ausblenden:
Schlagwörter:
Gene targeting; Genotyping; GluN1; Grin1; Hypomorphic allele; Southern blot
Zusammenfassung:
For the genetic alterations of NMDA receptor (NMDAR) properties like Ca2+-permeability or voltage-dependent gating in mice and for the experimental analysis of nonsense or missense mutations that were identified in human patients, single nucleotide mutations have to be introduced into the germ line of mice (Burnashev and Szepetowski, Curr Opin Pharmacol 20:73-82, 2015; Endele et al., Nat Genet 42:1021-1026, 2010). This can be done with very high precision by the well-established method of gene replacement, which makes use of homologous recombination in pluripotent embryonic stem (ES) cells of mice. The homologous recombination at NMDAR subunit genes (Grin; for glutamate receptor ionotropic NMDAR subtype) has to be performed by targeting vectors, also called replacement vectors. The targeting vector should encode part of the gene for the NMDAR subunit, the NMDAR mutation, and a removable selection maker. In these days, the targeting vector can be precisely designed using DNA sequences from public databases. The assembly of the vector is then done from isogenic NMDAR gene fragments cloned in bacterial artificial chromosomes (BACs) using "high fidelity" long-range PCR reactions. During these PCR reactions, the NMDAR mutations are introduced into the cloned NMDAR gene fragments of the targeting vector. Finally, the targeting vector is used for homologous recombination in mouse ES cells. Positive ES cell clones which have the correct mutation have to be selected and are then used for blastocyst injection to generate chimeric mice that hopefully transmit the Grin gene targeted ES cells to their offspring. In the first offspring generation of the founder (F1), some animals will be heterozygous for the targeted NMDAR gene mutation. In order to regulate the expression of NMDAR mutations, it is important to keep the targeted NMDAR mutation under conditional control. Here, we describe a general method how those conditionally controlled NMDAR mutations can be engraved into the germ line of mice as hypomorphic Grin alleles. By breeding these hypomorphic Grin gene targeted mice with Cre recombinase expressing mice, the hypomorphic Grin allele can be activated at specific time points in specific cell types, and the function of the mutated NMDAR can be analyzed in these - so called - conditional mouse models. In this method chapter, we describe in detail the different methodical steps for successful gene targeting and generation of conditional NMDAR mutant mouse lines. Within the last 20 years, several students in our Department of Molecular Neurobiology in Heidelberg used these techniques several times to generate different mouse lines with mutated NMDARs.
