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3'UTR constructs


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#1 it_never_ends

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Posted 26 October 2009 - 01:59 PM

Hi All,

We are a little tight on time right now, trying to roll a study. I have a few miRs of interest all predicted to target the same gene. Cloning the 3'UTR and then performing the studies might take a while. A quick google search gives 2 companies- GeneCopoeia and SwitchGear, that presumably sell pre-made 3'UTR constructs tagged to Luciferase reporter for my gene of interest.

I haven't noticed any publications citing their constructs. Just wondering if anyone had any experience with either of these 2 companies. Or if you knew any other that worked well for you.

Also, a related question, most Luc-reported studies use a microRNA expression plasmid co-transfected with their Luc-reporter plasmid. I was wondering if there is any reason that co-transfection with miR-mimics (eg: Pre-miRs from Ambion) are not popular for repression of Luciferase signal?

Any help is appreciated. Thanks!

#2 Functional Screens

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Posted 26 October 2009 - 03:00 PM

In general, the 3'UTR is in one exon and it should not be a big problem to use PCR to pull out from genomic DNA.

Regarding your co-transfection question, most people might want to generate stable lines to constitutively express miRNA, so they use vector for co-transfection. Also, it's easy to monitor the transfection efficiency with markers, e.g. GFP and RFP, on the miRNA expression vector.




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