Editing Retrovirus Production

1. On the day before transfection, plate 0.5-1 x10<sup>6 </sup>293T cells onto 6cm dishes in 4-5 ml DME/10%IFS. The cells are ready for transfection after 18-20 hours, or when they are about 25-30% confluent. Plate slightly more cells when making VSV-G pseudotyped viruses. For 10cm dishes, double the quantities. 

2. Pipette 6 ÎŒl GeneExpressoMax into serum-free medium (at room temperature) so that the final volume after the addition of the DNA will be 100ÎŒl. 

3. Add 2 ÎŒg total DNA (in TE). For instance, if using pCL vectors (Ampho, 10A1, or Eco; Naviaux (1996) J. Virol, vol 70, p 5701), use 1ÎŒg transfer vector (pBabe, pMSCV-IRESGFP, etc.) and 1 ÎŒg of the pCL vector. For making VSV-G retroviruses by triple transfection use 0.9ÎŒg gag/pol expression vector, 0.1 ÎŒg VSV-G expression vector, and 1 ÎŒg transfer vector. 

4. Do not vortex; swirl to mix. 

5. Incubate the GeneExpresso/medium/DNA solution at room temperature for 15 minutes. 

6. Transfer the mixture onto the 293T cells using a pipet. 

7. Incubate overnight at 37 C. 

8. On 3rd day following transfection feed the cells with 3-4 ml fresh medium. 

9. Harvest viral supernatants at 48 and 72 hours (or 36 and 60 hours). Ecotropic and GaLV viral titers at 72 hours will be will be approximately 20-40% less than at 48 hours. VSV-G viral titers are essentially the same at 48, 72, and 96 hours. Filter with a 45 uM syringe filter. 

10. In a typical viral infection protocol for adherent cells: use 3.5 ml of 36-48 hour viral sup, with 8 ug/ml polybrene, to infect 1-2 x10<sup>6</sup> cells (20-30% confluent) on a 10 cm dish. After 3 hours remove the sup and add fresh medium. 

11. Repeat the infection 24 hours later with the 60-72 hour sup. 24 hours after the second infection, split the cells; apply drug selection or sort the day following the split. Although two infections are optimal, one infection is often enough. 

Note: For collecting VSV-G viruses, use medium buffered with 10-25 mM HEPES to limit pH dependent cell killing by the VSV-G protein. With this protocol ECO virus titer is ~ 10<sup>7</sup>/ml, VSV-G, 10A1, and AMPHO viruses with titer is 10<sup>6</sup>/ml. 

[[Category:Protocols]]