RNA pol II driven shRNA expression - CMV driven shRNA expression (Dec/17/2005 )
Hello everyone
I am new to this field. Had tried to deliver my shRNA using retrovirus, but with little success.
Does someone has the experience with CMV promoter driven shRNA expression?
If I insert a reporter gene in front of the shRNA coding sequence (shRNA coding sequence is in the 3' UTR of the reporter gene) , then select for the reporter gene. Theoretically, every cell that expresses the reporter gene will also express the shRNA. In this way, the reporter gene itself will also become the target of the shRNA.
Here is the reference
http://www.pnas.org/cgi/content/full/102/37/13212
Hi WHR,
Why don't you try the kit from Invitrogen Block-iT Pol II miR RNAi Expression Vector kits, they offer such system based on the same principles on your attached PNAS paper. Alternatively Hannon's lab also supports another version of siRNA expression system (see their homepage for details).
What you are worried may be true. But considering the shRNA structure must be edited out from the original transcripts by Drosha in the nuclear before transported to the cytoplasma where most RNAi machinary works, it could be the chance that mature reporter gene mRNA in the cytoplasma without the ShRNA targeting sequence in their 3'-UTR, the only thing I am not sure is without the polyA, how stable the edited mRNA will be.
Good luck
Why don't you try the kit from Invitrogen Block-iT Pol II miR RNAi Expression Vector kits, they offer such system based on the same principles on your attached PNAS paper. Alternatively Hannon's lab also supports another version of siRNA expression system (see their homepage for details).
What you are worried may be true. But considering the shRNA structure must be edited out from the original transcripts by Drosha in the nuclear before transported to the cytoplasma where most RNAi machinary works, it could be the chance that mature reporter gene mRNA in the cytoplasma without the ShRNA targeting sequence in their 3'-UTR, the only thing I am not sure is without the polyA, how stable the edited mRNA will be.
Good luck
Hi schloss,
Thanks for your reply
I decide to insert a shRNA expression cassete into pIRES2-acGFP1 (BD bioscience) to generate a vector like this: CMV-IRES-acGFP-H6-mir5'-shRNA-mir3'-pA
In this way, either U6 or CMV promoter can control the expression of mir5'-shRNA-mir3'.
Furthermore, a puromycin resistance gene can be cloned downstream of CMV prmoter. Thus, the drug resistance and the shRNA will be directly linked.
Do you think this feasible?
Hi WHR,
Sorry for the toooooooooo late reply, I'd rather not use U6 promoter in between. Although U6 can lead the transcript for the mirRNA construct, but under the impact of CMV promoter it could not work perfectly. On the other hand, if you would like to keep both PURO and EGFP, my suggestion is like,
CMV-EGFP-mir5'-shRNA-mir3'-IRES-puromycin-pA
In this case you are sure the puromycin selected clone will be high possible of EGFP positive and expressing siRNA. Be careful of IRES, the transcript after it always less shows up in comparison with the first one direct after the promoter.
hopefully it is not too late
schloss
Sorry for the toooooooooo late reply, I'd rather not use U6 promoter in between. Although U6 can lead the transcript for the mirRNA construct, but under the impact of CMV promoter it could not work perfectly. On the other hand, if you would like to keep both PURO and EGFP, my suggestion is like,
CMV-EGFP-mir5'-shRNA-mir3'-IRES-puromycin-pA
In this case you are sure the puromycin selected clone will be high possible of EGFP positive and expressing siRNA. Be careful of IRES, the transcript after it always less shows up in comparison with the first one direct after the promoter.
hopefully it is not too late
schloss
Thank you very much,schloss,
I had constructed a vector which show good knockdown efficiency. I constructed this by mistake, however, it work!
CMV-puromycin-U6-mir5'-shRNA-mir3'-pA
When co-transfected with CMV-EGFP-TAA-shRNA target site-pA,
The construct mentioned above showed much more knockdown of EGFP than
U6-mir5'-shRNA-mir3' /pGEM-T
Even when the U6 promoter was deleted, the vector had no decrease in it's potency.
Next step, I have to find a way to deliver it into my cell of interest, which is hard to transfect.
hi laybe electroporation of cells is a possibility?
calcium phosphate is also an old but effective method.
calcium phosphate is also an old but effective method.
Thank you,fred,
I will have a try.
Hi WHR,
Could you please let me know the mir5' and mir3' flanking sequences you used for cloning.
- There is one from Hannon lab and Open biosystems
- Another one from Invitrogen- that they have used to design miRNA oligos and cloned within the 5' and 3' mir flanking sequences in their PolIImiRNA knockdown system.
Both the sequences are completely different from each other.
I want to clone CMV-miRNA into a lentivector, however the only sites I have are EcoR1 and Mlu1/Cla1. I do not see a direct way of cloning the transcription unit into the vector. Any advice?
Thanks,
Anita.
well pcr the cassette with appropriate restriction sites seems a good way ?
i am using the Lenti vector from invitrogen for cloning shRNA. they give u the entry vector to clone the shRNA. and its easy to clone it in. The problem with the vector is high titres. if u modify it, one can get high titres.
if u want to use cmv, then amplify with appropriate sites.