Hi all, I have many stable cell clones that originated from single cells. However, I found (by IF) that not every cell expresses the protein, and this percentage varies between clones.
I was wondering whether this is a result of gene silencing? And to what extent does gene silencing normally contribute to this phenomenon (i.e., not 100% cells express the protein of interest)? Thanks.
It is a very common problem and often caused by islands of methylation creeping into the promoter of the gene. Once way to reduce the rate at which it occurs is to add insulator elements (eg. gypsy insulator) on either side of your construct.
Differences in expression can also be due to differences in where the construct has been inserted into the genome and the copy number. With all your clones did you confirm chromosomal insertion? If not the gene could have been extrachromosomal and subsequently lost.
Robert Adolf Brinzer thanks indeed! I was wondering for cells ~20 passages old, what percentages of silenced expression should we expect? Some cell lines I have are really miserable, with 20-30% cells expressing the protein under IF.
However, because the clone originated from a single cell, so I would assume the insertion and copy number should be exactly the same within the population? I have done PCR and could get the amplification and have sent the band for sequencing, and I used lentivirus to generate the clones so the insert should be integrated in the genome.
By the way, do you happen to know anything about doxycycline inducible expression? Some people said doxycycline cannot 100% induce all the cells to express the protein under regulation, and my protein is also under dox regulation, I was wondering if that could also be a reason?
Did you have a control PCR where you try and amplify from your insert across the plasmid backbone to check for circular products? After about 16 passages you can expect to have a 90% reduction in expression level but it should be quite consistent across cell populations.
Is your viral vector mobilization incompetent? If not lentiviral vectors are known to excise from the insertion site.
Doxycycline is an antibiotic related to tetracycline and induction relies on the same regulatory machinery. You have your promoter with several spaced tet operon binding sites to which a transgenic TetR protein binds, blocking ribosome binding and therefore repressing transcription. When the antibiotic binds to TetR, the protein is released from the binding site allowing transcription. There are also systems that use modified TetR proteins that only bind in the presence of the antibiotic. Have you tried higher concentrations of doxycycline?
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