This is the first time I did retroviral transduction to generate stable cell line expressing the gene of interest. The recipe is as follows:
1. Produce retrovirus with backbone (pMXs-gene-HygR) + packaging (psPAX2-gag-pol)+ Env (pVSV-G) using normal 293T. I also did use plat-E cells and just transfect with backbone (done using 24 well plate).
2. In both cases, I did not measure viral titer, just transduce into VERO cells with different volume (500uL - 50uL) and wait 48h then start antibiotic selection.
3. All cells died 2-3 days after antibiotic selection, whether highest lentiviral load or lowest load.
I wonder what went wrong.
Also, I came across info on Lenti-CRISPRv2 and I just wondering how does the lentiviral CRISPR cas9 system works compared to traditional lentiviral knock in strategy.