lentiviral expression - transefection, low titier, protein expression (Jan/26/2010 )
I have problems in lentiviral vetors transfection, virus production and transduction.
The vector I used is pTRIP with IRES GFP , and I am able to observe GFP positve 293T cells(empty vector at least 90% and inserted vector only about 40%) when I cotransfected lentiviral vectors.
After 48~72hours, I collected virus supernatant,and I used the virus to infect 293T cells. GFP expression can be detected in empty virus, but poor or not detected in inserted virus. Besides, I harvested and lysed these 293T cells. I am not able to detect any protein expression.
I also used the empty virus to infect primary macrophages and 293T cells in the same time. The transduction efficiency is about 60% in 293T cells. Although I used 5 fold virus to infect primary macrophages, no or less than 1% GFP positve macrophages could be detected.
What could be the problem? Any suggestion?
Thanks a lot for your help!
Regarding differences in efficiency between 293T and primary macrophages, it's normal to get much less transduction in primary cells. Did you use polybrene for the transduction? If not, you should definitely be adding polybrene to the virus to increase your transduction efficiency. I use 6 ug/ml.