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.