Generation of the 1d2d-PSD-95-EGFP knockin mouse. A, Domain structure of PSD-95. The mutant 1d2d-PSD-95-EGFP contains mutations in the PDZ1/2 domains and EGFP at the C-terminus, as described in the Materials and Methods. B, Schematic diagram of the PSD-95 gene locus, the recombinant allele after homologous recombination, and the mutant allele after Cre recombination. The region from exon 5 to the middle of intron 8 was replaced by the corresponding region of mutant PSD-95 cDNA connected with the EGFP gene (see methods). B, BamHI. C. Southern blot analyses of mouse genomic DNA to confirm homologous recombination. Mouse genomic DNA was digested with BamHI and hybridized with a 3’ external probe, whose position is indicated in B. wt/wt, wild-type mouse; wt/KIΔNeo, heterozygous KI mouse after deleting the Neo cassette; wt/KI+Neo, heterozygous KI mouse containing the Neo cassette. D. Genotyping of the mouse by PCR amplification of the region from exon 4 to exon 8 to detect an ~300-bp size shift. wt/wt, wild-type mouse; KI/KI, homozygous KI mouse; wt/KI, heterozygous KI mouse. E, Immunoblot of brain extracts from wild-type (WT) and homozygous KI (KI) mice. The whole brain crude membrane fractions from WT (2 μg total protein) and KI (20 μg total protein) mice were analyzed with an anti-PSD-95 K28/43 antibody. F, Immunohistochemical staining of sagittal sections from the hippocampus of WT and homozygous KI adult mice with an anti-GFP antibody. Extensive staining was observed in the hippocampal fields of the KI mice, but not in the WT mice. Scale bar = 500 μm.