So, I am trying to determine whether my mice are homozygous or heterozygous for a floxed gene. I ordered the primers specified by the company which developed the mice and they amplify a 301 bp sequence in an intron of the gene located after the exon flanked by the loxP sites. WT mice should render this 301 bp product while floxed animals generate a 419 product because the amplified sequence now contains the down stream loxP site and the FRT site remaining after floxing the mice with flipase. I am only getting "homozygous" 419 bands even tho I am suppossed to have some heterozygous animals in my colonies. If i missed DNA from my floxed mouse with that of a WT mouse I see the 301 and 419 bands. When I repeated the PCR with old samples and with DNA from new animals (some of them should be heterozygous) and i either get only the 419 band or no band at all! I am thinking of sequencing the products but also would like to hear some thoughts as of why I cannot detect heterozygous WT/Flox with my PCR
Hey Ruben,
Is there any chance the mice started to be crossed homozygously at some point?
Are all your mice back-crossed to the same strains?
I have seen similar situations when sequencing primers overlap with the floxed sequences so they fail to recognize (or do it very little) the WT strain. However, you ruled this out with your tests using mixed template.
Maybe the company sold you embryos that were already homozygous.
Hi Sebastián,
they were given to me as heterozygous and homozygous animals who where then crossed so I was expecting some heterozygous mice. They are all same strain
Hi Ruben,
I don't know how urgent you need the mice but, just to be sure, I would cross one of your mice with a known WT and genotype the progeny. There you must have HET progeny. Even more if you get only HET progeny you may suspect you were given with Homozygous and Homozygous in HET's clothing ;).
Cheers,