:Science Advances. (IF=11.511). Ping Zheng,et.al. (2019). Crucial role of feedback signals from prelimbic cortex to basolateral amygdala in the retrieval of morphine withdrawal memory. [腺相关病毒, 成瘾, AAV1, AAV-WGA-Cre]  Injection site: BLA in mouse cortex  carrier:rAAV2/1-hSyn-Cre-EGFP  Virus titer: 1.13 × 1013 VG/mL  Injection volume:60nl  Observation time: 5 weeks  3.2 Cross synaptic labeling using WGA—AAV-WGA-Cre  Customer published articles:Science Advances. (IF=11.511). Ping Zheng,et.al. (2019). Crucial role of feedback signals from prelimbic cortex to basolateral amygdala in the retrieval of morphine withdrawal memory.Adenovirus, addiction, AAV1, AAV-WGA-Cre]  Injection site: PrL in mice  Carrier::AAV-hSyn-mCherry-IRES-WGA-Cre  Virus titer:4.28 × 1012 VG/mL  Injection volume:300nl  Observation time: 4 weeks

Details

Promoter optimization

Original link:Metaorganisms - Promoter Optimization  The current main research directions in basic research and development are the modification of lentivirus (LV) vectors, screening of adeno-associated virus (AAV) serotypes, optimization of specific promoters, comprehensive development of CRISPR/CAS technology with in vivo applications as the core, and the development of other latest biological research tools.  The promoter elements of viral vectors determine the specificity of viral expression. In order to conduct targeted research or gene therapy, we need to continuously develop and optimize promoters suitable for specific cells or tissues. At present, the R&D department of Heyuan Foundation focuses on optimizing and screening promoters for specific cells in different tissues or organs, and also welcomes cooperation projects in promoter research and development.List of commonly used promotersCAG/CBG/CMV/EF1aCommonly used broad-spectrum promotersGFAPAstrocytes (long promoters)hSynMature neuronsshortGFAPAstrocytes (short promoters)CaMKIIαProjection neuronGfaABC1DAstrocytesGAD67GABAergic interneuronsIba1/CX3CR1MicrogliamDLxGABAergic interneuronsMBPOligodendrocytefPVPV+interneuronNG2/NGL2Oligodendrocyte precursor cellsfNPYNPY+interneuronTHCatecholaminergic neurons(DA、NE、E)fSSTSST+interneuronsPRSx8Noradrenergic neuronsDATDopaminergic neuronsChATCholinergic neuronsL7pcp2Purkinje cell D1/D2Dopamine D1/D2 receptor neuronsFEVSerotonin neuronsHCRTOrexin neurons in the hypothalamuscTNTCardiac muscle cellsTBGliver  Specific promoter infection cases   AAV8-TGB-Fabp1[1](TGB liver specific promoter)  AAV-CaMKIIα-NpHR-EYFP[2](CaMKIIαDivine Shooting Meridian Element Specific Promoter)  AAV-Syn-CAPON-L-GFP[3](SynMature neuron specific promoter)  AAV-GFAP- AAV–Ascl1/mCherry [4](GFAPAstrocyte specific promoter)  reference:  [1] Pi H. et al., (2019) Long-term exercise prevents hepatic steatosis: a novel role of FABP1 in regulation of autophagy-lysosomal machinery. FASEB J. doi: 10.1096/fj.201900812R.  [2] Li YD, et al., (2017) A distinct entorhinal cortex to hippo-campal CA1 direct circuit for olfactory associative learning. Nature Neuroscience.  [3] Zhu L J, et al., (2014) CAPON-nNOS coupling can serve as a target for developing new anxiolytics. Nature Medicine.  [4] Liu Y G, et al., (2015) Ascl1 Converts Dorsal Midbrain Astrocytes into Functional Neurons In Vivo. J. Neuroscience.

Details

Disease research

Original link:Metabiology - Disease Research  Based on a deep understanding and research of viral vectors, more and more researchers are starting to use viral vectors for disease research. At present, virus vector tools are widely used in different tissues and organs. By using tool virus vectors, disease related genes can be targeted and tissue-specific modified and manipulated, which has become a technical means for researchers to conduct disease research and treatment.  Selection of viral vectors  The size of different viral vectors varies greatly, indicating significant differences in their gene capacity; The difference in immunogenicity means that its in vivo/in vitro application scenarios require subtle adjustments; The difference in expression time and cycle means that we need to make corresponding choices based on research needs.Table 1 Comparison of Biological Characteristics of Different VirusesLentivirusAdenovirusadenovirusRetrovirusenvelopehavenothinghavenothingParticle diameter80-100 nm20-30 nm90-100nm~100nmgenomedsRNAssDNAdsDNARNAGenome size9.75kb4.7kb36kb~5kbExpression start time48-72h72-96h24-48h48-72hExpression duration> 2 months> 6 months< 1 month> 2 monthsInternal diffusion abilitycommonlystrongstrongcommonlyImmunogenicitysecondaryExtremely lowstrongsecondaryTiter range108~109TU/mL1012-1013VG/mL1011-1012PFU/mL107~108TU/mLTiter detection methodQPCR integration of DNAQPCR non integrated DNAAntigen antibody immunostaining kitQPCR integration of DNAIntegration methodRandom high-frequency integrationDirectional low-frequency integration (rAAV non integration)Non integratedRandom integrationApplication scopeIn vitro cell infection, gRNA library screening, in vivoWidely used in vivo based on different serotypesHepatophilia, rapid or short-term expression, blood, heart, cell line, etc Specific infection only affects cells in the division stage Recombinant adeno-associated virus vector (rAAV) has always been a star vector for in vivo research. In addition to its advantages of extremely low immunogenicity, high safety, wide range of host cells, strong diffusion ability, and long expression time of genes in vivo, it also relies on its diversity of species.  The commonly used rAAV in current research is a mixed viral vector produced by combining the AAV2 genome with different capsid proteins, generally labeled as rAAV2/N (N represents different capsid serotypes). The recombinant virus has stable expression and gene integration ability of AAV2 type, while obtaining tissue infection affinity of different serotypes, exhibiting certain organ targeting specificity.Table 2 Tissue affinity of different serotypes of AAVSerotypeOrganizational affinityrAAV2/1Neurosystem (high titer cis synaptic), muscles, skeletal muscles, myocardium, smooth musclesrAAV2/2Nervous system, muscles, liver, vascular smooth muscle, eyesrAAV2/3Muscles, liver, lungs, eyesrAAV2/4Nervous system, muscles, eyes, brainrAAV2/5Neurological system, lungs, retina, liver, synovial jointsrAAV2/6Nervous system, lungs, muscles, heartrAAV2/7Muscles, liverrAAV2/8Neurological system, liver, pancreas, retina, adipose tissuerAAV2/9Nervous system, myocardium, lungs, retina, skin, muscles, adipose tissuerAAV2-retroNeurosystem (reverse non synaptic)AAV-PHP.eBCross blood-brain barrier (intravenous injection)AAV-PHP.STotal peripheral nerve (tail vein injection)AAV-PANPancreatic (intraperitoneal injection)AAV-LUNGPulmonary (tail vein injection)AAV-DJRetina, lungs, kidneys, in vitro infected cellsAAV-7m8retinaAAV-ShH10YRetinal Muller cellsAAV-Rh10Liver, Blood, Heart, Extracorporeal Infected CellsAAV-Anc80L65Inner ear, retina, skeletal muscle, liverAAV-SCH9SVZ area neural stem cells Small suggestion: If you are unsure which serotype to choose, you can try using Pandora's Virus (AAV trial kit) as a pre experiment to compare the infection effects of different serotypes on the target tissue and explore the optimal experimental conditions (injection method, injection site, virus dosage, etc.) in order to obtain more ideal experimental results. (Link to Pandora's Virus>>)liver  The liver is an important organ for regulating lipid metabolism and blood sugar metabolism, and its abnormal function will lead to fatty liver, hypercholesterolemia, hyperlipidemia, diabetes and other diseases.  1. Injection method  The use of viral vectors to infect the liver is generally achieved through tail vein injection.  Based on the years of experience of Heyuan Biotechnology, using AAV vectors to infect liver tissue generally selects AAV2/8 serotype. To infect the liver through tail vein injection, each mouse needs to be injected with a viral vector with a total titer of 1x1010.    Results of AAV infection in metabiotics  2. Application cases 2.1 Liver specific promoter TGB  Customers publish articles:FASEB Journal. (IF=5.391). Yang Y,et.al. (2018). Long-term exercise prevents hepatic steatosis: a novel role of FABP1 in regulation of autophagy-lysosomal machinery. [Non alcoholic fatty liver disease, autophagy lysosomes, overexpression of adeno-associated viruses]  Injection method: tail vein injection in mice  carrier:AAV8-TGB-Fabp1  Serotype:rAAV2/8  Injection volume:200ul,2× 1011 VG  Observation time: 1 week  2.2 Application of Cre loxp technology in mouse liver  Due to its high affinity for the liver, adenoviruses can infect over 90% of the liver, making them an effective carrier for liver specific infections. Injecting the Cre adenovirus (Ad Cre) into mice with loxp through the tail vein can achieve liver specific expression and knock out the target gene, thereby quickly obtaining liver specific gene knockout mice.  2.3 Application of CRISPR/Cas9 technology in mouse liver  By injecting CRISPR/Cas9 system related elements (Cas9 and sgRNA) into mice through adenovirus, specific expression in the liver can be achieved, and specific knockout of the liver can be achieved. This method is more suitable for testing in vivo experiments.pancreas  The pancreas is an organ with both internal and external secretion functions, closely related to digestion, blood glucose metabolism, lipid metabolism, etc., and is also one of the key target organs in metabolic research.  In practical research, there are two main ways to use virus vectors to infect the pancreas. One is to use AAV2/8 virus vectors to inject the virus into the pancreas through the bile duct; Another new method is to use the AAV-PAN virus vector newly developed by Heyuan Biotechnology to infect the pancreas by intraperitoneal injection:和元生物AAV-PAN感染结果heart  1. Injection method  The injection method of AAV has been adopted differently in different articles. Overall, it generally includes intravenous injection, intracardiac injection, myocardial targeted injection, and pericardial injection (located between the myocardium and the pericardium), among which intravenous injection includes tail vein injection, jugular vein injection, facial vein injection, etc.  Tail vein injection and intracardiac injection are more evenly distributed due to the virus flowing into the bloodstream. Intracardiac injection needs to be performed in combination with aortic clamping, and due to the need for assisted breathing during thoracotomy, the surgical procedure requires high requirements. When intramyocardial injection is performed, 2-5ul is injected at each point, resulting in higher expression near the injection site, but limited diffusion. In general, many experiments can be directly injected into the tail vein, but higher doses of the virus may be required. If intramyocardial injection is used, that is, small and multiple point injections, good infection expression effects can be achieved. However, the latter requires slightly higher technical difficulty and longer operation time.  结合新生鼠(出生2-10天)注射效果比较来看,新生鼠注射表达效果大大优于成年鼠,消耗病毒剂量也相对较低。新生鼠的注射,一般根据实验室的条件来看,可以选择颈静脉注射、面部静脉注射和心腔内(左心室)注射,目的是将病毒导入血液循环中。  2. Promoter selection  Some commonly used promoters such as CMV, CAG, alpha MHC, cTnC, etc. are often used to drive expression in cardiac tissue. Many efficient promoters, such as alpha MHC, are only used in transgenic mice and are not suitable for loading onto AAV vectors due to their large fragments. Through literature review, we found that some small promoters such as CMV have strong expression ability in cardiac tissue, especially for large fragments, which have a significant driving effect. However, if the target gene needs to be mediated for specific expression in the heart, it is recommended to use the cTnT promoter, which is often used for myocardial specific expression and has strong expression ability.  3. Application cases 3.1 Cardiac specific promoter cTNT  Customers publish articles:Yue Z, et al., (2019) PDGFR-b Signaling Regulates Cardiomyocyte Proliferation and Myocardial Regeneration. Cell Rep.Injection method:Carrier:AAV9Serotype: rAAV2/9Promoter: cTnTInjection volume: 200ul, 2 × 1011 VGObservation time: 1 week3.2 Cas9 heart specific transgenic mice  Scientists have constructed heart specific transgenic mice with eSpCas9 (1.1), which drive the specific expression of Cas9 in the myocardium through the promoter of the Myh6 gene (α MHC). The alpha MHC promoter is a commonly used promoter in heart specific transgenic mice. This tool mouse can solve the problem of Cas9 delivery and expression in the heart during the experimental process. Transgenic mice can maintain long-term high levels of Cas9 expression in the heart, which is precisely the crux of the low editing efficiency of the CRISPR/Cas9 system in the cardiac region.    Cas9 transgenic mice only need to express sgRNA in the heart through AAV delivery, which can achieve relatively high editing efficiency. Researchers injected sgRNA targeting the Myh6 gene exon into mice via loading onto an AAV vector to achieve heart specific knockout. The expression level of Myh6 was reduced by more than 50%, and mutant mice with myocardial hypertrophy and dilated heart were successfully obtained.lungs At present, AAV virus vectors are commonly used for viral infections in the lungs. There are two main ways to use virus vectors to infect the pancreas. One is to use AAV2/9 virus vectors, which deliver the virus through the respiratory tract through the nose or trachea (via the respiratory tract), with a reference dose of 1 × 1011VG virus and a volume of 50ul; Another new method is to use the AAV-LUNG virus vector newly developed by Heyuan Biotechnology, and inject it into the tail vein to specifically infect the lungs:                                                                              Result of pulmonary infection caused by Heyuan AAV-LUNGmuscle         At present, AAV virus vectors are commonly used for virus vectors in muscle tissue, as AAV can not only efficiently and specifically target muscle tissue, but also express persistently in muscle tissue. Adenovirus can easily cause a strong immune response when used and is easily cleared. Its expression time is relatively short and it is less commonly used for targeted expression in muscle tissue. Although lentivirus infection has a wide range of expression and can efficiently express in cultured muscle cells, its infection expression in muscle tissue is very limited. At present, the main serotypes of AAV carriers targeting muscles are AAV1, AAV6, and AAV9. Among them, AAV9 has the highest expression efficiency and is more likely to be highly expressed in muscles.  1. Injection method        The study of using virus vectors to infect muscle tissue is generally divided into systemic injection and intramuscular injection. Systemic injection has relatively uniform expression, but most viruses are absorbed by other organ tissues, resulting in fewer viruses targeting muscles. Intramuscular injection is a type of targeted injection that can efficiently express the virus near the injection site. However, the disadvantage is that the spread of the virus is very limited, so multiple injections are often required, usually between 5-10ul per point.  2. Application cases  Injection method: intravenous injectioncarrier:AAV-CMV-miniDystrophinSerotype:rAAV2/9  Observation time: 16 weeksadipose tissueAdipose tissue is composed of a large number of adipocytes, divided into white adipose tissue, brown adipose tissue, and beige adipose tissue. Adipose tissue is an important organ for storing redundant energy in the body, as well as an important secretory organ. It plays an important physiological role in blood glucose metabolism, lipid metabolism, chronic inflammation, scar repair, and other aspects of the body.In research, if we use a viral vector to infect adipose tissue, we generally use targeted injection. The rAAV2/8 serotype is generally selected when using AAV virus vectors to infect adipose tissue. In multi-point injection of adipose tissue, each point injects approximately 1x1010 total titers of the virus vector.  AAV infected fat

