We have produced a 3rd generation, self-inactivating lentiviral vector for the expression of our gene of interest under the EIF1alpha promoter. This vector also contains the GFP reporter gene under the CMV promoter. As a control, we have produced a transfer vector expressing an unrelated gene.
When we transfect this plasmid into 293T cells, we detect GFP expression in ~50% of the cells. However, when we transfect this plasmid into 293T cells along with packaging vectors (a plasmid expressing gag and pol genes, and a plasmid expressing VSV-G) we do not see any GFP+ cells. This happens with both transfer vectors, namely the one with our gene of interest and the one with the unrelated control gene.
When we co-transfect a 2nd generation lentiviral vector expressing either our gene of interest along with packaging plasmids, we do not have this issue.
Can anyone explain why we have loss of reporter gene expression when we co-transfect packaging plasmids?
Thank you!
It is possible that co-expressed proteins can precipitate GFP and suppress fluorescence.
Hi Henry. Thanks for your response. If by co-expressed proteins you mean my gene of interest, I do not think that is possible. We are studying a long noncoding RNA, which does not encode for any protein. In addition, the same effect is observed with the control vector that contains a random sequence.
If on the other hand you do not mean my gene of interest, then I am not sure exactly what you mean. Could you please elaborate?
Thanks!
Hi Fabio,
I understood your question as packaging vector - plasmid expressing gad and pol genes. If you are studying non-coding RNA, there could be some other complementary sequence problems.
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