Transfection - (Dec/30/2011 )
I am PhD student. I got struck with transfection of shRNA to my hESC using DOX inducible Lentivirus. I used PLVTHM and cut H1 promoter with shRNA bit into PLVCT-ttrKRAB vector which contains GFP.
When It was transfected into 293 (with packaging and envelop part) for producing virus using Lipofectamine 2000. I can see GFP on 293 cells. (about 90% of cells showed GFP). However when I harvested supernatant soup and transfect into my hESC (either concentration or non concentration), there were no GFP on my cells when DOX was added (even 5 days).
Since PLVCT KRAB vector doesn't contain any antibiotic resistant gene, I tried to put Zeocin resistant gene with CMV promoter in the part of plasmid (at the end). Unfortunately, my tranfected cell were all died after adding Zeocin.
I got struck with this problem for nearly half year. Do you guy have ever used those Lentiviral vector? Could you please give me some suggestion?
Many thanks and happy new year,
There are alot of abbreviations in your question, I understand some of them but have to guess at others, hESC, PLVTHM,
It sounds to me that you aren't getting good ligation into your vector. You say there are lots of GFP positive cells, but you have no antibiotic resistance gene, so how can you tell the difference between cells transfected with the empty vector with just GFP and those with the vector plus your shRNA insert? Usually if you are inserting using restriction enzymes you either use 2 different enzymes so that the vector cannot re-ligate to itself, or if you are only using one enzyme, treat the cut vector (but not the insert!) with an alkaline phosphatase enzyme like TSAP to prevent the vector re-ligating to itself without the insert.
there are lots of things that could go wrong, give more details on your techniques! It does sound like a problem with getting your inserts into the vector.
(also stupid question, but got to rule out the obvious first, you are reverse-transcribing your shRNA into DNA first before trying to stick it into the lentivector right?)