Details

Chemical Genetics Technology

Original link:Metabiology - Chemical Genetics Technology  Chemical genetics technology (also known as pharmacological genetics technology) is an important new technology that has emerged in recent years along with photogenetics. This technology modifies some biomolecules to interact with previously unrecognizable small molecules, achieving controllable and reversible control of their activity (compounds can be added or removed at any time to initiate or interrupt specific reactions). This technology has been widely applied in signal transduction, drug development, functional genomics, and other research areas. They are widely used to enhance or inhibit neuronal activity in a cell specific and non-invasive manner. Although DREADDs lack precise time control capabilities like photogenetics, they are most likely to require long-term regulation of neuronal circuits during disease treatment, and DREADDs are highly suitable for such applications. In addition, many FDA approved drugs target GPCRs, and DREADDs are modified GPCRs, so DREADDs may provide rich possibilities in drug development.  Currently, the modified biomolecules include nucleic acid hybridization, protein kinases, various metabolic enzymes, and G protein coupled receptors (GPCRs). There are many chemical genetics platforms based on GPCRs modification, such as Allele specific activation of genetically encoded receptors constructed in 1991, Receptors activated solely by synthetic ligands (RASSLs) constructed in 1998, and Engineering modified receptors (RASSLs). And the Designer receptors exclusively activated by designer drugs (DREADDs) constructed in 2007, DREADDs have become the most widely used chemical genetics technology, and this article will mainly discuss DREADDs technology.    图1.化学遗传学技术  1) DREADDs technology  DREADDs technology was invented by Bryan L. Roth et al., who altered the structure of the G protein coupled receptor acetylcholine receptor, which can only be activated or inhibited by a specific compound Clozapine N-oxide (CNO). The receptor of such changes selectively acts on different GPC R-cascade reactions, including Gq, Gi, Gs, Golf, and β - arrestin, with Gq DREADD and Gi DREADD being the most widely used. By expressing the above receptors in cells, the results produced by CNO vary.  Figure 2: Principles of action of several commonly used DREADDs receptors(Scott M. Sternson & Bryan L. Roth, Annu. Rev. Neurosci., 2014) 2) Common DREADDs receptors  1、Gq-DREADD和hM3Dq:  The initial Gq-DREADD, also known as hM3Dq, was modified from the human muscarinic acetylcholine receptor (mAchRs) subtype M3 (also known as hM3). Under normal physiological conditions, hM3 binds to acetylcholine and then couples with Gq class G protein coupled receptors, acting on the signaling pathway of phospholipase C, inositol triphosphate, and intracellular calcium ions (Figure 3).  Surprisingly, as long as the Y3.33C and A5.46 sites are mutated, the hM3 receptor cannot couple with acetylcholine, but will bind to CNO at a nanomolar concentration level. This mutated hM3 receptor is named hM3Dq (human M3 mucosal DREADD receptor coupled to Gq). Due to the conservatism of Y3.33 and A5.46 in different subtypes of human muscarinic acetylcholine receptors, M1 and M5 have also been successfully transformed into Gq-DREADD (hM1Dq and hM5Dq). However, so far, hM3Dq remains the most widely used Gq-DREADD.  The results of CNO induced hM3Dq vary among different cell types, for example: 1) In mature neurons, CNO induced hM3Dq results in depolarization of neurons, enhancing neuronal excitability, which is also the most commonly used function of hM3Dq, which is to promote the discharge activity of divine meridians; 2) In astrocytes, it has been reported that CNO induced hM3Dq results in increased release of Ca+from astrocytes, thereby altering the physiological conditions of the autonomic nervous system; 3) Outside the nervous system, there are also some studies, such as expressing hM3Dq in pancreatic beta cells. Acute CNO treatment promotes insulin release, while chronic CNO treatment leads to an increase in beta cell numbers; In liver cells, activation of hM3Dq increases blood glucose levels, possibly due to increased glycolysis and gluconeogenesis.    Figure 3. hM3 binds to acetylcholine as a signaling molecule to activate the Gq coupled GPCRs signaling pathway  2、Gi-DREADD和hM4Di:  Y3.33 and A5.46 are conserved in different subtypes of human muscarinic acetylcholine receptors, so scientists can also mutate the Y3.33 and A5.46 sites on M2 and M4 mAchRs. As downstream of M2 and M4 activate the Gi channel, Gi DREADDs are generated, named hM2Di and hM4Di, which can activate the signaling pathway regulated by Gi (Figure 4).  Gi coupled GPCRs can activate G protein inward rectifying potassium channels (GIRK), and under the action of CNO, hM2Di and hM4Di receptors are activated to inhibit neuronal firing activity, with hM4Di being the most commonly used Gi DREADD. There are also studies indicating that hM4Di can inhibit the release of neurotransmitters, thereby achieving the effect of inhibiting neuronal activity.    Figure 4: hM4 binds to acetylcholine as a signaling molecule to activate the Gi coupled GPCRs signaling pathway  For more chemical genetic tools on viruses, please search for...  3) Application Strategies of DREADDs Technology  The application of DREADDs technology using viral vectors generally includes the following key steps (Figure 5):  1. According to the experimental purpose, determine the appropriate DREADDs receptors. Generally speaking, activate neurons by selecting hM3Dq and inhibit neurons by selecting hM4Di;  2. Expressing DREADDs receptors in animals using viral vectors;  3. Design an experimental plan to administer CNO drugs to animals within an appropriate time window, activate receptors, and administer CNO through brain targeted injection, intraperitoneal injection, and water feeding;  4. Phenotypic detection, which detects changes in neuronal activity through behavioral or electrophysiological methods.    Figure 5. General strategy of DREADDs technology  4) DREADDs technology advantages  The application of DREADDs technology and photogenetics technology is very similar, and their purposes are also the same. So, how do we choose them in our daily research? The selection criteria are an analysis of the advantages and disadvantages of two technologies:  The advantages of DREADDs technology mainly include:  1. The experimental requirements are relatively low and the operation is simple: unlike optogenetics technology that requires fiber optics, laser controllers, etc., DREADDs technology only requires conventional pharmacological techniques such as injection or feeding of CNO;  2. Non invasive: Unlike optical genetic technology, which requires craniotomy surgery to embed optical fibers, it will not affect mouse behavior due to additional load, and can regulate the activity of specific brain regions and neurons in mice with complete free movement;  3. Realizing long-term activation or inhibition of neuronal activity: Due to the different principles of the two technologies, photogenetics technology relies on the opening of photosensitive channels, which require the flow of ions on the cell membrane to generate potential changes and affect neuronal activity. However, long-term ion reverse concentration differences require the consumption of a large amount of ATP (such as ion pumps), which can cause cell damage and death. In addition, the thermal effect of light stimulation can also damage cells. In contrast, DREADDs express a receptor that can continuously activate or inhibit neuronal activity for several hours without affecting normal cellular physiology;  4. High safety: CNO is a metabolite of the FDA approved drug Clozapine, which is relatively safe for in vivo use. In addition, many FDA approved drugs target GPCRs, and DREADDs are modified GPCRs, so DREADDs may provide rich possibilities in drug development.  Table 1 Comparison of advantages and disadvantages between photogenetics and chemical genetics.  Photogenetic technology DREADDs technology  Time accuracy can reach millisecond or even sub millisecond hour levels, enabling sustained activation or inhibition of neuronal activity for several hours without affecting normal cellular physiology  Through brain localization injection, specific promoters, and subcellular organelle localization peptides in space, photosensitive proteins can be anchored to target cells or organelles for operation, reaching the level of a single cell. DREADDs receptors can be anchored to a specific type of cell through localization injection and specific promoters  Surgical technique for implanting fiber optic cables in craniotomy requires fiber optic cables, laser controllers, etc., which is relatively difficult and non immersive. Conventional pharmacological techniques such as injection or feeding of CNO are sufficient  Application Cases of DREADDs Technology  1、hM4Di  ① Customers publish articles:Science. (IF=41.058). Mu D,et.al. (2017). A central neural circuit for itch sensation. [Adenovirus, itching, photogenetics, chemical genetics]    Injection site: PBN in mice  carrier:AAV-hSyn-HA-hM4Di-IRES-mCitrine  Serotype:AAV2/9  Virus titer:1.0× 1013 VG/mL  Injection volume:150nl  Observation time: 3 weeks  ② Customers publish articles:Neuron . (IF=14.319). Xu HF, et al. (2019) A Disinhibitory Microcircuit Mediates Conditioned Social Fear in the Prefrontal Cortex. [Social phobia, AAV, chemical inheritance]  Injection site: PrL in mice  carrier:AAV2/9-hSyn-DIO-hM4D(Gi)-mCherry  Serotype:AAV2/9  Virus titer:3.44× 1013 VG/mL  Injection volume:100-200nl  Observation time: 4 weeks

