Hi,
I'm trying to create a stable overexpression line with EF1a-FlagGOI-IRES-GFP using lentivirus packaged with pxPAX2 and pM2G. After transduction of 293T with 8ug/ml polybrene, I was able to get ~30% GFP+ cells despite significantly weaker fluorescence comparing to transfection. RNA was confirmed by qPCR with increasing level corresponding to virus added. However, I could not detect the protein both on western blot and immunostaining of both the gene and flag tag, whereas transfection showed significant expression in both methods. I'm pretty sure the gene is non-toxic and is not expressed in 293T endogenously. I've added 100uL of 10x concentrated virus to each 12-well and still no protein expression on day6.
The lentivirus would ultimately be used for MSC with protamine sulfate. Any idea what might went wrong?
Thankyou for the advice in advance.
Hi Adrian,
I follow your comments that "you could not detect the protein both on western blot and immunostaining of both the gene and flag tag" but I couldn't quite understand what you meant by "whereas transfection showed significant expression in both methods". You mean GFP???
If so, GFP depends on the IRES in your construction. I would suggest you to check the sequence of your flag-GOI because it seems you may have a frame shift. Framed shift would still render mRNA levels, likely detectable by the same primer set you have, but won't translate into your the desired protein. Since your tag is 5' to your GOI and is not detected may be is where you should check.
Hope it helps you to solve your problem.
Cheers,
I agree with Sebatian, and share his confusion about what you are comparing when you say 'transfection show significant expression by both methods.' Please elaborate.
As to the frameshift issue, I can only share the experience of others from the lab I did a post-doc in at Scripps. A post-doc had made a transgenic mouse line carrying a influenza hemagglutin gene. The idea was to study immune tolerance at both cellular and antibody levels. The mice were completely tolerant of the relevant virus HA protein at the cellular level, but not at all at the antibody level.
It turns out that during the generation of the construct, a 14 base pair Pst 1 fragment was lost, resulting in a frameshift and generation of a truncated protein, too small to generate an antibody response, but containing the major cellular target for HA in the mice. Something similar may be going on in your cell line.
Thankyou for the experience Gary Lee Gilmore and Sebastián Dupraz .
What I meant is GOI protein overexpression is detected using western blot and immunostaining on the FLAG tag and the protein itself in transfected 293T. The same method however yielded negative results in transduction. Found out the backbone is pHAGE-EF1a-IRES-ZsGreen, the specific plasmid I used is addgene:128803.
Sequencing is performed and there's no frameshift or any mutation in the ORF of maxiprep. Is it possible that Gen2 lenti is mutation prone with certain backbone?
Hi Adrian,
Now your problem is more clear, thanks for elaborating.
If transduction isn't working, then you should pay attention to the packing process of the lenti (Maybe you are transducing with a preparation enriched in cold viral particles). Alternatively the cargo of this lenti isn't integrating due to structural problems. For instance, are you sure the LTRs in the lenti plasmid are in order?
Cheers,
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