5' arm of homology > LoxP > Targeted exon > FRT > Neomycin > FRT > LoxP > 3' arm of homology
At great length I screened 180 colonies to find only 2 positive clones. These were electroporated and used to generate chimeras. The chimeras were confirmed and mated to B6 to confirm germline transmission. Floxed hets were identified and then mated to beta-actin driven cre to generate a systemic knockout.
So I should have mice that are:
cre positive, neomycin negative, deleted allele
cre positive, neomycin negative, wild type allele
cre negative, neomycin positive, floxed allele
cre negative, neomycin negative, wild type allele
BUT I have no mice that are cre positive, neomycin negative, with the deleted allele; instead I have mice that are cre positive, neomycin positive, wild type allele.
i.e. the presence of cre should result in recombination at LoxP sites and the neomycin lost along with the targeted exons.
I checked the LoxP sites of the construct before it was injected. I'm in the process of sequencing the LoxP sites in the current progeny.
Could it be the cre recombinase? I was under the impression that you only need one copy of cre for recombination to occur.
Has anyone ever come across this/have any ideas what may be happening??? Any suggestions would be gratefully received!
Edited by Final year phd at wits end, 09 September 2010 - 12:50 PM.