Details

Construction of stable cell lines

Original link:Harmony Biology - Construction of Stable Cell LinesThe expression of exogenous genes in cells can be divided into two categories. One is transient expression, where exogenous DNA/RNA does not integrate into the host chromosome. Although it can achieve high levels of expression, it usually only lasts for a few days; One type is stable expression (constructing stable transformants), where exogenous DNA is integrated into the host cell chromosome, allowing the host cell to express the target gene for a long time.The basic principle of establishing a stable cell line is to clone exogenous DNA into a vector with certain resistance, transfect the vector into the host cell and integrate it into the host chromosome, and screen using the resistance markers contained in the vector. The most commonly used resistance screening markers for eukaryotic expression vectors are neomycin, hygromycin, and puromycin. Cell lines that can stably express the target protein or stably silence specific genes are screened.Screening methodScreening stable cell lines for lentiviral infectionBy utilizing the integrated expression characteristics of lentiviruses to screen stable cell lines, this method overcomes the drawbacks of the long cycle of traditional plasmid cloning methods and can efficiently obtain stable cell lines in a short period of time.Advantages of using lentivirus to prepare stable strains(1) Cells have a wide range of applications and can be used for various mammalian cells;(2) The construction of stable transformation plants has a short time and a long expression duration;(3) Multiple fluorescent markers and resistance genes are available to meet the requirements of observation experiments.Stable cell line classification  Mixed clone stable cell lines  Mixed clone cell lines are directly screened for drug resistance after gene transfection, and the screened cells express resistance genes and target genes, but contain various different cell clones. The integration positions and expression levels of target genes vary among different clones.  The screening of mixed clone cell lines is relatively fast and cost-effective. When the transfection efficiency of the cell line to be constructed is high and the expression efficiency of the target gene is high, the expression effect obtained by the monoclonal cell line is not significantly different from that of the mixed clone cell line. In this case, multi clone cell screening is an ideal choice. Monoclonal stable cell line  Monoclonal cell lines are cell lines obtained by amplifying single cells containing stable integrated exogenous fragments. The expression level of the target gene integration site in each cell is highly consistent, but some phenotypes may be lost. Monoclonal cell line screening has a long cycle and high cost. For cell lines that require subcellular localization experiments, monoclonal cell screening is usually recommended for those that are difficult to infect and have low expression rates.Service ProcessList of constructed stable strains (partial)Chinese nameCorresponding stable plantCorresponding cultivation conditionsCell sourceAstrocytoma of the human brainU-87 MG-luc-mNeonGreen-PuroMEM+10%FBS+2ug/ml PuromycinChinese Academy of SciencesHuman glioma cellsU251-CMV-EGFP-PuroDMEM+10%FBS+2ug/ml PuromycinChinese Academy of SciencesHuman bladder transitional cell carcinoma cellsT24-EGFP-PuroRPMI-1640+10%FBS+2ug/ml PuromycinChinese Academy of SciencesHuman pancreatic cancer cell lineSW 1990-EGFP-PuroL-15+10%FBS+2ug/ml PuromycinChinese Academy of SciencesHuman neuroblastoma cellsSK-N-SH-EGFP-PuroMEM+10%FBS+0.4ug/ml PuromycinChinese Academy of SciencesHuman breast cancer cellsSK-BR-3-EGFP-PuroDMEM+10%FBS+2ug/ml PuromycinChinese Academy of SciencesHuman cervical squamous cell carcinoma cellsSiHa-CMV-EGFP-PuroDMEM+10%FBS+2ug/ml PuromycinChinese Academy of SciencesMouse neuroma cellsNeuro-2a-EGFP-PuroMEM+10%FBS+2ug/ml PuromycinChinese Academy of SciencesMouse neuroma cellsNeuro-2a-CMV-PuroMEM+10%FBS+2ug/ml PuromycinChinese Academy of SciencesHuman breast cancer cellsMDA-MB-468-EGFP-PuroL-15+10%FBS+2ug/ml PuromycinChinese Academy of SciencesHuman breast cancer cellsMDA-MB-231-CMV-EGFP-Luc-PuroL-15+10%FBS+2ug/ml PuromycinChinese Academy of SciencesHuman breast cancer cellsMCF7-CMV-EGFP-PuroMEM+10%FBS+2ug/ml PuromycinChinese Academy of SciencesMouse lung cancer cellsLLC-CMV-sfGFP-Luc-PuroDMEM+10%FBS+2ug/ml PuromycinChinese Academy of SciencesHuman chronic myeloid leukemia cellsK-562-CMV-EGFP-PuroRPMI-1640+10%FBS+2ug/ml PuromycinChinese Academy of SciencesHuman T-cell leukemia cellsJurkat-CMV-EGFP-PuroRPMI-1640+10%FBS+0.5ug/ml PuromycinChinese Academy of SciencesHuman endometrial cancer cellsishikawa-CMV-PuroRPMI-1640+10%FBS+2ug/ml PuromycinChinese Academy of SciencesHuman liver cancer cellsHuH-7-CMV-sfGFP-Luc-PuroDMEM+10%FBS+2ug/ml PuromycinChinese Academy of SciencesHuman liver cancer cellsHuH-7-Luc PuroDMEM+10%FBS+2ug/ml PuromycinChinese Academy of Scienceshuman colon cancer cellHT-29-EGFP-PuroMcCoy’s 5A+10%FBS+2ug/ml PuromycinChinese Academy of SciencesHuman liver cancer cellsHep G2-CMV-EGFP-Luc-PuroDMEM+10% FBS+2ug/ml PuromycinChinese Academy of SciencesHuman liver cancer cellsHep G2-CMV-PuroDMEM+10% FBS+2ug/ml PuromycinChinese Academy of SciencesHuman cervical cancer cellsHeLa-EGFP-PuroDMEM+10% FBS+2ug/ml PuromycinChinese Academy of SciencesHuman cervical cancer cellsHeLa-RFP-PuroDMEM+10% FBS+2ug/ml PuromycinChinese Academy of Scienceshuman colon cancer cellHCT 116-CMV-EGFP-Luc-PuroMcCoy’s 5A+10%FBS+2ug/ml PuromycinChinese Academy of SciencesHuman pharyngeal squamous cell carcinoma cellsFaDu-CMV-EGFP-PuroMEM+10%FBS+1ug/ml PuromycinChinese Academy of SciencesMouse colon cancer cellsCT26.WT-CMV-Luc-PuroRPMI-1640+10%FBS+8ug/ml PuromycinChinese Academy of SciencesMouse colon cancer cellsCT26.WT-CMV-PuroRPMI-1640+10%FBS+8ug/ml PuromycinChinese Academy of SciencesHuman renal clear cell carcinoma with skin metastasis cellsCaki-1-EGFP-PuroDMEM+10% FBS+2ug/ml PuromycinChinese Academy of SciencesHamster kidney fibroblastBHK-21 [C-13]-CMV-PuroMEM+10%FBS+2ug/ml PuromycinChinese Academy of SciencesHuman papillary thyroid carcinoma cellsB-CPAP-CMV-EGFP-PuroRPMI-1640+10%FBS+1%NEAA+2ug/ml PuromycinChinese Academy of SciencesMouse melanoma cellsB16-CMV-PuroRPMI-1640+10%FBS+1ug/ml PuromycinChinese Academy of SciencesMouse melanoma cellsB16-CMV-EGFP-PuroRPMI-1640+10%FBS+1ug/ml PuromycinChinese Academy of SciencesHuman non-small cell lung cancer cellsA549-CMV-EGFP-PuroF12K+10% FBS+2ug/ml PuromycinChinese Academy of SciencesHuman non-small cell lung cancer cellsA549-luc-mNeonGreen-PuroF12K+10% FBS+2ug/ml PuromycinChinese Academy of SciencesMouse breast cancer cells4T1-CMV-Luc-PuroRPMI-1640+10%FBS+1ug/ml PuromycinChinese Academy of SciencesMouse breast cancer cells4T1-CMV-EGFP-PuroRPMI-1640+10%FBS+1ug/ml PuromycinChinese Academy of SciencesHuman gastric cancer cellsMKN-45-CBh-luc2-mNeonGreenA-PuroRPMI-1640+20%FBS+2ug/ml PuromycinConcordHuman lung adenocarcinoma cellsNCI-H1975-CBh-luc2-mNeonGreenA-PuroRPMI-1640+10%FBS+2ug/ml PuromycinChinese Academy of Scienceshuman colon cancer cellHCT116-CBh-luc2-mNeonGreenA-PuroMcCoy’s 5A+10%FBS+2ug/ml PuromycinChinese Academy of Sciences

