I want to look at the effect of a mutant form of GEF (guanine nucleotide exchange factor) on the resultant downstream small GTPase activation in neuronal cultures. To do so, I am planning on infecting neuronal cultures with Sindbis Virus to deliver this mutant recombinant protein. What I want to know is how will the infection typically affect the endogenous GEF protein level in neuronal cultures?
Sindbis Virus-mediated expression of protein of interest is so strong that it might block the overall endogenous protein expression, not just the endogenous GEF, especially in vivo. Suppose your in vitro assay is to be performed before any toxic effect is seen, then it's a matter of whether that GEF has a feedback control of its expression level or not. So, it depends on the protein and the timing after the infection.
If you are just expressing the active-form or dominant-negative form, then the endogenous portion is not so much of a problem. If it's an inactive mutant, endogenous activity cannot be ignored. In some cases you really want to remove any confounding effect of the endogenous protein in your assays, I'd advise you use the knockdown (shRNA) in combination with expression of your mutant form that harbours the shRNA-resistant silent mutation. Hope it helps a bit.
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