3 replies to this topic

[batteryhorsestaple]

In my last lab we designed our genotyping PCR to give wt and floxed bands at different sizes to distinguish wt/het/hom floxed mice. We occasionally used to get another band a few dozen bp bigger than the floxed band which we called 'delta' (and we could get any combination of the 3 bands in one mouse).

My supervisor had explained how this extra band in generated in <5% of mice, and it was due to some kind of recombination event rather than a fault with the PCR. I've since forgotten how this extra band occurs and she's not answering her email (to me) anymore :s

Anybody have any idea what I'm talking about? It's happening in my new lab and I want to sound smart

[doxorubicin]

So when you say you can get any combination of the 3 bands in one mouse, this means you can even have all three bands from one mouse?  I don't know what kind of recombination you could be talking about.  Presumably the homologous recombination event happened only one time in the right place, so you have just one "cassette" in your genome.  If there was Cre recombinase mediated recombination of your gene, then your PCR wouldn't give any band.  I have seen slightly different sized bands from mice of different genetic backgrounds (e.g. 129Sv vs. Bl6).  To know for certain what the bands are you should probably just cut the bands out and have then sequenced.

[batteryhorsestaple]

We genotype from the tails but get Cre expression elsewhere - so there should be no cre-mediated excision in our genotyping samples. In most cases we can distinguish the loxP flanked gene from the wild-type but in a small number of animals there is (I think) some kind of* cre-independent recombination event when you breed certain combinations of lox/lox or lox/wt mice together. These are not new mice, the cassette is in the right place and working, just a small % of offspring have a funny 'extra' genotype.

*but I'm not sure, it was years ago when it was mentioned to me so I'm pretty hazy.

We just cull the weird ones, so I don't really need to know what it is - it's just nice to know, you know?

Edited by batteryhorsestaple, 26 March 2012 - 06:08 PM.

[sligeach]

hi battery horsestaple

I am currently genotyping some transgenic mice for the presence of a floxed allele and i have observed something almost identical indeed i have just today posted a question similar to yours on this forum. In my case i have now used three different primer sets which flank the floxed insertion allele and i often get a third allele which is 300bp longer than wild type but about 1000bp shorter than the floxed allele. I had thought something weird must have happened during recombination but i cant get a satisfactory answer so hopefully somebody may be albe to find an answer as it would be very much appreciated. I am intending to cut out a third allele band and get it sequenced just to see what exactly it is as i hate not knowing.