retrovirus Phoenix transfection - Help help (Nov/04/2008 )
I found there were lots of friends who have failed to transfect plasmid into taret cell by Phoenix-helper cells. Recently, I also had this trouble, but really didn't know the reason.
Strictly following the stanford protocol, I can see very strong GFP fluorescence in Phoenix cell after transfected with GFP palsmid. But after 48h incubation in 32-37, I used the fresh supernatant to tranfect targe cell (293, MCF7), I almost didn't get any GFP signal in the target cell after 48h. I also tried this by using polybrene but the result is the same. I found lots of friends had this problem. Who can tell me what have happened? Thanks a lot.
-rabbitwrong-
QUOTE (rabbitwrong @ Nov 4 2008, 04:50 PM)
I found there were lots of friends who have failed to transfect plasmid into taret cell by Phoenix-helper cells. Recently, I also had this trouble, but really didn't know the reason.
Strictly following the stanford protocol, I can see very strong GFP fluorescence in Phoenix cell after transfected with GFP palsmid. But after 48h incubation in 32-37, I used the fresh supernatant to tranfect targe cell (293, MCF7), I almost didn't get any GFP signal in the target cell after 48h. I also tried this by using polybrene but the result is the same. I found lots of friends had this problem. Who can tell me what have happened? Thanks a lot.
Strictly following the stanford protocol, I can see very strong GFP fluorescence in Phoenix cell after transfected with GFP palsmid. But after 48h incubation in 32-37, I used the fresh supernatant to tranfect targe cell (293, MCF7), I almost didn't get any GFP signal in the target cell after 48h. I also tried this by using polybrene but the result is the same. I found lots of friends had this problem. Who can tell me what have happened? Thanks a lot.
I'm not using Phoenix cells, but have a similar problem. I can see GFP expressed in my packaging line, but can't seem to infect target cells.
Right now I am testing infection with my supernatant (where I saw GFP expression, so I assume packaging/tranfection was ok) at different Polybrene concentrations and doing NIH-3T3s (supposedly easy to infect) in parallel in case my cell line is just plain difficult to infect.
Note that last time I saw a very low but significant background fluorescence of Polybrene in the GFP channel by FACS so be careful with controls!....
Anyone have any other suggestions? I get transfection of the packaging line. It isn't the best I've ever done (~10-15%) but I'm thinking it should be sufficient. What else to optimize????
-Mountainman-