Figure 2

Cre-loxP recombination and expression of the mutant GluN2A(N595Q) subunit. (A) PCR-genotyping of various tissues of the adult GluN2A+/flox-non-transgenic (C) and GluN2A+/flox-Cre transgenic (M) mice. The PCR products represent the wild allele (a, 599 bp), flox gene (b, 807 bp), and Cre-mediated-recombinant gene (c, 454 bp). (B) The mRNA and protein levels of the GluN2A subunit expressed in the GluN2A+/flox-NestinCre mice are comparable to those of control mice. (B-a) Northern analysis of whole brain RNAs of control (+/+, GluN2A+/+; +/flox, GluN2A+/flox; flox/flox, GluN2Aflox/flox) and GluN2A+/flox-NestinCre mutant mice (+/flox-NestinCre). GluN2A and G3PDH cDNAs were used as probes. (B-b) Western blots of whole brain homogenates of control and mutant mice were performed using rabbit polyclonal antibodies against GluN2A, GluN2B, and GluN1 subunits (Upstate Biotechnology). (C) RT-PCR analysis of RNA prepared from whole-brains of control (+/+, wild type; +/flox, GluN2A+/flox-non-transgenic; flox/flox, GluN2Aflox/flox) and GluN2A+/flox-NestinCre mutant (+/flox-NestinCre) mice. Brain RNA was analyzed by RT-PCR amplification followed by digestion of amplified DNAs by NcoI (N), common for both mRNAs, and EcoRI (E), specific for mutant mRNA. Lane U is undigested DNAs. The 521 bp-long PCR product (band a) was the expected size and completely digested by NcoI to predicted lengths (band c: 278 bp, and d: 243 bp), verifying that the PCR products were derived from GluN2A mRNA. Digestion with EcoRI gave rise to two bands (band b: 338 bp, and e: 183 bp) only in the PCR products from the mutant mice (+/flox-NestinCre). There results clearly indicate that detectable mutant GluN2A mRNA was not expressed prior to Cre-loxP recombination and mutant GluN2A mRNA was detectable only in the GluN2A+/flox-NestinCre mice.