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Classification of Viral Vector Systems

The classification of viral vector systems includes:

• Retroviruses
• Lentiviruses
• Adenoviruses
• Adeno-associated viruses

Virus Production Procedure

Depending on the intended application, unidirectional or bidirectional cDNA libraries can be generated. Complementary DNA is generated using oligo-DT primers or random hexamer primers.

1. Viral vectors were first constructed as plasmids in E. coli.
2. This was transfected into packaging cells along with several helper plasmids.
3. Inside the packaging cells, the vector DNA located between the LTRs is transcribed into RNA, and the viral proteins expressed by the helper plasmids are further packaged into viruses.
4. Live virus is released into the supernatant and can be used to infect target cells directly or in concentrated form.

When the virus is added to the target cell, the genetic material is shuttled into the cell where it is randomly integrated into the host genome. Any genes placed between the LTR or ITR during vector cloning are permanently inserted into the host's DNA along with the rest of the viral genome.

Virus Production and Transfection Steps

1. Construct viral plasmid vector

2. Transfect packaged cells

Transport plasmids, envelope plasmids and gag-pol plasmids were co-transfected with packaging cells (such as cells derived from HEK293T were highly transfected with different transfection reagents).

Some transfection reagents are available for the production of viral vectors:

• Broad Spectrum DNA Transfection Reagent

• Lentiviral Transfection Reagent

• Virus DNA Transfection Reagent

• Large RNA Transfection Reagent

3. Collect the virus pellets

The time to reach maximum virus concentration is usually 48-72 hours after incubation. The culture medium was removed and filtered through a 0.45μm filter to obtain culture supernatant containing short-term production of virus, stored at -70°C or -80°C, or immediately used for infection of other packaged cell lines.

4. Purified virus

5. Virus titer

The common method is to infect the target cells with the virus, which can be roughly quantified according to the presence of vector RNA or protein.

References

1. Kitamura T; et al. Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics. Experimental Hematology. 2003. 31(11): p. 1007-1014.
2. Xu W; et al. Evaluation of residual promoter activity in γ-retroviral self-inactivating (SIN) vectors. Mol Ther. 2012. 20(1): p. 84-90.
3. Perry C; et al. Lentiviral Vector Bioprocessing. Viruses. 2021. 13(2).
4. Bulcha J.T.; et al. Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther. 2021. 6(1): p. 53.

Related Sections:

• Transfection Reagents for Genetic Therapy
• Transfection Reagents for Drug Screening Study