I used the pInducer system to create stable cell lines, harbourig a tet-inducible shRNA against my GOI. After PURO-selection for single cell clones I checked for mRNA, protein expression and expression of RFP. I found some really good clones, showing up to 90% KD efficiency in both qPCR and Western after only 48h DOX treatment. Now, after several passages, I am not longer able to detect the KD on the protein level... Cells still express RFP and show good KD in mRNA levels - plus they show KD-specific phenotype - but no change in protein expression :/
Since, this is an inducible system I would not have expected adaptive effects...
Any suggestions, why this is happening and what I can do or try now?
Has anyone experianced similar things with the pInducer system?
I would be thankful for any help!!
Olesja
I hope you used retroviral/lentiviral system for creating stable cell lines. Better yet, clonal selections were employed.
Are you maintaining the Puro selection? "Although constant selective pressure may not be needed to maintain silencing of genes with a neutral or growth-promoting phenotype, it is possible that continuous selective pressure may be required for stable silencing of genes whose loss have a negative effect on cell growth or are essential for cell survival". (See: SHEILA A. STEWART, DEREK M. DYKXHOORN, DEBORAH PALLISER, et al,"Lentivirus-delivered stable gene silencing by RNAi in primary cells" RNA (2003), 9:493–501.
I would suggest to go back to your earliest frozen cells (master stocks), and grow up with Puro selection (of course after 1-2 days of culturing), check these for desired phenotype, and make further stocks while growing in Puro selection. A clonal selection strategy may also be considered at this point if you get the desired results, or go back all the way to new round of vector transduction, Puro selection, and clone isolation as by limiting dilutions method.
One other concern that can be envisioned is the possibility of any changes in the differentiation state of the cells involved, in particular when working with the primary cells. Culturing media and conditions, serum, DMSO, growth factors, matrix for adhesion/attachment, etc., can cause such changes.
I had same problem of my tet-on system, there is very good KD in mRNA level, but not protein level. Did you solve the problem? @Olesja Ritter
Even though in some cell lines I get good KD in protein level as well, but I had problem of getting the same phenotype in the inducible system as in my transient transduction system.
Did anyone had same issue when you switch your gene KD from constitutive and transient KD system to teton inducible system?
No I could not solve the problem. I had several "theories" but in the end the best was to start a second round of transfection and puro selection. I keep my clones constantly in puromycin now and I'm avoiding to keep them in culture for too long.
I can imagine that the discrepancy between mRNA and protein levels could be due to stabilized proteins. You can easily test this in an Anisomycin or Cyclohexamide experiment. If this is the case, prolonged tet-induction might be helpful. Try to induce for a longer time and keep in mind that tet is only stable for up to 2 days in cell culture medium.
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