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Promoters and vectors for shRNA? The need for specific vectors and promoters?


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4 replies to this topic

#1 lab_member

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Posted 14 June 2009 - 05:43 PM

Hi all,

I have been wondering why only certain vectors are specified to be able to take shRNA insert and they usually have the U1 or U6 promoter. I have always thought that as long as you put an shRNA insert downstream of any promoter, the RNA would be transcribed and the shRNA would be produced. However, seeing why only specific vectors are being 'specialized' for shRNA inserts...I think there must be a reason for this... I think I have never seen a vector said to be for shRNA having a CMV promoter.

So does anyone know is there specific criteria for a shRNA promoter and vector?

Thanks! :o

#2 Bomber

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Posted 14 June 2009 - 11:05 PM

Hi,

you might want to check the manual for pSilencer-plasmids from Ambion.
You can find some information about promoters etc.
They also have a shRNA plasmid containing a CMV.
Best regards

Edited by Bomber, 14 June 2009 - 11:05 PM.


#3 lab_member

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Posted 14 June 2009 - 11:59 PM

Hi Bomber,

Thanks for your suggestion. I didn't know of a CMV promoter shRNA vector before :-)

#4 Functional Screens

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Posted 15 June 2009 - 07:06 AM

There is something to do with the processibility and efficiency.
1. The shRNA are highly expressed with pol III promoters such as H1 and U6.
2. The pol III promoters make "short" hairpin RNA (shRNA) that needs "Dicer only" to process the shRNA, no Drosha involved.
3. Of course you can use pol II promoters such as CMV to express shRNA/RNAi, but "process/excise" shRNA in the pol II transcript needs Drosha. In order for Drosha to "efficiently process" the shRNA, the flanking sequences are important. That's why some companies make CMV promoter express miRNA flanking sequences for users to insert shRNA oligos. Personally, I think pol III promoters can do the job, no need to make it too complicated.
I hope I answer your question.

#5 lab_member

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Posted 16 June 2009 - 07:22 PM

Hi Functional Screens

Thanks for your reply! I found it very helpful! :-)




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