Hi all,
I am trying to generate some stable cell lines expressing either the WT or mutant version of my gene of interest (GOI). My lentiviral construct consists of a BSD gene for blasticidin selection, my GOI and a GFP gene. My GOI and GFP gene are under the same promoter. After lentiviral transduction, cells undergo sorting to select only cells expressing GFP and they are always grown in selection media.
While I am able to obtain my stable cell line expressing the WT successfully, the mutant cell lines always lose GFP expression after sorting. However, these cells are still able to survive on selection media. I did a western blot and determined that they are not expressing my mutant GOI. I did a PCR later and found out that my GOI is not integrated into its genomic DNA at all! But they are still growing under antibiotic selection! Is it possible that the cells preferentially kicked out my gene of interest and not the BSD gene? I am also puzzled why this only happens when I tried to generate stable cell lines of the mutants and not the WT GOI. Does anyone have any idea what could possibly be going on around here?
I apologise for the long qsn and thank you in advance.
Obviously, the transduction was successful or the cell would not be resistant to Bla. And the same GFP signal could be detected even to be sorted out, adding the rationale of successful transduction.
The question is, cells lost expression of GOI after several days. I don't know exactly the template for the PCR but I'd like to recognize it as genomic DNA, you need to show positive control of this procedure. And if the template is reverse transcribed cDNA, that's predictable.
My inference for this situation: there might be some epigenetic control mechanism for your GOI mutant expression, i.e. the cell had some feed back mechanism to totally block your GOI transcription, RNAi or mediating mRNA degradation when the mutant form expressed, if GFP downstream of the GOI, they could not be transcribed or expressed, either. And the resistant gene could be transcribed by another promoter.
What a life.
If there is negative selection for your mutant protein due to toxicity, such tricky clonal selections are possible (especially in case of large multi-cassette lentiviral vectors). I have also had situations where the protein expression was lost, while cells remained puro/hygro-resistant (never checked whether it was promoter suppression or GOI loss!!). If you really want to have reliable comparison of the WT and mutant proteins, the best solution would be to use an inducible expression system like Tet-on.
Hi,
I agrre there might be an epigenetic regulation. This is known for e.g. CMV promoter. If you want to be sure that your GOI is expressed you could think about cloning this into a GOI-T2A-Resistance manner. I have used this kind of approach with several hematopoiteic cell liines and it worked pretty good. In addition you could try sodium butyrate which could reverse the epigentic pattern (in case yo use the CMV promoter)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC20860/
Sven
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