Hmm, that does sound frustrating. We also use psPAX2 and pMD2.G and generally get pretty high titers with 4:3:1 (transfer:packaging:envelope) and concentration by ultracentrifuge.
There could be several problems.
1. Low viral titer/MOI
- During packaging, did you see appreciable fluorescence in the HEK293T cells? I usually see strong fluorescence during packaging (except as it turns out, when using a transactivated promoter).
- Have you checked the titer on HEK293 cells?
2. No tropism for your cell type.
3. GFP is out of frame or the fusion mRNA isn't being expressed. Does transfecting the transfer plasmid alone result in expression in your cell type?
I'd look at the viral titer first, and transfect some of the HEKs with the transfer vector at the same time. If it is infectious for HEK293 but not your target cells, I'd be suspect the tropism to be a problem. If you don't see fluorescence in either the infected or transfected cells, then something else might be wrong with your construct.
*edit, you could also look at p24 levels via ELISA to determine if you've produced viral particles.
** One other note, sometimes the virus takes >72 hours to integrate and express your GOI.
Edited by miST32, 24 February 2016 - 10:43 AM.