I have stable EpH4 cell line with my gene of interest knockout and I'm planning to introduce the mutant gene into it using HEK293 cell, with hygromycin selection. Here are some problems I encounter:
1. after the infection, only a small fraction of cells survived, maybe only 2%. comparing to 70% survival rate when infecting WT cell (no gene KO).
2. the remaining cells grow extremely slow. less than 20% confluent 15 days after expansion (with hygromycin for selection). Why it's growing so slow? Is it ok to remove hygromycin once all mock cells are dead?
3. I managed to get one stable cell line with mutant gene. However, when I did WB, nothing showed up. It's probably not the problem with WB since the other to control I did were all fine. And I saw protein on the membrane after transfer, but when I blotted it, nothing at all!! even GAPDH and beta-tubulin!!! Anyone has same problem with WB?
4. still with that stable cell line with mutant gene integrated into the genome, I extract genomic DNA, did PCR to amplify the gene I integrated in. There is a 10bp deletion right in the middle of the gene. How could it be? because the cripsr? but guided RNA could not target that deleted sequence.
Please, take into consideration that a DNA vector, a transfection reagent, expression of an antibiotic resistance (trans)gene, expression of a reporter (trans)gene, and selection by acute/chronic antibiotic treatment may evoke cellular responses that affect the biochemical processes under investigation. In the following review, we demonstrate that an assumption that empty vector-transfected cells preserve the cytogenetic and phenotypic characteristics, and represent the adequate control in transfection experiments is not universally valid. We exemplify a number of studies, which reported obvious genomic, transcriptomic and phenotypic changes of tumor cells after transient/stable transfection of an empty vector. Finally, we conceptualize that the diverse experimental manipulations (e.g., transgene overexpression, gene knock out/down, chemical treatments, acute changes in culture conditions, etc.) may act as a system stress, promoting intensive genome-level alterations (chromosomal instability, CIN), epigenetic and phenotypic alterations, which are beyond the function of manipulated genes.
Please see the following manuscript for details:
Stepanenko AA, Heng HH. Transient and stable vector transfection: Pitfalls, off-target effects, artifacts. Mutat Res. 2017 Jul;773:91-103. doi: 10.1016/j.mrrev.2017.05.002.
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