Details

Knockout cell line construction

Original link:Genomics - Knockout Cell Line ConstructionClustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 is an adaptive immune defense mechanism formed by the long-term evolution of bacteria and archaea. The CRISPR/Cas9 system can be used for editing various genomes. The CRISPR/Cas9 system recognizes the cleavage region through small guide RNA (sgRNA) and uses Cas9 endonuclease to perform predicted cleavage in the middle of the recognition site, causing DNA double strand breaks and random mutations after cell error repair. The CRISPR/Cas9 system has high mutation efficiency, flexibility, simplicity, short cycle, and low cost, and has been widely used in gene editing research. The application areas of CRISPR/Cas9 system are gene knockout strain construction and gene knockout animal research.The company can provide project services including: CRISPR/Cas9 experimental protocol design, CRISPR/Cas9 gene knockout vector construction, CRISPR/Cas9 gene knockout vector virus packaging, CRISPR/Cas9 gene knockout cell line constructionService features:1. To customize a specific gene knockout cell line, only the gene name needs to be provided;2. Quickly obtain effective sgRNA with high gene editing efficiency;3. Short experimental period;4. CRISPR/Cas9 lentivirus packaging services can be provided as neededOperation processDesign, vector construction, and activity verification of sgRNA targeting the target sequence2Co transfection of target cell lines and preliminary screening of mixed clones3 Preparation of monoclonal antibodies, screening and identification of cell genotypes using monoclonal antibodies4 Amplification of positive cell lines

