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overexpress mature microRNA by siRNA vector

miRNA

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#1 Xiaoting Hong

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Posted 14 August 2013 - 12:05 PM

Hi, i am trying to overexpress microRNA in cells. I am wondering if i can using those siRNA vectors to express the mature microRNA(PSIREN or p Silence). For those vectors is H1/U6 promoter and then express the shRNA , usually designed as sense strands+loop+antisense strand+TTTTTT. Can i just replace the sense strands of siRNA with mature miRNA sequence ( since they are almost the same) to get overexpress of mature microRNA?



#2 pcrman

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Posted 14 August 2013 - 12:17 PM

For optimal expression and function of miRNAs, the proven strategy is to clone the precursor-miRNA (pre-miRNA) into a vector. The typical size of a pre-miR is around 70 bp, Usually people include  ~100 bp flanking sequences, resulting in an insert of ~270 bp in the vector. 

 

The cloning method you mentioned does not work well for miRNA because miRNAs do not form perfect complementary stem and cannot be efficiently processed by dicer if you just add an artificial loop.



#3 Xiaoting Hong

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Posted 14 August 2013 - 01:43 PM

Thank you, pcrman. But my aim is to express C.elegant miRNA (cel-miR-67)in human cell. I am studying the exosome transfer of microRNA, so i do not care about its function. Rightnow i am using the miRNA mimic, those chemically synthetic double-stranded RNAs, but i am trying to build a stable expressing cell line.

One of my concern is if i express the pre-miRNA of C.elegant miR , can it be processed correctly in human cells?

 

And also i am not quite understand about" miRNAs do not form perfect complementary stem and cannot be efficiently processed by dicer ". I can design the shRNA with mature miRNA sequence and make it full perfect complementary. That would be mature sequence of miR(24base of cel-miR-67)+artifical loop+ complementary antisense strand+poly T. 

 

Thanks again!







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