Virus Production

* This product is for research use only. Not intended for use in the treatment or diagnosis of disease.

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:

Product Information
Product NameBroad Spectrum DNA Transfection Reagent
Catalog NumberBT-000023
Shelf Life1 year.
StorageStore at 4°C.
Cell Densitiy and ConfluencyIdeally, cells should be ≥80% confluent prior to transfection.
CharacteristicsDNA transfection reagent for various cells.
Low cytotoxicity.
High transfection efficiency.
Product Information
Product NameLentiviral Transfection Reagent
Catalog NumberBT-000028
Shelf Life6 months, when properly stored and handled.
StorageStore at 4°C.
Cell Densitiy and ConfluencyOptimally, ~90% confluency.
CharacteristicsHigh transfection efficiency in HEK293 cells.
High titer lentivirus production.
Serum compatibility, no need to change any medium during transfection.
Product Information
Product NameVirus DNA Transfection Reagent
Catalog NumberBT-000027
Shelf Life6 months, when properly stored and handled.
StorageStore at -20°C.
Cell Densitiy and ConfluencyRecommended 80-95% confluence at the time of transfection for adherent HEK 293T/17 cells, 2 x 106 cells/ml for suspension 293-F cells.
CharacteristicsEfficient DNA delivery.
Flexible-compatible with different recombinant viruses and cell culture systems.
No animal sources.
Reliable-stable high virus titer production.
Culture format versatility.
Product Information
Product NameLarge RNA Transfection Reagent
Catalog NumberBT-000040
Shelf Life1 year.
StorageStore at 4°C.
Cell Densitiy and Confluency60%-80% while transfection.
CharacteristicsLow cytotoxicity.
High transfection efficiency -enable efficient RNA transfection in a variety of cell lines to ensure successful experiments.
Serum compatible -transfection in the presence of serum without changing media.
Suitable for all sizes of RNA -especially large RNA and CRISPR guide RNA.

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.

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