Hello researchers,
i need some help, i am packaging Lentivirus( having my transgene) in HEK293T cells, as I will collect the supernatant and concentrate my lentivirus,I also want to check overexpression/expression of my gene/protein in HEK293 T cells after collecting supernatant....is this possible? if yes how can I proceed? and which assay I will likely use for this? qPCR? WB? kindly guide me in detail.
Thanks
Looking at both gene expression and protein blot would present a compelling evidence if you have the resources for both. PCRs are convenient to get an idea to see if Western blot is even worth doing as a general screening tool, though you don't always see a good correlation between gene expression vs protein so keep that in mind. For PCR, you also need to ensure the reference gene is stable across your protein overexpression group vs control to make a cross comparison possible. If you had a lot of comparisons/groups this would be a great screening tool to identify what's worth running Western blots. At the end of the day, as you are dealing with protein expression, protein data is the trump card and the most convincing evidence that your vector has an effect on protein translation. Western blot would be suitable for that. Even if gene-level data suggests there is enhanced expression of some protein, you will need to confirm marked protein expression as a result of your OE treatment.
Yes, you can measure the gene expression of your cargo cassette in 293T cells, Unless you are using a cell type-specific promotor.
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