Hi,
My CRISPR vector carries an RFP tag. I can observe fluorescence when I package the lentivirus using 293T cells. After collecting the lentivirus and determining the titer by qPCR, I re-transfected 293T cells with the virus (MOI: 10). But I couldn't see the fluorescence, I'm not sure which step is wrong. Thanks a lot.
Hi Tianyu:
Just to clarify, by "re-transfected 293T cells" I assume you mean you are "infecting" the 293T cells in which you had "transfected" the plasmids by the virus you produced. If that is the case, I believe you are observing superinfection resistance which happens due to surface downregulation of the receptor used for the infection process. To confirm that, you should produce the virus in 293T cells via transfection and then infect a fresh batch of 293T cells, NOT the ones you already transfected. If you are still unsuccessful, then you should check which envelop you are using to pseudotype the virus and use cells for infection that have the receptor for that env. Normally, VSV-G Env is used for pseudotyping lentiviral vectors and VSV-G Env allows infection of a wide variety of cells.
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