(A) GluN3B and membralin genes arranged in tail-to-tail fashion with overlapping 3′ coding regions of both genes. Blue and brown boxes indicate predicted exons of GluN3B and membralin genes, respectively. The entire GluN3B coding region and part of the 3′ coding region of membralin between the 5′- and 3′-arm (green arrows) in the WT allele were replaced by a Neo/PGK cassette in the DKO allele by homologous recombination. Spe I restriction sites, Southern blot and Northern blot probes, and primers used for PCR genotyping are indicated. (B) Genotyping by Southern blot analysis of SpeI-digested genomic DNA and by PCR. Mouse genomic DNA from 12 littermates from a single breeding pair of heterozygotes was subjected to Southern blot (top panel) and PCR analysis (bottom panel). Hybridizing bands or PCR fragments corresponding to WT and DKO allele are indicated. Both methods identified 4 WT (#6, 7, 8, and 10), 6 heterozygote (#1, 2, 3, 5, 9, and 12), and 2 homozygote (#4 and 11) mice. (C) Northern blot analysis of total RNA from null (KO) and WT mice using probes 1 and 2, derived from the last exon (XI) or exon VII-X of membralin, respectively (see A). Probe 1 generated a hybridization band (∼2.8 kb) in WT but not in DKO mice. Probe 2 generated a ∼2.8 kb band in WT and a ∼3.4. kb band in DKO mice.