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CRE mediated recombination problems

CRE mES Cells

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#1 Shrin

Shrin

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Posted 31 October 2012 - 04:00 PM

Hi,

I have a mES Cells line in which my gene is floxed by to different lox sites (LoxP and lox511), for directional replacement. I would like to replace it for a different loxP-Mut-Lox511 cassette. To optimize the system, I am using, right now, a loxP-GFP-lox511 construct. I am doing electroporation with CRE plasmid (under EF1 promoter) along with my LoxP-GFP-Lox511construct. I am pretty sure that the elctroporation efficiency is high (90 %) checked by DsRED expression. Unfortunately, I am not able to get any green clone. I have tried electroporation with linearized plasmids and with super-coiled plasmids, with the same negative result. I have checked the sequence of my CRE and LoxP-GFP-lox511 they are fine. Even without green cells I have done gDNA PCR to check GFP, but I could not detect GFP in gDNA. It is clear that my recombination is not working but I have no idea why.
Could anybody give me any advice?

Thanks a lot.

Sam




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