improper recombination in transgenic mice - (Dec/11/2012 )
I am currently working on genotyping some transgenic mice. These mice are meant to have a neomycin cassette inserted into a specific genomic locus and also they are meant to contain cre restriction sites approximately 200-400bp either side of this neomycin cassetete. To my surprise there seems to be something strange going on within the genomic locus where the neomycin cassette is meant to be inserted as i have used three different sets of primers and in many animals i get a band which is approximately 300bp longer than one would expect for wild type but much shorter than one would expect for a correct insertion of the neomycin cassete and cre restriction sites.
I was therefore wondering has anybody ever encontered something similar where something seems to have gone wrong during recombination with one correct floxed neomycin cassette yet the second allelle seems to be some sort of mutant that is neither wild type nor correct floxed allele. My plan is to hopefully in the near future cut out one of these strange bands and get it sequenced to confirm or disprove what im thinking but id like to hear if people have any comments or suggestions.
Have these mice ever been crossed with a Cre-expressing line? If so, it's possible that there is a leaky cre expression that may have gotten into the germ line. Are these males or females or both showing the third band?
Another thing that comes to mind is specificity of the primers. Try increasing the annealing temp by a few degrees to see if the band goes away.