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Recommendation of Li Ji Biological Slow Virus Reagent at the end of the article; Forwarding and likes collection8mL of lentivirus concentrate；And also prepared for our buddiesDisney/Universal Studios tickets (two tickets for one person!!), Huawei Bluetooth headsetLottery, the draw will start on April 8th. Come and participate!
Slow viruses are often used as gene delivery and editing tools, with extensive applications in fields such as gene therapy, gene expression regulation, and disease model construction
main features
1. Stable gene transfer and expression: Slow virus vectors can stably integrate foreign genes into the host cell genome and transmit them to offspring cells during cell division. This integrated gene delivery method ensures the long-term stable expression of exogenous genes in host cells.
2. Controllable gene expression levels: By adjusting the construction and design of lentiviral vectors, it is possible to regulate the expression level of exogenous genes. For example, different promoters, enhancers, and regulatory elements can be used to control the transcription level of genes, thereby achieving regulation of target gene expression levels.
3. Multifunctional applications:Slow virus vectors can not only be used for gene delivery and gene expression regulation, but also for various applications such as gene editing, gene therapy, and cell engineering, such as CRISPR/Cas9 mediated gene editing, CAR-T cell therapy, etc. Through the mediation of lentiviral vectors, gene editing tools or specific gene sequences can be imported into target cells to achieve precise editing and regulation of genes, thereby changing the functions and characteristics of cells and achieving gene repair, mutation, or knockout functions.
Let's take a look at how to use lentiviral vectors to manipulate gene regulation from multiple perspectives
1、 Slow virus vector mediated overexpression and point mutations of coding genes
★Article Title：Gain-of-function mutations in the catalytic domain of DOT1L promote lung cancer malignant phenotypes via the MAPK/ERK signaling pathway
★Publishing journals：Science Advances
★Author's affiliation：Shenyang Pharmaceutical University
In this article, the author identified a series of functional acquired mutations in the histone methyltransferase DOT1L. The strongest one is located in the catalytic DOT domain R231Q, which can enhance the substrate binding ability of DOT1L. in addition，R231Q promotes cell growth and drug resistance of lung cancer cells in vitro and in vivo。Mechanism studies also indicate that the R231Q mutant specifically activates the MAPK/ERK signaling pathway by enriching H3K79me2 on the RAF1 promoter and epigenetic regulation of downstream target expression. The combination of DOT1L inhibitor (SGC0946) and MAPK/ERK axis inhibitor (binimetinib) can effectively reverse the phenotype induced by R231Q. The above research results reveal the functional acquired mutations of epigenetic enzymes and provide promising insights for precise treatment of lung cancer patients.
Figure 1. Mutation at the R231Q site of histone methyltransferase DOT1L can enhance its substrate binding ability and promote tumor growth
Infected cells | Human large cell lung cancer cells |
Regulatory methods | Gene overexpression (full length/point mutation) |
Carrier information | LV-DOT1L-WT/LV-DOT1L-R231Q |
2、 Slow viral vector mediated overexpression of non coding gene circRNA
★Article Title：CircCRIM1 suppresses osteosarcoma progression via sponging miR146a-5p and targeting NUMB
★Publishing journals：American Journal Of Cancer Research
★Author's affiliation：Jiangsu University School of Medicine
CircCRIM1 is a circular RNA formed by reverse splicing of the CRIM1 gene. Recent studies have shown that CircCRIM1 has multiple functions in the tumorigenesis of various malignant tumors, including osteosarcoma (OS). The author of this article investigated the role and mechanism of circCRIM1 in OS progression. Screening for differentially expressed circRNAs (including circCRIM1) in OS and human osteoblasts (hFOB1.19) by retrieving the GSE96964 circRNA expression microarray dataset. RT qPCR was used to detect the expression levels of circCRIM1, its sponge miRNA, and target genes.The effects of circCRIM1 on OS cell proliferation, migration, and invasion were studied through in vitro functional acquisition experiments.Evaluating the in vivo function of circCRIM1 on OS by measuring subcutaneous and in situ tumor growth in nude mice。Overexpression of circCRIM1 inhibits the migration, invasion, proliferation of OS cells in vitro and the growth of OS tumors in vivo。In terms of mechanism, the author identified miR146a-5p as a sponge miRNA of circCRIM1 through bioinformatics prediction, and confirmed their interaction and co localization through reporter gene detection and FISH analysis. This interaction leads to an increase in the expression of downstream target gene NUMB, which will result in inhibition of the Notch signaling pathway. The author further demonstrated that overexpression of miR146a-5p can reverse the anti-tumor effect induced by circCRIM1 in OS cells. The above research results support the role of circCRIM1 as a tumor suppressor in OS through sponge mediated miR146a-5p and its downstream target NUMB.
