inducible shRNA vector system - (Apr/09/2009 )
I am just seeking for an effective inducible shRNA vector system for controlled knockdowm expression. Early on I tried the single-tTS-shRNA system from Clontech which is one vector containing TetR and inducible shRNA. Unfortunately, however, the leakiness and low-effiencey of H6 promoter always existed. Later on I found one major reason is that the TetR expression driven by CMV promoter is not enough for inhibiton of teton on the same vector. There has been many trial on inducible shRNA system. but the few company such as Block it Enter system in invitrogen, has commercial vector besides clontech. I am afraid that the drawback is still on.
Hope somebody give some suggestions. You are greatly appreciated.
I thought I am going to use the single vector tet-on system to do inducible RNAi as described in this paper which claims the system works better than the traditional two vector system with less leakiness and high induction. They have made their vector available at addgene.org.
Thank you so much, PCRMAN, I have downloaded and am reading the paper for the possibility of applycation on my project.
the tet-on single lentiviral vector is antoregulatory system, I can't find the TetR may express enogh to inhibit shRNA expression in the absence of dox compared with single-tTS-shRNA system in clontech; otherwise, I hope to use the non-viral vector system in my project because I have no experienc on lentivirus in our lab. Thank you for your time.
pcrman on Apr 9 2009, 10:09 PM said:
There is another single lentivirus system, deposited at ATCC, pSLIK (single lentivirus for conditional knockdown!). PMID: 18470652. I've used it myself and the induction works really great, no GFP without dox and strong increase in expression within 24 hrs when it's included (start to see it at 12 hrs). Our only problems have been 1) our cells were resistant to G418, so we switched Neo for Puro, but then find that Puro expression from the IRES in our cells is quite low without dox. When dox is in the medium, I think there is read through and so Puro increases and the selection works. Just another couple of things to think about and bear in mind when making a choice.
BTW, we use the functionally tested tet-free FBS from Clontech - Clontech says it is really important to use it for low background, not sure if that's just marketing though :-).
thank you for mentioning the pSLIK which probably is an better system than the one (Nature Method) I mentioned. The paper that describes this system is acutally this one (PNAS) http://www.pnas.org/content/103/37/13759.abstract not the one with the PMID:18470652. According to the PNAS paper, "Although
I have finally choosed to use the pLVUT-tTRKRAB vector for my inducible shRNA designation. However, I cant get cloning site for it available so far. After looking through the previous post in phpbb, the same question has been suspended for long time but no response. If you experienced on this vector cloning, could you please give some suggestion?
Thank you very much in advance.
Sorry do not have a reply, just another question - has anyone tried the pTRIPZ vector from OpenBiosystems? Its supposed to miRNA adapt the shRNA of interest (make a shRNAmir) and induce downregulation via addition of Doxycycline. It also has a RFP marker that is expressed when the shRNA is induced.
I was going to use the CLontech system originally but now based on this thread I am not so sure...
Appreciate your feedback,
I am new to the forum. I have followed your discussion with the TET inducible shRNA systems and I would like to know if anyone knows or has experience with IPTG inducible shRNA system. Thank you