I am looking for primer sets that can differentiate mice containing inducible cre's (ERcre's) from mice containing normal cre's using standard PCR genotyping.
Well obviously you would design primers homologous to the ERT2 part. That can of course differ between different mice based on the actual Cre-ERT2 construct that was used to make the transgenic mice.
I used these ones to detect VE-cadherin-CreERT2 when I had to cross these mice with other that contained my lox allele of interest, but also carried Emx1-Cre.
ERT2 fw 5'-G T G C C T G G C T A G A G A T C C T G
ERT2 rc 5'-A G A G A C T T C A G G G T G C T G G A
Of course it is also best to have a specific PCR for your constitutive Cre e.g. if you know at least the promotor sequence. Otherwise you can simply PCR for the Cre-ORF that should give a band for both Cre and Cre-ERT2 and compare that with your specific ERT2 PCR to sort out those mice crrying constitutive Cre and Cre-ERT2.
Thanks, Christian! I will try that.
Bob, we received some mice from a lab that we thought were simply heterozygous for a floxed allele, which I confirmed with genotyping. So I bred those mice to my cre of interest. Once I did the genotyping, I knew something was wrong because I had too many cre+ mice (our primers detect ERcre and straight Cre's). So I emailed the lab, and they said that it's likely that there was an ERcre in the animals that they gave us. So I confirmed that there was a cre in the delivered animals (I should have done this first just to be safe).
So now I want to differentiate between ERcre and our Cre so I don't have to start over again with all cre- mice.
You could cross the original mouse to background (C57) and select Flox/+ Cre - mice and then interbreed littermates (Flox/+ X Flox/+) or Flox/+ X Cre; +/+. You would only be one generation behind, but clean.
Hi guys,
Has anybody tried CreERt2 primer sets for qPCR?! I am referring to primers bridging the interface of Cre and ERt2 you wrote about. I have a GFAP-CreERt2 mouse; hence want to verify transgenic expression exclusively in the brain but we are detecting endogenous Cre expression despite using Qiagen RNA extraction kit with DNase.
Thanks a lot!
Nadja
@Nadja Bjelopoljak did you find the primer? I am looking too, but I can find the right probe!
Thanks
Valeria
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