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Fail lentiviral transduction. And what is Lenti-CRISPRv2

lentiviral transduction vero crispr cas9

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#1 Thomson

Thomson

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Posted 21 July 2020 - 08:34 PM

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. 

 

Thanks!

 







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