Details

Cellular Functional Experiments

Original link:Metabiology - Cellular Functional Experimentscell proliferation       Cell proliferation, apoptosis, and cell cycle are important phenotypes in tumor research, and are one of the problems solved by molecular biology and pharmacology research. By overexpressing or interfering with a certain gene in cells, studying the impact of a gene on cell proliferation ability, further studying the function of the gene, or conducting drug treatment on cells, studying the effect of drugs on proliferation.Experimental Principles (CCK-8)Cell proliferation is an important life characteristic of organisms, and cells proliferate through division, which is the basis for the growth, development, reproduction, and genetics of organisms. There are many research methods for cell proliferation, mainly including CCK8/CellTiter Glo ™ Wait for the method.Cell Counting Kit-8(abbreviationCCK-8)The reagent can be used for simple and accurate analysis of cell proliferation and toxicity. The basic principle is that the reagent contains WST-8 [chemical name: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfonobenzene) -2H-tetrazole monosodium salt], which is reduced by dehydrogenase in cells to a highly water-soluble yellow formazan product (Formazan die) under the action of electron carrier 1-methoxy-5-methylphenazine sulfate dimethyl ester (1-methoxy PMS). The number of generated Jia Zan substances is directly proportional to the number of live cells. Therefore, this characteristic can be directly used for cell proliferation and toxicity analysis.Advantages of CCK-8 method:  1. Easy to operate, avoiding the influence of human factors during the cell counting process;   2. Low cell dosage, high detection sensitivity, and stability;   3. The enzyme-linked immunosorbent assay (ELISA) reader can be used repeatedly, and the detection time is flexible, without affecting the subsequent experiments of cells;  The Formazan produced by CCK-8 is water-soluble and does not require liquid exchange, making it particularly suitable for suspended cells. Compared with MTT method, it is more convenient and safe.  Purpose: To investigate the effect of target genes on cell proliferation ability or the effect of drugs on cell proliferation ability  Materials: Target cells, stable transgenic strains (empty space, target gene)  Steps: Cell collection - Cell laying - (drug treatment) - Cell culture for 0, 6, 24, 48, and 72 hours, then add CCK-8 treatment and detect with an enzyme-linked immunosorbent assay (ELISA) readerExperimental Principles(CellTiter-Glo™)  ATP adenosine triphosphate (ATP adenosine triphosphate) participates in various enzymatic reactions in organisms and is an indicator of live cell metabolism. Its content directly reflects the number and state of cells. During the experiment, an equal volume of CellTier Glo was added to the cell culture medium ™ Reagents measure the luminescence value. In the light signal and system, the luminescence value is directly proportional to the amount of ATP, which is positively correlated with the number of live cells. Therefore, cell viability can be obtained by detecting the ATP content.  Advantages of CTG method:  Compared with ordinary MTT and CCK8 methods, the detection reagents of the CellTiter Glo? Luminescent live cell detection system have the highest sensitivity and longer signal duration. This system has been widely used in the field of life science research, such as activity detection of some bioactive factors, large-scale screening of anti-tumor drugs, cytotoxicity tests, and tumor radiosensitivity determination;  2 CellTiter-Glo ™ The reagents are compatible with commonly used culture media in cell culture, such as RPMI1640, MEM, DMEM, and Ham's F12, and are not affected by phenol red and organic solvents, with small errors and high accuracy.  Purpose: To investigate the effect of target genes on cell proliferation ability or the effect of drugs on cell proliferation ability  Material: Target cells  Steps: Cell collection - Cell laying - (drug treatment) - Cell culture for 0, 24, 48, 72, and 96 hours, then add CellTiter Glo ™ The solution was shaken with a microplate shaker for 5 minutes and left at room temperature for 10 minutes - the fluorescence value was detected by an enzyme-linked immunosorbent assay (ELISA) readerCell apoptosis  Apoptosis is a focus of tumor and developmental research, as well as a hot topic in pharmacological research. Apoptosis refers to the process in which a cell, under certain physiological or pathological conditions, follows its own program and autonomously terminates its life. It is an active, highly ordered, gene controlled process involving a series of enzymes. The process of cell apoptosis can be roughly divided into the following stages: receiving apoptotic signals → regulating the interactions between apoptotic molecules → activating proteolytic enzymes (Caspases) → entering a continuous reaction process.  Basic Principles  Cell apoptosis detection was performed using the Annexin V/PI dual staining method. Annexin V is a Ca2+dependent phospholipid binding protein with a molecular weight of 35-36kD, which can bind specifically to phosphatidylserine (PS) with high affinity. In the early stages of cell apoptosis, PS can flip from the inner side of the cell membrane to the surface of the cell membrane and be exposed to the extracellular environment. Annexin V can be labeled with fluorescein (FITC or PE) or Biotin, and labeled with Annexin V as a fluorescent probe. Flow cytometry or fluorescence microscopy can be used to detect the occurrence of cell apoptosis.  7-AAD, similar to propidine iodide (PI), is a nucleic acid dye that cannot penetrate the complete cell membrane. However, in the late stage of apoptosis and dead cells, 7-AAD can penetrate the cell membrane and cause the nucleus to become red stained. Therefore, by matching Annexin V with 7-AAD, cells in the early and late stages of apoptosis can be distinguished from dead cells.  Objective: To obtain cell lines overexpressing a certain gene through lentiviral infection, and detect the apoptosis of normal cells, control group cells, and gene overexpression group cells using Annexin PE and 7-AAD double staining methods, in order to study the effect of genes on cell apoptosis.  Materials: Plasmids and cell lines  Step: Cell preparation - Collect cells - Annexin PE and 7-AAD for double staining - Flow cytometry detectionCell invasion and migration  The ability of cells to enter the circulatory system is an important research object during the development of tumors. The related signaling pathways can mainly be divided into controlling cell adhesion and controlling the cytoskeleton. The migration and invasion of cells are mainly related to the cytoskeleton and adhesion. The changes in the invasion and migration ability of tumor cells are usually detected using Transwell. Cell scratch is also a method for measuring the motility characteristics of tumor cells, but its application is limited due to the inability to distinguish between normal proliferating and migrating cells.      Transwell experiment  A permeable membrane (usually polycarbonate membrane) at the bottom of the Transwell chamber is placed in a well plate, with the chamber referred to as the upper chamber and the culture plate referred to as the lower chamber. Due to the permeability of polycarbonate membranes, the components in the lower culture medium can affect the cells in the upper chamber. Transwell can be used to study the effects of components in the lower culture medium on cell growth, motility, and other factors.  Tumor migration experiment: studying the migration ability of tumor cells or the migration ability of tumor cells under specific circumstances. Commonly used are 8.0 and 12.0 µ m membranes. Tumor cells are seeded in the upper chamber, and FBS or certain specific chemokines are added to the lower chamber. Tumor cells will run towards the lower chamber with high nutritional content. Counting the number of cells entering the lower chamber can reflect the migration ability of tumor cells.  Tumor invasion experiment: studying the invasive ability of tumor cells or the invasive ability of tumor cells under specific circumstances. Apply a layer of matrix adhesive on the polycarbonate membrane to mimic the extracellular matrix. Tumor cells are seeded in the upper chamber, while FBS or certain specific chemokines are added to the lower chamber. Cells must digest the matrix before migrating from the upper chamber to the lower chamber. Count the number of cells entering the lower chamber to determine their invasive ability.  Purpose: To investigate the effect of overexpression/interference of target genes on cell invasion and metastasis ability  Materials: Normal cells, control lentivirus, overexpressed gene lentivirus  Steps: Cell Resuscitation - Transwell Planking - Cell Culture and Staining - Photography  Principle of Scratch Experiment  Tumor cells still have the ability to migrate in vitro. The cell scratch method is one of the methods used to determine the motility characteristics of tumor cells. It draws inspiration from the in vitro cell induced wound healing experimental model, scratches the wound on single-layer cells cultured in vitro, and then compares the migration ability of tumor cells in different experimental groups.  Purpose: Using screened stable strains (control virus, overexpressed gene virus), statistical analysis of scratch width was conducted on three groups of cells: empty cells, control stable transformation, and target gene stable transformation, to study the effect of genes on cell migration ability.  Materials: Virus and cell strains, target cells sourced from customers, stable strains and meta construction  Steps: Cell laying - Marking - Cell culture and photography - Statistical analysisCell cycle detection  The cell cycle refers to the entire process that a cell undergoes from the completion of one division to the end of the next division, divided into two stages: interphase and division. The cell cycle reflects the rate of cell proliferation, and the measurement of individual cell cycles can be done using time-lapse photography, but it cannot represent the cell population cycle. Therefore, other methods are often used to measure the population cycle.  principle:  The DNA content varies at different stages of the cell cycle. Typically, the G0/G1 phase of normal cells has the DNA content of diploid cells (2N), while the G2/M phase has the DNA content of tetraploid cells (4N), while the DNA content of the S phase is between diploid and tetraploid cells. PI, also known as propidium iodide, can bind to intracellular DNA and RNA. After RNA is digested by RNA enzymes, the fluorescence intensity of PI bound to DNA detected by flow cytometry directly reflects the amount of DNA in the cell.  Therefore, when using flow cytometry PI staining to detect intracellular DNA content, the cell cycle phases can be distinguished into G0/G1 phase, S phase, and G2/M phase, and the percentages of each phase can be calculated using special software. Therefore, when using flow cytometry PI staining to detect intracellular DNA content, the cell cycle phases can be distinguished into G0/G1 phase, S phase, and G2/M phase, and the percentages of each phase can be calculated using special software.  Materials: Target cells, viruses  Steps: Cell preparation - Sample collection - Machine testing - Data analysisClone formation experiment  Principle: When a single cell proliferates for more than 6 generations in vitro (for about 1 week or more), the cell population formed by its offspring is called a colony or clone. Each clone contains more than 50 cells, ranging in size from 0.3 to 1.0mm. The survival rate of cell inoculation only represents the number of cells that adhere to the wall after inoculation, but not all cells that adhere to the wall may be able to proliferate and form clones. Only cells with both adherent and proliferative activity will form clones. The clone formation rate reflects the strength of a cell's independent survival ability and is used to evaluate the population of cellsDependability and proliferation ability. Due to different biological characteristics of cells, there is also a significant difference in cell clone formation rate. Generally, the clone formation rate of primary cultured cells is weak, while the passaged cell lines are strong; The formation rate of diploid cell clones is weak, while the transformed cell lines are strong; Normal cell clone formation rate is weak, while tumor cells are strong. The clone formation rate is related to the inoculation density to a certain extent. When measuring the clone formation rate, the inoculated cells must be dispersed into a single-cell suspension and directly inoculated into a culture dish for more than a week. Check at any time and terminate the culture when the cells form clones.  Objective: To study the effect of genes on cell population dependence and proliferation ability through statistical analysis of the number of clone formation in the target gene overexpression/interference cell group, empty cell group, and negative control group (empty virus).  Materials: Viruses and cell strains  Steps: Planking - Cell Culture - Observe Clones, Stain, and Calculate Clone Formation Rate

Details

Fluorescent enzyme reporter gene testing

Original link:Heterobiosis - luciferase reporter gene detection  Heterobiosis luciferase reporter gene detection is an ideal reporter gene because mammalian cells do not contain endogenous luciferase, which immediately generates functional luciferase once transcription is complete. Single reporter gene experiments are often influenced by various experimental conditions, while dual reporter genes provide a baseline for the experiment through co transfected "controls" as internal references, which can minimize the impact of external factors such as cell activity and transfection efficiency on the experiment, making the data results more reliable. The Dual Luciferase ® reporter gene detection system expresses both firefly luciferase and sea kidney luciferase in cells simultaneously, without homology and corresponding to different reaction substrates, therefore there is no cross interference. Thanks to the super strong optical signal and ultra-high signal-to-noise ratio.  MiRNA target gene validation  MiRNA mainly acts on the 3 'UTR of the target gene (degrading or inhibiting translation). The 3' UTR (wild-type and binding site mutant) sequence of the target gene is constructed into the 3 'end of the reporter gene F-Luc in the vector. By comparing the changes in reporter gene expression (decreased or unchanged luciferase activity) after overexpression of miRNA, the site of action between miRNA and the 3' UTR of the target gene is determined.  Objective: To verify whether miRNA has a regulatory effect on the target gene 3'UTR.  Material: Plasmid pMIR-REPORT Luciferase gene 3'UTR (Wt); PMIR-REPORT Luciferase gene 3'UTR (Mut); PRL CMV (H321, Promega); 293T cell line  Step: Target prediction - Construct plasmid - Transfect cell detection - Report gene detection (Luciferase activity detection) - Statistical analysis  Result display:   Study on promoter activity  Transcription factors are protein molecules with special structures that regulate gene expression, also known as trans acting factors. Some transcription factors only bind to specific sequences in their target promoters, which are called cis factors. The DNA binding domain of transcription factors covalently binds to cis factors, thereby inhibiting or enhancing gene expression.  Transcription factors mainly act on the promoter of target genes by replacing the promoter region sequence of the target gene with the promoter of the reporter gene F-Luc. By co expressing transcription factors, changes in the expression of the reporter gene are determined to identify the binding site between transcription factors and target gene promoters, as well as their effects on the target gene.  Purpose: To investigate the effect of transcription factors on the promoter activity of target genes  Materials: Experimental plasmid (pGL4.10 gene promoter); Control plasmid; PRL CMV (E2261, Promega); transcription factor plasmid; 293T cell line  Step: Target prediction - Construct plasmid - Transfect cell detection - Report gene detection (Luciferase activity detection) - Statistical analysis  Result display:  The customer needs to provide information:  1. Target gene name  2 target gene species  3 names of regulatory factors (miRNA or transcription factors)  Provide to customers:  1. Prediction results of binding sites  2 Plasmids and Their Construction Report  3 Double Fluorescent Enzyme Detection Reports

