Generation of the γ2F77/I77 gene switch mouse version (γ2 knockdown mouse).

<p><b>A,</b> Targeting strategy. A full length γ2 cDNA was placed into the 5′UTR of exon 1 of the GABA_AR γ2 subunit gene. The γ2 cDNA encodes the I77 version of the γ2 subunit. The 5′loxP contains a BglII restriction site. For the initial targeting a frt-flanked neomycin resistance gene (neo) was placed after the γ2 cDNA. The neo cassette contains a second loxP site at the end, the 3′loxP site. The 5′frt site contains another BglII site. Black bars indicate the positions of Probe A and B. Arrows are PCR primer positions. Black triangle, loxP sites; black diamonds, frt sites; B, BglII site; S, SalI site; Sp, SpeI site; TGA, STOP codon; UTR, untranslated region; HA, hemagglutinin epitope; polyA, polyadenylation signal. <b>B,</b> Southern blot analysis of BglII digested tail biopsies hybridized with probe A. Lane 1, 4, 8 and 9 are samples from WT mice. Lane 2, 3 and 5–7 show samples from heterozygous targeted mice. The 5.1 kb band represents the 5′ BglII fragment. <b>C,</b> PCR analysis of mouse tail DNA. Lane 1 shows a WT genotype, lane 2 a heterozygous and lane 3 a homozygous (for the mutation) genotype. The PCR amplification is across the 5′loxP site.