hello
I am trying to target a gene using artificial microrna technology. I am planing to put the artificial microrna on the 3'utr of EGFP, the transcription will be done using cmv promoter and the vector will be pcDNA 3.1.
is it possible to use this design? how to design the artificial microrna? is it necessary to add flanking regions to the artificial microrna? there is a tool to design the artificial microrna?
What is your specific goal? Gain-of-function or target validation? in vitro or in vivo? In vitro, you could simply work with miRNA mimics. Mimics just simulate mature miRNAs of your interest. Once transfected into e.g. cells they incorporate with RISC and you can study the phenotype.
my purpose is to knockdown a gene using artificial microrna and study its function in EPC cell.
Hi Adham
there are several ways to over express a microRNA in a cell. you can use microRNA mimics which is a quite efficient method. but you can also clone microRNA precursor into an expression vector like pcDNA. in my experience it is better to add 50-100 nucleotides to precursor sequence. also don't fuse precursor sequence to GFP, sometimes it could affect microRNA processing system. check out our work in this paper it might help: http://www.sciencedirect.com/science/article/pii/S0378111915014602
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