Inducible RNAi - Has anyone tried the new tet inducible systems? (Jun/29/2005 )
i'm wanting to get some advice on the new tet-inducible RNAi systems. i've had good success using pSuper.retro.puro to make stable cell lines in Jurkat and HeLas. i now need to find an inducible system, because my new target when knockouted out causes cell cycle arrest. there is a system from invitrogen (too expensive), bd science (unsure price), and pSuperior (adapted from pSuper).
i'm unfamiliar with using tet repressor cells, i've heard from my PI that the whole process can be laborious, to say the least, but i also have seen some commercial cells available. any suggestions? of course my PI would prefer for me to design the inducible system and the tet repressor cells, myself, but with some arm twisting i may be able to purchase. especially if i have some hands-on advice and suggestions! thanks guys!
unfortunately no one has replied, so i'm going to reply myself and give an update. have purchased part of the system and i am designing another part. i am testing my stabley expressing tet repressor cells and cloning/sequencing a lentriviral vector that now has the operator upstream of the promoter. it's kind of a gruelling process, but i think within the month i should have the system up and running and have my first set of pilot experiments done. (checking for basal expression of the hairpin and inducible expression) if anyone has done this before and has helpful suggestions feel free to comment--- like timeframes from induction of cells to when knockdown will occur or what i should use as a target to establish how good the system is...
Hi, Twirling sheep:
I am going to do the same experiment. Which company did you purchase the inducible system?
I am thinking about the pSuperior. Thanks.