Details

Cell line in stock

Original link:He Yuan Biotechnology - Cell Line in Stock  Heyuan Biotechnology has a rich cell inventory, with tumor cell lines as the main feature, covering hundreds of available tumor cell lines. At the same time, there are also stable cell lines that have been constructed to express fluorescence, resistance, or carry Luc for customer selection. The cell bank management is professional and standardized, and the technical team is specialized in cell culture, conservation, identification, and testing.  The stock list of cell lines is as follows (partially):Item numberCell nameSpecies and genusculture mediumChinese nameHYC3001293T/17peopleDMEM+10%FBSHuman embryonic kidney cellsHYC3099FaDupeopleMEM+10%FBSHuman pharyngeal squamous cell carcinoma cellsHYC3111Hep 3B2.1-7peopleMEM+10%FBSHuman liver cancer cellsHYC3112Hep G2peopleMEM+10% FBSHuman liver cancer cellsHYC3117HuH-7peopleDMEM+10%FBSHuman liver cancer cellsHYC3129AsPC-1peopleRPMI-1640+10%FBSHuman metastasis of pancreatic adenocarcinoma cellsHYC3131HCT 116peopleMycCoy’s 5A+10%FBShuman colon cancer cellHYC3132HT-29peopleMcCoy’s 5A+10%FBShuman colon cancer cellHYC3139LS 174TpeopleDMEM+10%FBSHuman colon adenocarcinoma cellsHYC3141HCT-15peopleRPMI-1640+10%FBSHuman colorectal adenocarcinoma cellsHYC3154786-O [786-0]peopleRPMI-1640+10%FBSHuman renal clear cell adenocarcinoma cellsHYC3161SV-HUC-1peopleF12K+10%FBSImmortalized human ureteral epithelial cellsHYC3164HuT 78peopleIMDM+20%FBSHuman T-cell leukemia cellsHYC3165COLO 320DMpeopleRPMI-1640+10%FBSHuman colorectal adenocarcinoma cellsHYC3172UM-UC-3peopleMEM+10%FBSHuman bladder transitional cell carcinomaHYC3187MDA-MB-436peopleL-15+10%FBSHuman breast adenocarcinoma cellsHYC3189SiHapeopleMEM+10%FBSHuman cervical squamous cell carcinoma cellsHYC3198ES-2peopleMcCOY’s 5A+10%FBSHuman ovarian clear cell carcinomaHYC3205MDA-MB-231peopleL-15+10%FBSHuman breast cancer cellsHYC3211BT-549peopleRPMI-1640+10%FBSorDMEM+20%FBSHuman breast ductal carcinoma cellsHYC3217A549peopleF12K+10% FBSHuman non-small cell lung cancer cellsHYC3221NCI-H292peopleRPMI-1640+10%FBSHuman lung cancer cells (lymph node metastasis)HYC3222MSTO-211HpeopleRPMI-1640+10%FBSHuman lung cancer cell lineHYC3223SK-MES-1peopleMEM+10%FBSHuman lung squamous cell carcinoma cellsHYC3225NCI-H1395peopleRPMI-1640+10%FBSHuman lung adenocarcinoma cellsHYC3228NCI-H1975peopleRPMI-1640+10%FBSHuman lung adenocarcinoma cellsHYC3235MG-63peopleMEM+10%FBSHuman osteosarcoma cellsHYC3236SW 1353peopleL-15+10%FBSHuman chondrosarcoma cellsHYC3238MNNG/HOS Cl #5 [R-1059-D]peopleMEM+10%FBSHuman osteosarcoma cellsHYC3241U-87 MGpeopleMEM+10%FBSAstrocytoma of the human brainHYC3244SK-N-SHpeopleMEM+10%FBSHuman neuroblastoma cellsHYC3246U251peopleDMEM+10%FBSHuman glioma cellsHYC3249CCRF-CEMpeopleRPMI-1640+10%FBSHuman acute lymphoblastic leukemia T lymphocytesHYC3254RajipeopleRPMI-1640+10%FBSBurkitt's lymphoma cellsHYC3256Jurkat, Clone E6-1peopleRPMI-1640+10%FBSHuman T-cell leukemia cellsHYC32576T-CEMpeopleRPMI-1640+10%FBSHuman T-cell leukemia cellsHYC3259K-562peopleIMDM+10%FBSHuman chronic myeloid leukemia cellsHYC3262RehpeopleRPMI-1640+10%FBSAcute non-B non-T lymphocytic leukemia in humans

Details

Animal experiments

Original link:Metaorganisms - Animal ExperimentsThe Animal Experiment Department of Heyuan Biotechnology was established in 2014 and has an advanced small animal SPF level animal room in China. It has an Experimental Animal Management Committee (IACUC) and holds the "Experimental Animal Use License" SYXK (Shanghai) 2017-0003 for SPF level mice and rats issued by the Shanghai Science and Technology Commission. The SPF area covers an area of 350m2 and can accommodate over 5000 mice and rats simultaneously, providing customers with approximately 50-100 batches of animal experimental project services. All animal rooms are equipped with independent ventilated animal IVC and automatic differential pressure regulation systems, real-time temperature and humidity monitoring systems, and one-way flow routes for separating people, animals, and objects, strictly ensuring the environment inside the barrier.All experimental operators in the Animal Experiment Department of Heyuan Biology have received strict training management and possess corresponding job qualification certificates. At the same time, the animal room is also equipped with professional management personnel to strictly ensure the cleanliness of the environment inside the barrier and the quality control of the animals.At present, Heyuan Biotechnology can provide one-stop services for the breeding and identification of mice and rats, animal model construction (including tumor, nervous system, metabolic disease models), pharmacological and pharmacological evaluation, behavioral and pathological testing, providing a good scientific research platform for the mechanism research of various diseases and the development of new drugs.Rat and mouse breeding services: The Animal Experiment Department of Heyuan Biology can provide rat and mouse breeding and identification services. According to customer needs, provide the most professional mating and feeding plan to improve breeding efficiency and save feeding costs.Advantages:·Comfortable and clean feeding environment·Strict quality control management system·We attach great importance to animal welfare and raise high-quality SPF grade rats and mice·Obtaining a large number of offspring mice with the same genotype in a short period of time at once

Details

Tumor animal model

Original link:Metaorganisms - Tumor Animal ModelsTumor animal models play a crucial role in basic research on tumor occurrence, development, metastasis, and drug development and treatment. Therefore, tumor research cannot be separated from the establishment of animal models. There are several commonly used tumor models in scientific research, including subcutaneous tumor formation, in situ tumor formation, and tail vein injection modelSubcutaneous tumor modelA tumor animal model established by directly seeding tumor cells (or tumor tissue) subcutaneously in mice. This tumor model is a simple and important tool for preclinical evaluation of drug efficacy in vivo, and also plays an important role in the study of tumor pathogenesis and drug action.Subcutaneous tumor formationThis model is easy to operate, allowing for intuitive observation of tumor growth and convenient detection of important data such as animal weight, tumor growth curve, and tumor weight;In situ transplantation modelIn situ transplantation refers to the transplantation of tumor cells or tumor tissue blocks into the tissues and organs of immunodeficient animals, resulting in the formation of tumors and spontaneous metastases.The method is to directly inoculate diluted tumor cells into the submucosa of the organ. Alternatively, tumor cells can be subcutaneously inoculated to a volume of around 500-800mm3, and the tumor tissue can be removed and cut into small pieces of 1-3mm3. The prepared cancer tissue block can be implanted under the serosa of the organ, and the serosa can be sutured to form a heterologous in situ tumor.Commonly used in situ vaccinations include: brain, liver, muscle, and breast pad in situ.                                  The in situ transplantation model can better simulate the microenvironment of tumor cell growth in vivo, and simulate the process of tumor growth and even metastasis.Tumor metastasis model by tail vein injectionAfter injection into the tail vein, tumor cells first enter the arterial blood circulation system through the pulmonary capillary network, which can cause multiple metastases throughout the body. However, due to the viscosity and tendency of tumor cells to aggregate, they are generally trapped in the microvessels of mouse lungs, mainly forming lung metastases, which may later lead to metastasis to distant organs. Mainly used to establish tumor metastasis (blood pathway) models, blood cancer models, or tumor lung metastasis.Tail vein injection of breast cancer cellsTumor metastasis is the most common cause of tumor mortality in clinical practice, but the mechanism and process of tumor metastasis are very complex and currently not fully understood. Establishing a metastasis model can better simulate the occurrence and development process of tumors in humans, and has higher predictive power for drug screening and development.The Heyuan Shanghai Animal Experiment Platform can provide various tumor animal model constructions, pathological sectioning, drug efficacy testing, small animal imaging, and other services such as subcutaneous tumor formation, in situ tumor formation, and tail vein injection metastasis tumor formation.

