Cloning beginner and clueless! - (Jan/09/2014 )
Sorry but this is really basic knowledge that I lack!
I've just started a new postdoc and have been asked to design a reporter mouse. I have never done a single cloning/genetics/gene editing/even transfection before in my life and don't know where to start. The plan from my supervisor is to use a IRES-GFP targeting vector to insert gfp into the keratin 14 gene using the CRISPR/cas system. All we want to do is be able to sort the gfp expressing cells out.
What considerations are to be made when deciding where to put this vector? I don't really know how it works, could I insert the gfp after the final exon (after the stop codon) and before the 3'UTR? In this case would the homology arms just be the sequence upstream encompassing the final intron and exon etc and then downstream the 3'UTR and downstream sequence?
Would be very grateful if anyone could help me with this!
I think that is a common method. You remove the stop codon of your gene and put GFP in frame with its own stop codon. I think it is a good idea to do this before the 3'UTR. However, you won't know until you express this construct, whether or not the GFP insertion has effects on UTR conformation, function, etc.