Rudhard, York; (2001) Conditional activation of the N598R point mutation in the NR1 subunit of the NMDA receptor in knock-in mice. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The NMDA-type glutamate receptor is involved in synaptic plasticity. NMDA receptors are heterooligomers of the NR1 together with any of the NR2 (NR2A-D) subunit variants. Introduction of the point mutation N598R within the channel-lining segment M2 of NR1 abolishes voltage-dependent Mg²⁺-block and Ca²⁺-permeability of the NMDA receptor channel. The NR1 N598R mouse model, which had been obtained earlier in the group of Dr. Ralf Schoepfer, carries the N598R point mutation and a floxed neo cassette within intron 18 of the NR1 gene. This NR1 (N598Rneo) allele is essentially inactive. However, Cre-mediated excision of the floxed neo cassette yields an active NR1 (N598R) allele. The presented work has two parts: 1.) Analysis of mice with global expression of the NR1 N598R mutation. Global activation of the mutation - through breeding with the 'Deleter' mouse - results in lethality shortly after birth in mice carrying one mutant and one wildtype NR1 allele. Thus, the NR1 N598R mutation is dominant negative lethal. The impact of the NR1 N598R mutation on the formation of the whisker-related somatotopic map in the brainstem of newborn mice was investigated. A comparative histochemical study involving mice with five different genotypes is presented. A part of this work has been published in abstract form (Rudhard et al., 2000). 2.) Generation of a Cre knock-in mouse line, in which Cre is co-expressed with the kainate receptor subunit 1 (KA1). The targeting vector was assembled in yeast by homologous recombination with a YAC carrying the KA1 gene. The following elements were inserted in the 3'UTR of the native KA1 gene: internal ribosome entry site (IRES), followed by an EGFP-Cre translation unit, followed by flirted selection markers. Chimeric mice have been generated (performed by Eurogentec, Belgium) and are being bred for germline transmission for further analysis.

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