I have been trying to make a stable cell line expressing my GOI using lentiviral transduction of HEK293T cells.

To do so I ordered a lentiviral transfer plasmid from addgene which had a gene in it already. The gene is followed by an P2A sequence and then the puromycin resistance gene.

I have used this plasmid to make a lentivirus and transduce cells. This was no problem, the cells survived puromycin and expressed the protein.

But now for my GOI, I removed the present gene from the plasmid using restricition enzymes and added my GOI. Then I packaged the lentiviruses and transduced my cells. After adding puromycin, the mock transduced cells died and the transduced once survived. But, immunofluorescence staining was negative for my protein.

What puzzles me is that the puromycin resistance gene has no promotor of its own and uses the promotor and startcodon of my GOI and is expressed, but my GOI is not translated.

I checked with PCR and the gene for my GOI is present in the cells. The sequenceshow no mutations and the gene, P2A and puromycine resistance gene are all in frame.

I also tried transfecting HEK293T cells with the exact same GOI and then staining is positive.

Does anyone have a possible explanation and solution for this problem?