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).

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