What is the reason for using U6 promoter for shRNA vectors? can we replace it with cmv promoter ( for shRNA without flanking by mir30)
Pol III genes and promoters do not get polyadenylated and thus are ideal for making small nuclear RNAs such as shRNA hairpins. Pol II transcription requires a poly-A site to terminate the mRNA and process it correctly, which is not what you want in an shRNA. Thus, my prediction is that the CMV promoter which recruits Pol II will not be very effective.
U6 promoter is bound by RNA pol III, which synthesizes shRNAs, tRNAs, rRNA 5S etc., whereas CMV promoter is recognized by RNA pol II to synthesize mRNA (generally longer products)
CMV is not optimal for your application. I don't know, however if it won't work at all. U6 is the most optimal promoter to produce shRNAs, while CMV is used to express whole genes.
Best.
Pol III genes and promoters do not get polyadenylated and thus are ideal for making small nuclear RNAs such as shRNA hairpins. Pol II transcription requires a poly-A site to terminate the mRNA and process it correctly, which is not what you want in an shRNA. Thus, my prediction is that the CMV promoter which recruits Pol II will not be very effective.
Pol III promoters are popular since they allow expression of noncoding RNA molecules with precise 5' and 3' sequences (i.e. no 5' cap or 3' polyA tail). The fact that U6 transcripts are restricted to the nucleus (unlike pol II transcripts) is important to facilitate processing of the miRNA-like precursor hairpin by nuclear Drosha and subsequent steps of miRNA maturation. This strategy ensures both high knockdown efficiency and minimizes exposure of dsRNA in the cytoplasm, which can trigger interferon response.
Still, if you are interested in using pol II to drive shRNA expression (e.g. for inducible RNAi), I'd suggest checking out the PRIME vector system from the Elledge lab.
Article Stegmeier, F, Hu, G, Rickles, RJ, Hannon, GJ and Elledge, SJ...
Hi, Zaree
I hope you are doing well, U6 promoter usually used for shorter transcript as it is pol III dependent, so its commonly used for sgRNA, shRNAs and so on .... whereas CMV promoter is Pol II deepened which used mainly for longer transcript as mRNAs or florescence proteins as eGFP, RFP and so on ....
Short hairpin RNAs (shRNAs) transcribed by RNA polymerase III (Pol III) promoters can trigger sequence-selective gene silencing in culture and in vivo. The CMV enhancer can enhance U6 promoter activity and increase the production of shRNA . Thus, this enhanced U6 promoter is useful choices of shRNA sequences preclude the selection of a highly efficient RNAi target region
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