Hi Nitin,

First I would suggest to titer your supernatant, depending of your envelope protein (ex: anti-p24 titration), to know if you do product particles or not.

If you don't, maybe double-check your transfected plasmids (ex: restriction and sequencing). You can also check your packaging protein production by WB.

If you do product particles, the titer can be too low but a transduction issue would be surprising.

Hello Nitin,

do I understand it correctly, that the viral transduction of your MCF7 cells has worked before too? Maybe one of the following suggestions could be helpful for you:

- titer the produced virus

- determine MOI for viral infection of MCF7 cells

- mycoplasma and STR test for your cell lines to validate them

- depending on the transfection protocol of HEK293T cells increase efficiency (e.g. if calcium phosphate transfection use chloroquine and/or sodium butyrate in the protocol)

Thanks Benjamin and Janine for your suggestions.

Ben, if I understand it correctly, you are mainly suggesting to check for viral production by WB against an envelop/packaging protein (like p24)? Please correct me if I am wrong. Also Ben, how do I know if p24 is actually coming from the viral particles or from the transient transfections of pachaging plasmids?

Janine, we routinely check our cells for Mycoplasma but I will have a look when we did it last time. Regarding titre and MOI, I thought you need to have at least some viral production, which, in our case, is nil.

Let me work with your suggestions and see. Will keep you posted.

Thanks

There are ELISA kits to titer viral productions. For HIV derived lentiviruses, p24 is the capsid protein detected and quantified. You shouldn't find p24 in your supernatent except in viral particles.

I think WB is maybe the last thing to do after checking method and plasmids' sequence.

What do you use to transfect your HEK 293T ? Maybe replace your solution and try with a new one, this is the easiest checking to be done.