I have created a stable transfection of a mouse mammary cell line 4T1. However my gene of interest is not being expressed in the 4T1 cell line. On the other hand it is expressed strongly in 293 cells.
I had a look at the promoters of the gene of interest and the resistance. Neomycin is under a SV40 promoter (and the stables are resistant to 1000ug/ml G418), but the gene of interest is under a human CMV promoter.
The manufacturer says it will express genes in mammalian cell lines, but does not say what type.
Is the answer simply that the mouse cell line can't drive the human CMV promoter? And all I have is a flask full of g418 resistant mutants?
Some cells have mechanisms to control levels of particular proteins post-translationally. This may be limiting the overexpression potential in your clonal lines. Check by qPCR to assess whether your transgene is transcribed or not. Check by blots using the tags to see if any of the transgene is expressed at the protein level. Perhaps your endogenous protein is suppressed such that total levels of protein looked unchanged. Important to distinguish between this possibility versus repression or disruption of your CMV-GOI cassette.
CMV can be silenced, but other phenomenon can lead to transgene repression as well. Bear in mind that with random plasmid integration followed by Neo selection, there is no guarantee that the entire plasmid was properly integrated into the genome in a way that preserves your CMV-GOI cassette. Moreover, if your transgene confers a disadvantage in terms of cell survival and/or proliferation, you will introduce a selective pressure for low expressing cells, no matter what promoter is chosen.
Some options to circumvent this problem are to use inducible expression as well as empty viral vectors in lieu of plasmid integration.
In order to figure out what is going on in your system, you should run some blots and immunofluorescence experiments to assess what level of expression, if any, is present for you transgene in the G418-resistant 4T1 cells.
Hi Daniel,
Thanks for your reply. I have already done two cell extractions, and loaded equal protein (15ug) to the western blot, from both transfection and non transfection cell lines, and see no difference in protein expression.
Hence the question about gene expression of a murine protein, under a human CMV promoter
Is the protein expressed endogenously? Are you using an epitope tag? These factor into the sensitivity of the Western blot.
Yes, the protein is normally expressed endogenously, we are attempting to over express it. It is also Flag tagged and MYC tagged. I can detect the endogenous form with no difficulty.
Some cells have mechanisms to control levels of particular proteins post-translationally. This may be limiting the overexpression potential in your clonal lines. Check by qPCR to assess whether your transgene is transcribed or not. Check by blots using the tags to see if any of the transgene is expressed at the protein level. Perhaps your endogenous protein is suppressed such that total levels of protein looked unchanged. Important to distinguish between this possibility versus repression or disruption of your CMV-GOI cassette.
Thanks! Will the same assay (we use Taqman) that works for the qPCR, work on the transgene? Ie with the tags?
Is it also true that 293 cells have strong expressions of CMV promoters. I see heaps of the protein in transient transfection.
Yes, your Taqman assay should be suitable for the transgene, provided the cDNA used in the transgene cassette is mouse. Not surprisingly that you observed gang-buster expression in the 293s. They transfect at very high levels and churn out a ton of protein from CMV-based constructs. So while nice for transient transfections, they are poor indicators for how your gene cassette will behave in other target cell types.
Hi, Andrian, I agree with Daniel, not all cells lines can expressed genes in the same manner, even if you use same promoter. If there is a problem with expression, I suggest you also try other promoter. Alternatively, 4T1 cell have a negative selection against your protein. You can prove this point by using the other promoter like SV40. Good luck!
Another factor to consider here is cDNA size. Large cDNAs that lack introns are poorly expressed from stable lines, but work fine in transient transfections such as in 293s. If your cDNA is larger than 2kb, I'd suggest adding a synthetic intron to boost gene expression in your stable cell lines.
It's funny you should say that. My gene alone is 8kb... It's a 250kDa protein
sorry Bastian, iPad auto correct messed up your name. Thanks for your suggestion
Ok then, Adrian. I'd lay very strong odds that the problem here is cDNA size and not the CMV promoter. Evidence in the literature suggests that the beneficial effect of introns on gene expression is non-additive, so you should probably try to insert two rather than just one. You can either take advantage of natural introns if your gene contains short ones (500bp or less) or engineer in some 100bp artificial introns using PCR.
This interests me greatly. After your suggestions, I've done a bit more reading, and now think perhaps its not integrating into the genenome.
Hi Adrian,
Have only worked with mouse cancer cells in regards to your problem, but this is what I suggest if you’re unsure about integration:
No 1: find out if the gene is integrated or not. Do genomic DNA extraction and traditional PCR. Make sure your primers are suitable for the task and use wild type DNA as a negative control.
If the gene is there: Check for RNA expression by reverse transcription PCR. If you have the gene integrated, but no RNA expression, it could be an epigenetic problem (gene silencing). Of course, it doesn’t exclude other problems such as those that have already been discussed, but it will tell you about whether the gene integrated, but isn’t being transcribed, or transcribed, but not translated.
You should also check for transient expression 24-48 hrs after transfections, then continue to test periodically while the cells are under G418 selection (up to as much as daily!!). You may find that it integrates, then disappears. If so, it could be an indication of the cells disliking the product and chops up the gene (fragmentation). In this case you may need to look for another cell line or modify your construct. Enhanced Green Fluorescent Protein (EGFP) is well liked by most cells, and when fused to your protein of interest, it can help make stable expression.
So since I posted last, I Checked RNA expression by qPCR, and found a very strong increase in gene expression in stable transfected cell lines when compared to WT (>100fold increase)... Im going to perform a chamber slide assay to see if there is protein expression that I have not seen in my western. This suggests that the gene is integrated and transcribing. any more thoughts?
You could be missing it on a Western if the protein gets stuck in an insoluble fraction or if it is unstable in the 4T1 cells and is rapidly degraded or cleaved at a site near your epitope. IF is an excellent idea to help figure out what is going on.
In addition, depending on the sensitivity of your qPCR, you might be detecting pre-mRNA that never matures and exits the nucleus. If you don't see any signal by immunofluorescence, this could be a potential explanation.
Hi, Adrian, I would suggest to you try first western dot. In this case can avoid problem with large protein size (separation, transfer etc) and have at least preliminary results. If you dot results will be positive, you can try IF because you will be sure you have expression and that antibody recognize protein. Good luck!
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