lentivirus transduction - no GFP after trasduction (Apr/15/2008 )
hi
i need help. I am trying to knockdown my gene of interest via shrna-mir, but i am having trouble with the transduction. My vector has GFP so when i did transfection for viral production, i saw about 70-90% GFP but when, concentrated the virus and trasduce the virus with the aid of polybrene after 24hrs i did not see GFP so basically i dont know if my transduction worked. I am currently using 293T cells for both step and no knockdown...
-I use 3 vector systeam VSVG, CMVAR and mir vector, i dont know how to troubleshoot it
i need help. I am trying to knockdown my gene of interest via shrna-mir, but i am having trouble with the transduction. My vector has GFP so when i did transfection for viral production, i saw about 70-90% GFP but when, concentrated the virus and trasduce the virus with the aid of polybrene after 24hrs i did not see GFP so basically i dont know if my transduction worked. I am currently using 293T cells for both step and no knockdown...
-I use 3 vector systeam VSVG, CMVAR and mir vector, i dont know how to troubleshoot it
It's probably because your GFP is in 3' end.You can put cover-slip in your plate and take them after 24h and do immuno-staining with GFP antibody.
i need help. I am trying to knockdown my gene of interest via shrna-mir, but i am having trouble with the transduction. My vector has GFP so when i did transfection for viral production, i saw about 70-90% GFP but when, concentrated the virus and trasduce the virus with the aid of polybrene after 24hrs i did not see GFP so basically i dont know if my transduction worked. I am currently using 293T cells for both step and no knockdown...
-I use 3 vector systeam VSVG, CMVAR and mir vector, i dont know how to troubleshoot it
It's probably because your GFP is in 3' end.You can put cover-slip in your plate and take them after 24h and do immuno-staining with GFP antibody.
But if there is no GFP signal, then it's most likely that there is no GFP protein, so the staining wouldn't work either...right? I'd suggest doing PCR and RT-PCR for the GFP to see if it is present in the genome and if it is transcribed.
i need help. I am trying to knockdown my gene of interest via shrna-mir, but i am having trouble with the transduction. My vector has GFP so when i did transfection for viral production, i saw about 70-90% GFP but when, concentrated the virus and trasduce the virus with the aid of polybrene after 24hrs i did not see GFP so basically i dont know if my transduction worked. I am currently using 293T cells for both step and no knockdown...
-I use 3 vector systeam VSVG, CMVAR and mir vector, i dont know how to troubleshoot it
If the virus is successfully produced, the packaging cells should die in 2-3 days.
did you titre the virus?
i've found that even if it looks like the transduction works, it's not a guarantee that there was enough virus produced... and that a transfection would work.
V
I second this. GFP in your packaging cell line tells you that your construct can direct expression of GFP, but gives no indication as to whether packaging of infective virus is successful. One post doc in our lab also made the mistake of assuming that GFP in the packaging cell line meant his virus would be okay. In short, it turns out his packaging went very poorly, and we're starting again from the packaging step, and this time around we'll make sure he checks the titre of the virus before using it in experiments!! 
Ginger
i've found that even if it looks like the transduction works, it's not a guarantee that there was enough virus produced... and that a transfection would work.
V