Figure 2. Overexpression of circCRIM1 inhibits the migration, invasion, proliferation, and growth of osteosarcoma cells in vitro and in vivo
Infected cells | Human osteosarcoma cells |
Regulatory methods | Non coding gene overexpression (circRNA) |
Carrier information | LV-circCRIM1 |
3、 Slow virus vector mediated non coding gene LncRNA interference
★Article Title：Long noncoding RNA AGPG regulates PFKFB3-mediated tumor glycolytic reprogramming
★Publishing journals：Nature communications
★Author's affiliation: Sun Yat sen University
Tumor cells typically reprogram their metabolism to achieve rapid proliferation. The role and potential mechanisms of long non coding RNA (lncRNA) in metabolic remodeling remain elusive. Through screening, the author found that lncRNA AGPG is necessary for increased glycolytic activity and cell proliferation in esophageal squamous cell carcinoma (ESCC). Mechanistically, AGPG binds and stabilizes 6-phosphofructose-2-kinase/fructose-2,6-diphosphatase-3 (PFKFB3). By blocking APC/C-mediated ubiquitination, AGPG protects PFKFB3 from proteasome degradation, leading to its accumulation in cancer cells, which then activates glycolytic flux and promotes cell cycle progression. AGPG is also a transcriptional target of p53; The absence or mutation of TP53 can trigger significant upregulation of AGPG. It is worth noting that,Inhibiting AGPG can significantly impair tumor growth in patient derived xenograft (PDX) models.In clinical practice, AGPG is highly expressed in many cancers, and its high expression level is associated with poor prognosis, indicating that AGPG is a potential biomarker and cancer treatment target.
Figure 3. Knocking down AGPG inhibits tumor formation, proliferation, and downstream gene expression
Infected cells | Human esophageal squamous cell carcinoma cells |
Regulatory methods | Non coding gene interference (LncRNA) |
Carrier information | LV-shAGPG |
The Vpack slow virus packaging system independently developed and patented by HarmonyBio overcomes the problems of virus non virulence and low titer, ensuring more accurate lncRNA expression, brighter vector fluorescence expression, and more stable virus virulence
Figure 4. Expression and Infection Effect of LncRNA in the VPack System of Metaorganisms
4、 CRISPR/Cas9 technology mediated gene knockout
★Article Title：A novel protein RASON encoded by a lncRNA controls oncogenic RAS signaling in KRAS mutant cancers
★Publishing journals：Cell Research
★Author's affiliation: Nanjing University
The author reported that RASON is a novel protein encoded by LINC00673, which is a positive regulator of oncogenic RAS signal transduction. RASON is abnormally overexpressed in pancreatic ductal adenocarcinoma (PDAC) patients, promoting proliferation of human PDAC cell lines in vitro and tumor growth in vivo.CRISPR/Cas9 mediated knockout of Rason in mouse embryonic fibroblasts inhibits KRAS mediated tumor transformation.The deletion of Rason gene eliminated the carcinogenic gene KRAS driven pancreatic cancer and lung cancer in LSL-KrasG12D Trp53R172H/+mice. Mechanistically speaking, RASON directly binds to KRASG12D/V and inhibits endogenous and GTP enzyme activating protein (GAP) mediated GTP hydrolysis, thereby maintaining KRASG12D/V in an active state of GTP binding. In treatment, RASON knockout will make KRAS mutant pancreatic cancer cells and patient derived organs sensitive to EGFR inhibitors. The above research results indicate that RASON is a key regulatory factor in the KRAS signaling transduction of oncogenes and a promising therapeutic target for KRAS mutant cancers.
Figure 5. Knockout of Rason in CRISPR/Cas9 mediated mouse embryonic fibroblasts inhibits KRAS mediated tumor transformation
Infected cells | Mouse embryonic fibroblasts |
Regulatory methods | CRISPR/Cas9 Knockout |
Carrier information | LV-sgRason-spCas9 |
5、 CRISPRa technology achieves endogenous transcriptional activation
In addition to gene knockout, CRISPR technology can also activate the transcription of endogenous genes. The use of CRISPR/Cas9 technology for gene knockout, insertion, and replacement relies on the ability of Cas9 protein to cleave DNA. However, if two splicing sites, RuvC1 and HNH, are artificially mutated, the cleavage ability of Cas9 protein is lost to become dCas9 protein, but it can still bind to gRNA to form complexes. DCas9 and gRNA complexes can bind to specific positions in DNA. Based on this, researchers connect some transcriptional regulatory elements, such as transcriptional activated VP64, VP16 elements, to the back of the complex, and anchor the complex upstream of the gene transcription initiation region to achieve transcriptional activation of specific genes.
Figure 6. SAM technology achieves endogenous gene activation
Chavez A,et al,.Nat Methods,2016
Figure 7. mCherry expression significantly increased after adding gRNA
Overall, lentiviral vectors have stable gene transfer and expression capabilities in gene regulation, enabling controlled and specific expression of exogenous genes, providing powerful tools and platforms for gene research, gene therapy, and cell engineering.
HarmonyBio is fortunate to provide the slow virus packaging service mentioned in this article
We provide the above design and customization of virus vectors to package and purify virus particles services with MetaBio.
Recommendation of reagents related to the Yuan Li Ji lentivirus
*For more products, please refer to the official website of Heyuan Liji：www.life-ilab.com
Slow virus activity
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Activity scope：National end customers related to biology
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