Can you receommend a vector? - (Jun/15/2011 )
I am doing some mutation analysis. I have generated 4 mutants and a wild type and have been trying to transfect my cells and generate single cell stable clones. The problem is that the gene is under the control of the CMV promoter and we are getting constitutive silencing, this even happens when using a GFP reporter, where there is no phenotype. We can maintain expression (as detected by western and qPCR) of the transgene for 8-12 cell passages, but then the CMV becomes silenced. We are working in Raw 267.4 cells, a macrophage line which are apparently notorious for this.
So far i am looking for a human promoter within the plasmid which is inducible by the addition of a drug or something similar.
If anyone has any experience of this or has a suggestion for a plasmid into which i can clone my mutants then it would be great to hear from you.
I have seen studies demonstrating the silencing of viral promoters in some cell lines. Generally the solution is to use a native promoter such as PGK, EF1-alpha, or actin. Since RAW cells are mouse derived, you might ant to check into the actiivty of thee promoters since the more popular version will come fomr human sequences, but in human cells I've seen data showing EF-1a is the strongest of these promoters.
Hope this is helpful,