Details

Animal models of metabolic diseases

Original link:Metabolite - Animal Model of Metabolic DiseasesThe Animal Experiment Department of Heyuan Biology can provide animal models of obesity, diabetes, cardiovascular diseases, inflammation and other metabolic and circulatory diseases in rats and mice.1. Liver injury modelLiver disease is a general term for all diseases that occur in the liver. It includes infectious diseases, tumor diseases, vascular diseases, metabolic diseases, toxic diseases, autoimmune diseases, genetic diseases, intrahepatic biliary lithiasis, etc. Infectious diseases include viral infection, bacterial infection, parasitic infection, etc., such as viral hepatitis, liver echinococcosis, etc. Tumors are divided into benign tumors and malignant tumors, such as liver cancer, hepatic hemangioma, hepatic lipoma, hepatic sarcoma, etc. The application range of animal models for liver disease is very wide.Carbon tetrachloride induced liver injury model: A mouse model was established by intraperitoneal injection of different doses of carbon tetrachloride (CCl4) to induce acute liver injury in mice, and changes in plasma transaminase levels were detected.Cholestasis type mouse liver injury model: Inducing a cholestasis type mouse liver injury model through bile duct ligation.2. Establishment of renal ischemia-reperfusion injury modelChronic renal failure (CRF) model: Hemodynamic changes in residual nephrons after 5/6 nephrectomy in rats lead to hyperfiltration proteinuria in residual nephrons, resulting in CRF characterized by glomerulosclerosis.Acute kidney injury (AKI) model: AKI is a common critical illness involving multiple disciplines in clinical practice, with a prevalence rate of 3% to 10% in general hospitals and 30% to 60% in intensive care units. The mortality rate of critically ill patients is as high as 30% to 80%, and about 50% of surviving patients have permanent kidney injury. The prevention and treatment situation is severe. Establishing a stable AKI animal model is crucial for early detection and prevention of kidney disease research.3. Colitis modelThere are many types of gastrointestinal diseases, including ulcerative colitis, chronic enteritis, gastric bleeding, and gastric perforation, among which ulcerative colitis is currently a research hotspot. Constructing a stable animal model of ulcerative colitis is beneficial for studying and elucidating the pathogenesis of ulcerative colitis with unclear etiology and developing therapeutic drugs.DSS induced mouse ulcerative colitis model: Seven days after modeling, the mouse colon shortened, intestinal wall thickened, intestinal mucosal congestion, local bleeding, and obvious ulcerative surface were observed, with significant lesions in the cecum and distal colon. The HE staining results showed that the DSS group had mucosal loss, incomplete glandular tissue, extensive infiltration of inflammatory cells, and typical inflammatory changes. Weight changes: 2.5% DSS induced weight loss, decreased appetite, and poor activity in mice.4. Arthritis modelInduction of arthritis rat and mouse models using complete Freund's adjuvant. Arthritis refers to an inflammatory disease that occurs in the joints and surrounding tissues of the human body, caused by inflammation, infection, degeneration, trauma, or other factors. It can be divided into dozens of types.The animal model of compound drug-induced arthritis has the advantages of simple and feasible modeling method, minimal impact on animals, and easy and time-saving operation. After successful modeling, the animal joints showed significant swelling, which is similar to the symptoms of human arthritis. The modeling process is simple, stable, and the clinical, pathological, and immunological changes are similar to those of human arthritis, making it an ideal animal model for arthritis.5. Cardiovascular disease modelCoronary artery ligation induced myocardial ischemia models in rats and mice, including efficacy testing services for acute myocardial infarction, chronic myocardial infarction, chronic heart failure, and myocardial ischemia-reperfusion, mainly including modeling, efficacy testing, efficacy evaluation, etc.

Details

exosomes

Original link:Metabiotics - ExosomesExosomes are a type of extracellular vesicles (EVs) secreted by cells into the extracellular space. They are 30-150nm in size, have a double-layer membrane structure and a saucer like shape, and contain abundant contents (including nucleic acids, proteins, and lipids), participating in molecular transmission between cells. Extracellular vesicles are widely present in cell culture supernatants and various bodily fluids, including blood, lymph, saliva, urine, semen, milk, etc. They are also present in tissue samples, such as brain tissue, muscle tissue, adipose tissue, etc.Summary of brain tissue separation method: Cut brain tissue into thin slices, place them in a centrifuge tube and add digestive fluid for digestion. After water bath, gently invert repeatedly, and then use a pipette to intermittently and slowly blow and suck until digestion is complete. Then add to the culture medium based on digestion, mix well, and place on ice. Perform a series of differential overspeed centrifugation processes, including impurity removal, membrane filtration, superionization, etc. Finally, resuspend the extracellular vesicles with PBS, and identify them using transmission electron microscopy (TEM), nanoparticle size tracking molecules (NTA), and marker WB.Figure 1 Extracellular vesicle secretionAll cells can secrete exosomes, but there are significant differences in the quantity and content of exosomes secreted by different cells, which also determines that each type of exosome performs different functions. Extracellular vesicles are widely involved in intercellular material transport and information transmission, regulating cellular physiological activities. Meanwhile, extracellular vesicles have functions such as antigen presentation, immune escape, induction of normal cell transformation, and promotion of tumor occurrence and metastasis; In addition, extracellular vesicles can also serve as "natural nanoparticles" for drug delivery.What are the databases related to exosomes?The exoRBase database collects and describes all long RNAs in human blood exosomes, including circRNAs, lncRNAs, and mRNA.The EVpedia and Vesiclepedia databases summarize information on proteins, mRNA, miRNA, lipids, and other information found in different vesicle studies.The ExoCarta database mainly includes 286 research results from several species, including humans, rats, mice, sheep, etc., involving protein, mRNA, miRNA, lipid and other information.Sample preprocessing and extracellular vesicle separation and preservation:Extracellular vesicles exist in various bodily fluids of humans or animals, and we can choose different sample sources for relevant extracellular vesicle research. Due to the fact that exosomes are vesicular vesicles containing lipid bilayer membrane structures originating from the invagination of the cytoplasmic membrane, distributed in the extracellular matrix. In order to obtain high-purity extracellular vesicles, it is necessary to ensure the effective removal of all cell debris and other unwanted impurities.1) Cell culture supernatant             2) Plasma/Serum3)尿液                           4)脑脊液5)卵泡液                        6)宫腔液7)胆汁                            8)羊水9)腹水                           10)胸腔液不同的实验样本采集时需要注意的地方不一样,如细胞培养上清在收集样本前需换成无外泌体血清培养、血浆样本采集时用一定不能用肝素抗凝管、尿液收集时需要加抑菌剂等。从生物体液中分离外泌体的各种方法已经被开发出来,主要根据外泌体的大小、密度、免疫特性等特点进行操作。分离出高纯度的外泌体是我们后续开展外泌体研究的关键步骤,目前差速超速离心是外泌体分离方法中公认的“金标准”,也是高分文章中首选的分离方法。 外泌体提取在短时间(一周之内)使用,可以放在4度保存,如果长时间保存可以放在-20度或-80度保存。也可以将外泌体进行分装,分别放在-20度或-80度。外泌体检测方法:外泌体分离之后,需要经过一系列鉴定才能确定分离的是外泌体。鉴定方法从物理特征到表面分子标志物,多角度进行鉴定。l 透射电镜鉴定法:简称TEM,适合外泌体双层囊膜超微结构观察,即通常为茶托型或一侧凹陷的半球形。图二 透射电镜l 纳米颗粒跟踪分析法:简称NTA,该方法能保证外泌体原始状态、检测速度快,检测后能提供外泌体粒径和浓度信息。 图三 NTAl Western blot分子标志物检测:外泌体标志蛋白包括四跨膜蛋白家族,如CD9、CD63和CD81;细胞质蛋白,如肌动蛋白(Actin)和钙磷脂结合蛋白(Annexins);参与生物功能的分子,如凋亡转接基因2互作蛋白X(ALIX)、肿瘤易感基因101蛋白(TSG101)、热休克蛋白(HSP70、HSP90),以及细胞分泌的特异性蛋白。 图四 Western Blot(Tian Su et al., ACS Nano. 2019)外泌体高通量检测外泌体内含有与细胞来源相关的蛋白质和核酸,可以运输蛋白质、mRNA、miRNA、lncRNA、circRNA等进入受体细胞,参与细胞间通讯。不同细胞来源的外泌体所含有的蛋白成分和RNA不太相同,可作为多种疾病的早期诊断标记物,也能作为靶向药物的载体进行疾病治疗。1. miRNA高通量测序2. mRNA高通量测序3. lncRNA芯片(人、小鼠)4. ceRNA芯片(人、小鼠)5. 蛋白质组分析(iTRAQ、TMT、Label-free)外泌体标记或示踪:l 亲脂染料标记外泌体:目前已发表的外泌体文章中,外泌体大多使用亲脂性染料进行标记,体内和体外都有较多应用。亲脂性染料主要分为两大类,第一类是PKH67(绿色荧光)/PKH26(红色荧光),由于它们可以与外泌体的脂质双层膜稳定结合,所以染色效果较好,应用较广泛。 图五 PKH67标记的外泌体与神经元之间相互作用(Juan Carlos Polanco et al., Acta Neuropathol Commun. 2018) 图六 PKH26标记的外泌体与MDA‐MB‐231细胞共培养(Mengyu Yu et al., Cancer Sci. 2019)第二类是Di系列的亲脂性染料,包括DiI(橙色荧光)、DiO(绿色荧光)、DiD(红色荧光)、DiR(深红色荧光)。其中DiR的红外荧光可穿透细胞和组织,在活体成像中用来示踪。 图七 DiI标记的外泌体通过静脉注射观察在体内器官的分布情况(Laura Otero-Ortega et al., J Cereb Blood Flow Metab. 2018)l 慢病毒介导CD63-GFP表达:将外泌体的特定蛋白CD63和绿色荧光蛋白GFP的表达元件构建成质粒再包装到慢病毒中,随后用此慢病毒感染细胞,使细胞分泌的外泌体带有绿色荧光。 图八 用GFP标记的外泌体分别与SH-SY5Y、BV2和DRG细胞共培养(Rui Ren et al., Artif Cells Nanomed Biotechnol. 2019) 图九 注射有CD63-GFP的外泌体后观察第1天(D1)和第5天(D2)的荧光(Rui Ren et al., Artif Cells Nanomed Biotechnol. 2019)外泌体功能研究:将标记的外泌体加入受体细胞培养基中,与受体细胞进行共培养,观察细胞的功能变化,如细胞增殖、迁移与侵袭、细胞凋亡等;或者将外泌体注射入动物模型中,观察动物表型变化和检测动物相关指标。 图十 DiR标记的外泌体静脉注射小鼠结肠癌肿瘤模型(Gaofeng Liang et al., J Nanobiotechnology. 2020)  图十一 外泌体的功能研究(Tian Fang et al., Nat Commun. 2018)我们的优势:1) 实验周期短;2) 价格优惠;3) 完善的售后服务;4) 项目经验丰富,成功率几乎100%;5) 完整的一套实验服务。我们提供的服务:1) 差速超速离心分离;2) 透射电镜拍摄(TEM);3) 纳米粒径跟踪分析(NTA);4) Western Blot检测蛋白标志物;5) 外泌体高通量测序/芯片;6) 外泌体标记或示踪;7) 外泌体细胞功能检测;8) 外泌体动物注射;9) 外泌体动物模型验证等。

