How to confirm whether the cell line was successfully transfected with two plasm - Co-transfection of two plasmids to 293 cells (May/05/2008 )
To understant how two genes (gene A and gene may work together, we made a 293 cell line transfected with two plasmid (one contains gene A and another contains gene at the same time (Fugene + plasmid A + plasmid . The backbone (BiGi) of plasmid A and plamid B is the same, and both plasmids is doxycycline inducible. Now we have some clones that are both gene A and gene B inducible. But there are two problems.
1, Now to test if the plasmid is actually in the cell line, we performed a PCR with forward primer in the plasmid backbone, the reverse primer in the gene A or gene B. We used template from these stably tranfected cell line's DNA and cDNA. The PCR give us a positive result from reaction with cDNA template but not DNA. This suggest that the inducible plamid does exist in the cell line but not detectable in the genomic DNA. Then where are they?
2, Then I need to prove that the gene A and gene B inducible cell line is actually not a mix of two cell lines. I mean it is not a mix of one cell line with inducible gene A and the other with inducible gene B. Histochemistry may be a way to prove this, by showing both gene strongly stained in the same cells when inducing the cell line with doxycycline. But I am wondering whether it is enough, because even some cells are positive for both genes detected by histochemistry, it doesn't discriminate from the endogenous gene A and B expressed in the untransfected 293, although endogenous gene A and B protein expression is very low in 293 cells.
Is in situ hybridization doable based on the sequence info you have for the clone A and B? Presumably, you could design probes cover two exons. This can be used in conjunction with re-cloning by limited dilution.
genehunter-1, thanks for your reply. I agree that re-cloning (sub-cloning) is necessary.
293 cells express low level the gene A and B, then I introduced the plasmid carrying gene A and B into 293 cells to make a inducible cell line. I am wondering if in situ hybridization will be at the same situation as histochemistry because they both can't discriminate the endogenous and transfeced gene. I think the ideal tequnique need to do the following:
1, to discriminate the the endogenous and transfeced gene.
2, to discriminate the transfected gene A and transfected gene B.
The only difference is at the cloning site of plamid A and plamid B (it means the junction site of plasmid backbone and gene A or . Don't know whether it's going to discriminate plasmid A vs B, as well as endogenous vs transfected gene, if the probe cover the junction site.
Do endogenous gene A and B contain intrans? Most do.
Yes, the endogenous gene have intron.
IF (immunofluorescence) should be able to detect if the individual cells are overexpressing the desired constructs when treated with tetracycline/doxycycline. Granted the cells that were not induced will have a bit of signal as you mentioned they express both genes but at low levels. When you induce the cell line you should be able to detect a major increase in staining when compared to the mother line or non-induced cells. This way you can look at individual cells to see if both genes are being overexpressed in the same cell. This way you can also detect if you have a mixed population of expressing cells.
I think you are right, I will compare gene expression from the mother cells, uninduced transfected cells and induced transfected cells.
Anymore idea is appreciated.