Stable transfection of Pro-apoptotic genes - How is it possible? (Jul/16/2008 )
Hi,
I did a stable transfection of my gene, which is suspected to be pro-apoptotic.
Whole populations were used for future functional assays, like TUNEL, Annexin-FITC etc.
when I presented some of my data, I was questioned by this faculty member, who strongly feels that apoptotic genes will just kill the cells in the long run, how is ever possible to get stable clones if they die out?
I'm not too sure, are there papers out there where there are clones for Bax, Bad, etc?
The only weak answer I came up with was that my gene was not a strong apoptotic inducer.
What suggestions would everyone here come up with? For any apoptotic genes?
I did a stable transfection of my gene, which is suspected to be pro-apoptotic.
Whole populations were used for future functional assays, like TUNEL, Annexin-FITC etc.
when I presented some of my data, I was questioned by this faculty member, who strongly feels that apoptotic genes will just kill the cells in the long run, how is ever possible to get stable clones if they die out?
I'm not too sure, are there papers out there where there are clones for Bax, Bad, etc?
The only weak answer I came up with was that my gene was not a strong apoptotic inducer.
What suggestions would everyone here come up with? For any apoptotic genes?
Don't know the literature about apoptosis etc.
Are you sure your gene is expressed (protein detected with western)?
Maybe it indeed is a very weak inducer, but then you wouldn't notice with Annexin (as this has very aspecific binding as it is).
For a stronger inducer one could try inducible expression (after stable transfection with an inducible promotor before your gene of interest).
In my opinion, it would be very hard to have a cell line stably overexpressing a pro-apoptotic protein.. And even if your protein is a weak inducer, it still is an inducer, and since eukaryotic cells tend naturally to die in absence of survival signals, the result would most certainly be apoptosis.
Why do you want a stable cell line? Transient transfection would not be sufficient? Or knockdown approaches?
I did a stable transfection of my gene, which is suspected to be pro-apoptotic.
Whole populations were used for future functional assays, like TUNEL, Annexin-FITC etc.
when I presented some of my data, I was questioned by this faculty member, who strongly feels that apoptotic genes will just kill the cells in the long run, how is ever possible to get stable clones if they die out?
I'm not too sure, are there papers out there where there are clones for Bax, Bad, etc?
The only weak answer I came up with was that my gene was not a strong apoptotic inducer.
What suggestions would everyone here come up with? For any apoptotic genes?
most pro-apoptotic gene products must be triggered; if your protein is not triggered or mutated to a constitutive trigger you may succeed; I, for instance, succeeded in stable overexpression of the phosphomimetically activated form of a pro-apoptotic kinase: the apoptotic rate is always higher than of parental cells but not killing all cells; during the clonal selection process, the instable transgene cells are already eliminated by apoptosis
My two cents,
I agree with madrius. Is transient transfection not suitable? I do some work with the pro-apoptotic proteins you mentioned (and their anti-apoptotic Bcl-2 family members) and read a lot of literature of people working with transient transfections. Good literature. (i.e. http://www.ncbi.nlm.nih.gov/pubmed/1860661...ubmed_RVDocSum) is an example of some literature. I don't know if your working with mice but there is really good stuff out there. Interested as to why you want a stable transfection? Hope this helps