I infected MEL cells with lentivirus encoding my protein of interest (Flag tagged) with a BFP marker. So the construct goes EF1a promoter-gene-Flag, PGK-BFP-puro.
I selected the cells with puro for 1-2 weeks and a BFP-positive population has grown up from it. But when I do a western blot on these cells, my Flag-tagged protein does not show up.
How is this possible? Is this a clone that has very low expression of my gene (it is a large gene, 3.1kb)? Could the lentivirus have integrated only the BFP part, and my gene is either not integrated or cut off?
Other info:
The size of the insert between the LTRs is 7.8kb. Transfecting this plasmid into 293TN cells results in high expression of the gene by WB, and the 293TNs are visibly BFP-positive during virus production.
Hello Kelly,
Lentivirus transduction should not result in integration of only part of the backbone. However rearrangements are likely especially for large and repetitive constructs during proviral particles production (you might produce particles not 100% functional for your gene, but preserving intact PGK-BFP-Puro). Should this happen however, it would only affect a fraction of your virus, and you should still have overexpression in some of the transduced cells... which seems is not the case.
Most likely:
Double check the sequence of your plasmid. Mutation in the ORF of your gene could have occurred while cloning. You will need to sequence all the ORF to check that premature stop-codons are not actually occurring.
Most-unlikely, but possible:
If sequence is ok, then the problem must be elsewhere. You can perform qRT-PCR on your GOI in transduced (BFP+ cells). If the gene is overexpressed with respect to control cells you know that the lentivirus is working properly. The problem in this case may rely on post-translational modifications of your GOI. Is not always possible to overexpress a gene. There may be strong regulatory mechanisms controlly the protein levels of your GOI and, despite the amount of the RNA, your protein can be continously degraded.
This happened to me a couple of time (either with transduction or transfection). You may want to make a transduction and overexpression test in a routinely used cell line such as 293. When I have tried this the virus was working properly, this will clarify that your target cell most likely possess mechanisms to downtune your overexpression. Usually 293 are messed-up at several levels of regulation, so you should be readily be able to overexpress your protein without problems.
In addition if you are not yet doing so, use a Flag antibody for your protein expression assays. Many antibodies are just not specific for the protein they should target.
Hope it helps
Cheers
Francesco
Francesco,
Thank you for your response! I think I'll try the qPCR first... it's possible that the protein is there but my Flag antibody has gone bad. The WB was ambiguous and the band might not be that strong with this weaker EF1a promoter as opposed to something like CMV.
I also wonder whether the puromycin selection has selected for a particularly low-expressing clone? This is a polyclonal population (I never did FACS to get single clones), and maybe the cells that strongly express my GOI or have more than one insertion divide more slowly, and were out-competed by another population with fewer inserts. I may try to do this again and FACS sort a few days after starting the selection to try to get a high-expressing single clone.
Kelly
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