Figure 1.

Targeting the NMDA receptor NR2A subunit gene in mice. (a) The NR2A-H128S mutant allele was created by homologous recombination. The scheme shows the WT NR2A allele, the targeting vector, the targeted allele and the H128S knock-in allele. The CAT codon encoding histidine 128 (H128) in exon 1 was replaced by the TCC codon encoding serine (S), and a loxP-flanked (‘floxed’) neo cassette was introduced 3′ from exon 1 to select for embryonic stem cells harboring the knock-in allele. The final mutant allele was obtained after excision of the neo cassette by a Cre recombinase treatment of embryonic stem cells. White box, exon 1; star in white box shows the H128S mutation; dark line, intronic sequences; Ba, BamH I; Bs, Bsu361; Sp, SpeI (restriction sites); triangles, loxP sites; neo box, neomycin-resistance cassette; gray bars, probes for Southern blot analysis; lines above gene indicate expected labeled DNA fragments in Southern blot analysis. (b) Southern blot analysis of WT and targeted alleles in the selected embryonic stem cell clone. Genomic DNA was digested with Bsu361 and BamHI, and hybridized with 5′and 3′ external probes, respectively or the neo probe. Expected bands at each size as indicated in a are obtained. (c) Genomic DNA sequence analysis using tail biopsies from WT (left panel) and knock-in homozygous mutant (right panel) mice, showing the replacement of the CAT codon by the mutated TCC codon. (d) Genotyping of the NR2A-H128S knock-in line. PCR analysis using mutation-specific primers (strategy on left) reveals WT (280 bp) and mutant (315 bp) alleles (right). White arrow, forward primer for WT DNA, black arrow, forward primer for mutant DNA, gray arrows, reverse primer. Analysis of genomic DNA from WT NR2A, homozygous NR2A-H128S and heterozygous NR2A WT/NR2A-H128S mice is shown. The 35-bp differences between mutant and WT alleles results from the remaining loxP site in the mutant allele.