Hi All-
Despite having success using the 2nd generation packaging system for lentiviral production the boss thought the titre was too low so we transitioned to a 3rd generation system. Big mistake because we have had zero luck with this method--so we are going back to what worked. We currently don't have this fusion protein in a 2nd gen transfer vector and I've seen several places say that it is possible to use your 3rd gen transfer vector with a 2nd gen packaging system.
Has anyone here done that with any success? Which method did you use to transfect your 293T cells? Any help would be welcome.
If it helps we are using the 3rd gen transfer vector pCSC-SP-PW (aka pBob-GFP). The entire construct is 9.5Kb and the fusion protein is 1Kb.
Thanks!
From AddGene--"A 3rd generation transfer vector can be used with a 2nd generation packaging system, but a 2nd generation transfer vector cannot be used with a 3rd generation packaging system."
-mharr36-
Follow up:
Never got a response from anyone....
But using a CaPO4 ppt I was successful in generating lentivirus using my 3rd generation transfer vector with a 2nd generation packaging system!
-mharr36-
