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Ilebo Biotechnology (Shanghai) Co., Ltd
Slow virus transfection experiment
NegotiableUpdate on 03/04
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Overview
The lentiviral transfection experiment is a technique that utilizes modified lentiviral vectors to stably introduce exogenous genes into host cells. Slow viruses belong to retroviruses and have the ability to infect dividing and non dividing cells. They can integrate target genes into the host genome and achieve long-term stable expression. This technology is widely used in fields such as gene function research, gene therapy, and the preparation of induced pluripotent stem cells (iPS cells).
Product Details
1Slow virus transfection experimentTechnical Definition and Core Values
lentiviral transductionIt is the use of modified lentiviral vectors to deliver exogenous genes to target cells, achievingLong term stable expressionGene transfer technology. Its core advantages include:
Broad spectrum cellular applicabilityCan infect both dividing and non dividing cells (such as neurons, stem cells, and primary cells), breaking through traditional transfection limitations.
Efficient integration of expressionVirus RNA is reverse transcribed into DNA and integrated into the host genome to achieve long-term expression of exogenous genes.
low immunogenicityDelete viral pathogenic genes (such as HIV)tatTherev）Significantly reduce the host immune response.
Convenience of operationWithout the need for complex transfection reagents, cells can be directly infected through viral particles.
Terminology analysis：
TransfectionUsually refers to non virus mediated nucleic acid introduction (such as electroporation, liposomes).
TransductionSpecifically referring to virus mediated gene transfer, lentiviral technology belongs to this category.
IISlow virus transfection experimentMolecular mechanism: from virus packaging to gene integration
（1） Composition of lentiviral vector system
| component | function | technology evolution |
|---|---|---|
| transfer plasmid | Carrying exogenous genes (such as cDNA, shRNA) and packaging signals (PSI) | Third generation carrier deletiontatUsing CMV promoter to drive transcription |
| Packaging plasmid | Expressing viral structural proteins (Gag, Pol) | Split into two plasmids, gag/pol and rev, to reduce the risk of recombination |
| Enveloped protein granule | Expression of VSV-G protein (vesicular stomatitis virus G protein) determines host range | Replace the natural envelope of HIV and expand the types of infected cells |
| helper plasmid | Provide Rev protein to promote the efflux of non spliced RNA | Enhance virus production |
（2） Gene delivery process within host cells
The virus entersVSV-G protein binds to target cell membrane receptors (such as LDLR) and mediates membrane fusion.
Reverse transcription and nuclear insertionVirus RNA is reverse transcribed into cDNA in the cytoplasm and transported into the nucleus via the pre integration complex (PIC).
genomic integrationVirus integrase randomly inserts cDNA into host chromosomes to achieve long-lasting expression.
3、 Standard Operating Procedures and Technical Optimization
（1） Virus packaging and purification (using 293T cells as an example)
| step | Key Operating Points | Quality Control Standards |
|---|---|---|
| Plasmid co transfection | Transfect 293T cells with a four plasmid system (transfer+packaging+encapsulation+adjuvant) and collect the supernatant within 48-72 hours | Plasmid purity>1.8 (A260/A280), no endotoxins |
| Virus concentration | Ultrafiltration (50000 × g) or PEG precipitation method can increase the titer by 10-100 times | Concentrated titer>1 × 10 ⁸ IFU/mL |
| Titer determination | Flow cytometry (fluorescent reporter gene) or qPCR (viral genome copy number) | Functional titer (TU/mL)>10 ⁷ is considered qualified |
（2） Transfection and screening of target cells
Optimization of infection conditions：
Add Polybrene (8 μ g/mL) to enhance virus adsorption.
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Multiple Infection Index (MOI) test: Typically MOI=5-20 (adjusted according to cell type).
Stable strain screening：
Antibiotic screening: Treat with puromycin (1 μ g/mL) for 7-14 days to clear untransfected cells.
Monoclonal amplification: Obtaining homogeneous cell lines using limited dilution method.
key techniques：
Non dividing cells (such as neurons) need to extend the infection time to 72 hours.
It is recommended to use low MOI (≤ 5) for primary cells to avoid toxicity.
