why I cannot test expression in transduced cells - Can test expression in transfected cells, but not in infected cells (Oct/01/2010 )
I am working on a human BS gene which encodes a type II transmembrane protein. First, I cloned BS gene (containing kozak sequence and a HA epitope tag at N (or C)-terminal) into vector pCDNA3 or pBABE-MN-E-GFP. The BS expression can be tested by western blotting in cell lysate of 293T transfected with plasmid.
Second, I made pseudotyped MLV particles by transfecting pBABE-MN-E-GFP containing the cDNA of BS, transduced Jurkat T cells, and then sorted the cells with the top 10% GFP fluorescence intensity by flow cytometry. I can test a strong GFP expression but cannot test the expression of BS by western blotting in stable transduced Jurkat T cells. I do not know what’s happened? I can test the expression in transfected cells, but why I can not test the expression in transduced cells. Can anyone give me some advice? SOS!!
Thank you so much!!
Have you tested you viral vector in transcfection assays in 293T? I mean, have you made sure that the plasmid you work with (pBABE) does have the correct insert? This vector has an IRES for the GFP expression, which means that your insert does not necessarely needs to be expressed for GFP to be expressed. I would first confirm that the pBABE indeed has the correct insert. Then, you could check the reading frame if the plasmid does contain your insert.
Yes, I did. I can test the expression in 293T cells transfected with PBABE containing my target gene.
You wrote "stably transduced cells". Is it possible that your protein get degraded overtime, and/or that the cells lost the transgene overtime? Have you tried looking at the protein sooner, after something like 48 hrs? Plus, did you blot with an anti-HA or anti-BS antibody? Have you tried both constructions (HA tag at C or N-term) in the jurkat cells?