I have been trying to transduce a few murine cell lines with a with a lentivirus for the past month and I just dont know where could be the problem. My lentivirus is IRES-GFP so I know that my transfection of 293T cells is good (GFP+ cells after 24 hours). Here's the protocol I follow:
Day 0: plate 2e5 293T on a 6 well plate.
Day 1: add 1ug or lentivirus, 1ug of each packaging plasmids along with Fugene HD with a 3:1 ratio (9ul of Fugene). I transfect my 293T in 1ml of media complete with antibiotics.
Day 2: (24hrs after) add 2ml of media for a total of 3ml.
Day 3 (48hrs after) collect the 3ml of old media with virus and add 2ml of fresh complete mdia. Put the old meida in 4C.
Day 4: Collect the 2ml of media and pool with old media, filter through a .45um filter and add to my cell lines.
I add 1ml of the media containing virus and 8ug/ml to my cells lines (5e4 cells/well on 24 well plate). Spin infect at 37C for 99mins. The next day I see no GFP.
Any help would be appreciated.