Details

Non coding RNA

Original link:Metaorganisms - Non coding RNA  Non coding RNA refers to RNA that does not encode proteins, including miRNA, lncRNA, circRNA, piRNA, etc. Non coding RNA functions in many ways, interacting with proteins, DNA, and RNA to participate in various cellular activities, including gene activation and silencing, RNA splicing, modification, and editing, and protein translation.  1. miRNA: miRNA is a type of non coding single stranded RNA molecule with a length of approximately 22 nucleotides encoded by endogenous genes, which participates in post transcriptional gene expression regulation in animals and plants. Each miRNA can have multiple target genes, and several miRNAs can also regulate the same gene.Figure 1 miRNA maturation and mode of action (Mikhailidis DP, Athyros VG. Nat Rev Cardiol. 2014 Feb; 11 (2): 72-4)  Overexpression of miRNA: Constructing miRNA precursor sequences into viral vectors, which can be used for transient transfection of cells or packaged into viruses for stable strain screening and animal level overexpression of miRNA.  MiRNA knockdown: Design a TuD RNA blocking fragment or sponge fragment targeting miRNA, and then construct the fragment into a viral vector. This vector can be directly transfected into cells for miRNA knockdown, or packaged as a lentivirus for animal level miRNA knockdown.  MiRNA target gene prediction: miRNA recognizes the 3'UTR region of target genes through base complementary pairing, and guides the silencing complex to degrade or inhibit the translation of target mRNA based on the degree of complementarity. Therefore, the 3 'UTR region of the target gene can be constructed onto a reporter gene containing luciferase, and the effect of miRNA on the target gene can be determined by comparing the changes in fluorescence values detected by miRNA overexpression or inhibitor with the reporter gene. Further determine the site of action between miRNA and the target gene 3'UTR by site directed mutagenesis of the 3'UTR region.  Common miRNA databases:  (1) MiRBase( http://www.mirbase.org ): miRbase is an online miRNA database developed by researchers at the University of Manchester, which provides a comprehensive database of published miRNA sequence data, annotations, predicted gene targets and other information, and is one of the most important public databases for storing miRNA information.  (2) Targeted Scan( http://www.targetscan.org/ )Targeted Scan predicts target genes by searching for conserved 8mer and 7mer loci that match each miRNA seed region.  (3) TarBase( http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=tarbasev8%2Findex )The TarBase database   spent about 10 years manually collecting target genes of experimentally validated miRNAs, including those in humans, mice, fruit flies, worms, and zebrafish.  (4) MiRWalk( http://www.ma.uni-heidelberg.de/apps/zmf/mirwalk/ )MiRKalkis is a comprehensive database that provides predicted information on miRNAs from humans, mice, and rats, as well as validated binding sites located on their target genes.  2. lncRNA: lncRNA is the abbreviation for long non coding RNA, which is an RNA molecule with a length between 200-100000 nt. Most lncRNAs exhibit significant spatiotemporal expression specificity during tissue differentiation and development; The conservation of sequences is relatively low, with only about 12% of lncRNAs found in organisms other than humans.  According to the position of lncRNAs relative to protein coding genes on the genome, they can be classified into seven types: promoter associated, intronic, bidirectional, sense, antisense, 3'UTR associated, and intergenic. This positional relationship is of great help in inferring the function of lncRNAs.  Figure 2 Classification of lncRNAs (Huang X, et al.Cancer Lett. 2018 Jan 28;413:94-101.)  Generally speaking, lncRNAs mainly regulate gene expression from the following three levels:  (1) Epigenetic regulation: lncRNAs recruit chromatin remodeling complexes to specific sites, thereby mediating the silencing of related gene expression.  (2) Transcription regulation: including the following: 1) The transcription of lncRNA can interfere with the expression of neighboring genes; 2) LncRNA can interfere with gene expression by blocking the promoter region; 3) LncRNA can interact with RNA binding proteins and localize them to gene promoter regions to regulate gene expression; 4) LncRNA can regulate the activity of transcription factors; 5) LncRNA can also regulate gene expression by modulating basic transcription factors.  (3) Post transcriptional regulation: lncRNA can regulate gene expression at the post transcriptional level by forming a double strand with mRNA.  Overexpression of lncRNA: Constructing lncRNA sequences into viral vectors, which can be used for transient transfection of cells or packaged into viruses for stable strain screening and animal level overexpression of lncRNA.  LncRNA interference: Design interference fragments targeting lncRNA and construct the fragments into viral vectors. This vector can be directly transfected into cells for lncRNA interference, or packaged as a lentivirus for animal level interference of lncRNA.  Knockout of lncRNA: Knockout of lncRNA using CRISPR/Cas9 technology. Design multiple sgRNA sequences at both ends of a certain lncRNA fragment to achieve large fragment deletion and achieve knockout effect. Construct the sgRNA sequence into a viral vector, then load the Cas9 sequence into another vector, and subsequently infect the target cells with both viruses for knockout.  Searching for lncRNA target genes: The method is similar to the miRNA dual luciferase assay described above.  3. circRNA: circRNA, also known as circular RNA, is a type of non coding RNA molecule that objectively exists in living organisms without a 5 'end cap and a 3' end poly (A) tail, and forms a circular structure through covalent bonds.    The main characteristics of circRNA are:    (1) CircRNAs are produced by special variable splicing and are abundant in the cytoplasm of eukaryotic cells, mainly derived from exons, with a small portion of circRNAs derived from introns present in the nucleus.  (2) The expression level has species, organization, and time specificity.  (3) CircRNA has a closed circular structure and is not easily degraded by nucleases, making it more stable than linear RNA.  (4) Has a certain degree of sequence conservation.  (5) Plays a regulatory role at the transcriptional or post transcriptional level.  (6) The vast majority of circRNAs are non coding, but there are also a few that can be translated into peptides.  CircRNAs can be divided into three categories based on their sources: exon derived circRNAs, intron derived circRNAs, and retained intron circRNAs.  The mechanism of action of circRNA:  MiRNA molecular sponge, circRNA contains a large number of miRNA binding sites and has miRNA sponge function, indirectly regulating the expression of downstream target genes of miRNA.  Regulating gene transcription, circRNA can also regulate protein function by binding to RNA binding proteins, such as inhibiting gene transcription by binding to transcription factors.  Although circRNAs belong to non coding RNAs, there are also a few circRNAs that can encode peptides and exercise regulatory functions through these peptides.  Overexpression of circRNA: Similar to the lncRNA steps mentioned above, but due to the impact of loop formation efficiency, overexpression of circRNA is more difficult compared to overexpression of ordinary genes.  CircRNA interference: the same steps as the lncRNA method mentioned above.  CircRNA knockout: the same steps as the lncRNA method mentioned above.  CircRNA target gene search: using the same miRNA and lncRNA dual luciferase detection method as described above.  Our advantages:  1) Rich project experience and high success rate;  2) Price discounts, enjoy discounts;  3) Short experimental period;  4) Comprehensive after-sales service.  The services we can provide:service content Service Form 1Service Form 2miRNAOverexpressionPlasmid constructionSlow virus/adenovirus/adenovirusmiRNAinterferePlasmid constructionSlow virus/adenovirus/adenoviruslncRNAOverexpressionPlasmid constructionSlow virus/adenovirus/adenoviruslncRNAinterferePlasmid constructionSlow virus/adenovirus/adenoviruslncRNAKnock outPlasmid constructionSlow virus/adenovirus/adenoviruscircRNAOverexpressionPlasmid constructionSlow virus/adenovirus/adenoviruscircRNAinterferePlasmid constructionSlow virus/adenovirus/adenoviruscircRNAKnock outPlasmid constructionSlow virus/adenovirus/adenovirusDual luciferase assayPlasmid constructionComplete outsourcing services

Details

NEWS CENTER

apply for trial
More
About Life-ilab Biotech
Company Introduction
  The company was formerly known as Shanghai Liji Biotechnology Co., Ltd., established in 2015. Heyuan Liji was jointly established by Heyuan Biotechnology (stock code: 688238) and Liji Biotechnology in 2023. It is a domestic biological reagent company that integrates research and development, production, and sales. Currently, it has a research and development team of several doctors from Fudan University, Jiaotong University, and Tongji University, as well as a young, professional and daring sales team. It has also established multiple authorized agent systems in more than 40 cities across the country. Li Ji Biological Reagent, Casting a Century Old Classic! We will continue to devote ourselves to the innovative research and development, high-quality production, and thoughtful service of biological experimental reagents, and contribute our modest efforts to the advancement of scientific research!
More
 
600 +

Independent products

700 +

Publish SCI

5000 +

Serve customers

10000 +

Area occupied

Contact Information

Landline Phone Number:021-50778506 
Service Manager:18616108315

Address:4th Floor, Building 27, Lane 908, Ziping Road, Pudong New Area International Medical Park, Shanghai

E-mail:lsj0027@obiosh.com

Copyright (C) 2015-2025 Heyuan Liji (Shanghai) Biotechnology Co., Ltd. All Rights Reserved

沪ICP备16001244号-1

Tel

18616108315

WeChat

QQ

Apply

Top