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Original link:Metaorganisms - Gene Overexpression
In 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 Carrier
According 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 system | eukaryotic expression vector | Lentiviral vector | Recombinant adenovirus vector | Recombinant adenovirus vector |
Vector gene characteristics | Double stranded DNA plasmid | RNA retrovirus | Single stranded DNA virus | Double stranded DNA virus |
Exogenous gene expression time | Expression starts at 24 hours and lasts for 3-7 days | Start expressing 2-4 days and maintain stable expression over a long period of time | Expression starts from 7-14 days | Starting from 1-2 days to express |
Insert fragment size | 8kbabout | 4kbabout | 2.8kbabout | 4kbabout |
Stable cell line screening | Difficult to operate | sure | may not | No, express it instantly |
Cell experiment | Cell lines with low transfection efficiency in some cases | Preferred, broad-spectrum, high infection efficiency | Cell lines, some serotypes have low transfection efficiency | Broad spectrum, high infection efficiency |
Animal experiments | unsuited | Suitable, based on observation time and injection site | First choice, based on observation time and injection site | Highly immunogenic, choose carefully |
Titer range | 无 | 108~109TU/ml | 1012-1013VG/ml | 1010-1011PFU/ml |
Introduction to common components in carrier diagrams
| Component Name | type | purpose |
Initiator | CMV、CAG、EF1a、U6etc | Expression of broad-spectrum or specific target genes |
hSyn、CaMKIIαetc | Specific promoters initiate the expression of target genes in specific tissue cells | |
Protein label | 3xFLAG、HA 、6xHis、Mycetc | Fusion expression with the target gene, commonly used to detect the expression level of exogenous proteins |
Fluorescent labeling | EGFP、mCherry、mNeonGreenetc | Can be fused or non fused with the target gene for detecting transfection and infection effects |
Resistance gene | Puro、Neo、Hygro、BSDetc | Eukaryotic resistance, commonly used for screening stable strains |
Amp、Kan | Prokaryotic resistance, commonly used in prokaryotic bacterial culture and plasmid amplification | |
Connecting components | Linker | Used to connect two genes and commonly used for fusion expression, it can reduce the impact of fusion expression on protein folding and function |
IRES、2A | Used to connect two genes to achieve non fusion expression | |
Other components | WPRE | Increase the expression of target genes |
Carrier selection (partial)
The selection of viral vectors should refer to lentiviruses, adeno-associated viruses, adenoviruses, retroviruses, etc. according to the experimental requirements.
| Carrier category | number | Carrier element |
Overexpression of lentivirus | GL127 | pSLenti-CMV-EGFP-3xFLAG-WPRE |
GL121 | pSLenti-EF1-EGFP-CMV-MCS-WPRE | |
GL107 | pSLenti-EF1-EGFP-P2A-Puro-CMV-MCS-3xFLAG-WPRE | |
GL122 | pSLenti-EF1-EGFP-F2A-BSR-CMV-MCS-WPRE | |
GL124 | pSLenti-EF1-Luc2-F2A-Puro-CMV-MCS-WPRE | |
Adenovirus overexpression | H225 | pADV-mCMV-MCS-3xFLAG |
H201 | pADV-mCMV-EGFP-3xFLAG | |
H213 | pADV-mCMV-3xFLAG-EGFP | |
H204 | pADV-mCMV-mCherry-HA | |
Overexpression of adenovirus | AOV024 | pAAV-CMV-EGFP-P2A-3xFLAG-WPRE |
AOV025 | pAAV-CMV-mCherry-P2A-3xFLAG-WPRE | |
AOV064 | pAAV-hSyn-EGFP-P2A-3xFLAG-WPRE | |
AOV065 | AAV-hSyn-mCherry-P2A-3xFLAG-WPRE | |
H9429 | pAAV-CAG-EGFP-3xFLAG-tWPA | |
H9525 | pAAV-CMV-EGFP-3xFLAG-tWPA | |
H10881 | pAAV-CAG-EGFP-3xFLAG-tWPA | |
H10886 | pAAV-CMV-EGFP-3xFLAG-tWPA |
Service Introduction
Based 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
