I've identified some microRNAs that I think are interesting. I would like to over express them with a 35S promoter but don't know where to begin? I have a destination vector with the promoter, so what is the best way to get the microRNA sequence next to it in a way that will be expressed.
Please give some more info: what system do you plan to use for the overexpression?
Hi Kenneth
We use the invitrogen gateway system in our lab, so i was thinking about using the pMDC32 destination vector as this contains a 35S promoter and a terminator. I'm not sure how to get the miRNA in. I assume i'll have to clone the the miRNA into an entry vector such as pdonr221 (http://plasmid.med.harvard.edu/PLASMID/GetVectorDetail.do?vectorid=5). Will i have to insert the whole miRNA gene complete with all its folds or can i simply use the 24bp's that interfere with gene expression?
thanks in advance, Richard
You will want to insert at least the full hairpin precursor so that the cellular machinery can process it into the correct mature miRNA.
Dear Richard ,
In our lab we generate artificial MicroRNA for any gene by submitting gene ID in wmd3 web microRNA design tool. It generate a list of amiRNA. You have to choose the top one which have been highlighted by green colour. Now this microRNA is used to generate oligo using the same tool. there will be 4 different oligos to be synthesized. Now using these oligo overlapping PCR is done using pRS300 vector as template. further the Gateway cloning process can be done using BP and LR reaction to get microRNA to express in pMDC32.
If you need any more info then let me know, i will attach a whole protocol of this. (generally we do it for plant system)
good luck
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