Recombined ES cell clones were microinjected into C57BL/6 blastocysts and gave rise to male chimeras with a significant ES cell contribution. These chimeras were bred with C57BL/6J mice expressing Cre recombinase to produce the KCC2 double point mutant heterozygous line devoid of the neomycin cassette. For each line, F1 genotyping was performed by PCR and Southern blot. PCR primers hybridizing upstream (5′-GTGGTTCGCCTATGGGATCTGCTACTC-3′) and downstream (5′-AGACAAGGGTTCATGTAACAGACTCGCC-3′) of the neomycin cassette allowed identification of the KCC2 endogenous, double point mutant allele harboring the neomycin cassette, and double point mutant devoid of the neomycin cassette [298 base pairs (bp), 1946 bp, and 387 bp, respectively]. The Southern blot hybridized with an external probe allowed identification of the wild-type allele (14.1 kb) and the double point mutant allele (4.6 kb).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.