Hello Che-Wei, I have no experience with RPE-1 cells, but used to use lentiviral vectors on various cell types. I don`t think it is the virus itself, although if it concerns you can try to reduce the virus load. What MOI do you use?

I am not sure what sh-Luc stands for. You could check the toxicity of the transgene by delivering the plasmid by another method. Can RPE-1 cells be transfected by lipofectamine?

How do you purify and concentrate your LV preparation? Could there be impurities in there?

Hello Che-Wei,

LVs can be toxic to some cell type if you concentrated them and/or add polybrene in high concentrations. There are a lot of different factors that will influence LVs toxicity. 2nd harvest of LV are usually less toxic since they have less cellular debries.

More surely you have a combination of LVs (VSVg) toxicity and sh-Luc toxicity. If your LVs have very high titre, overexpression of the shRNA could also be toxic. If that is the case, try to use lower MOIs

Hi che-whei did you finally solve the problem? I face the same problem but using huh78 cells