Dear Xiaolu,

I use both the two-plasmid system (pSPAX2 and pMD2.G) and three-plasmid system (pGag-pol, pRev, VSV-G (pMD2.G)). The titer of lentiviruses using two-plasmid system is indeed higher than the three-plasmid system.

What’s more, polybrene can also enhance the transfection efficiency. You can use it at the concentration of 6-8 μg/ml. For polybrene sensitive cells, this step can be skipped.

Genemedi got a rich experience in lentivirus production, you could find more information on https://www.genemedi.net/i/lentivirus-packaging

Hope this may help you.

pSMART from Lucigen works fine. It is a little hard to clone though.

I have changed to packaging vectors psPAX and pMD2G as people suggested, the viral titer has increased to about 10 fold as much as Delta and VSVG. You can try them too, which can be found in Addgene.

I suggest using straight viral sup. Run a small titer if possible because too much VSV-G packaged virus may induce syncytia formation (read toxic). Also, we found that a 30 min pre-treatment of BX795 can at least double transduction efficiency of primary cells (http://www.ncbi.nlm.nih.gov/pubmed/22779406) . Sometimes poor transduction efficience can never be overcome, however your 293 cell results suggest you have a good packaging so it should be possible. Primary cells are just a pain. Good luck!

I know people in our lab use spinoculation protocol to infect jurkats.... that might help you. you should try pretreatment with BX795 as suggested by steven followed by spinoculation may give you better infection......... good luck with your experiments.

psPAX2 + pMD2.G (w/w 2: 1) works much better than deltaR8.2 + VSV-G. Packaging virus in 6-well plate may give higher titer than doing it in 10-cm plate. Try different ratio of pLVX : packaging plasmid mix, w/w 1:1 works the best to me. If everything is going right, should be able to get > 1 x 10^7 IU/ml virus in the sup, no need to concentrate the virus, but spinoculation with 5-8 ug/ml polybrene definitely helps.