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NMDA Receptors

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Cover of 'NMDA Receptors'

Table of Contents

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    Book Overview
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    Chapter 1 NMDA Receptors in the Central Nervous System
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    Chapter 2 Quantification of NMDAR Subunit Genes Expression by qRT-PCR
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    Chapter 3 Genetic and Functional Analysis of GRIN2A in Tumor Samples
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    Chapter 4 Detection of NMDARs Antibodies in Encephalitis
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    Chapter 5 Magnetofection™ of NMDA Receptor Subunits GluN1 and GluN2A Expression Vectors in Non-Neuronal Host Cells
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    Chapter 6 Transfection in Primary Cultured Neuronal Cells
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    Chapter 7 Selective Cell-Surface Expression of Triheteromeric NMDA Receptors
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    Chapter 8 Functional Analysis of Recombinant Channels in Host Cells Using a Fast Agonist Application System
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    Chapter 9 GluN2B Subunit Labeling with Fluorescent Probes and High-Resolution Live Imaging
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    Chapter 10 Design of Light-Sensitive NMDARs by Genetically Encoded Photo-Cross-Linkers
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    Chapter 11 Gene Targeted Mice with Conditional Knock-In (-Out) of NMDAR Mutations
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    Chapter 12 Electrophysiological Investigation of NMDA Current Properties in Brain Slices
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    Chapter 13 Analysis of Functional NMDA Receptors in Astrocytes
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    Chapter 14 GluNs Detection and Functions in Microglial Cells
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    Chapter 15 NMDA Receptor Activity in Circulating Red Blood Cells: Methods of Detection
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    Chapter 16 NMDA Receptors as Voltage Sensors
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    Chapter 17 Development of a Computational Approach/Model to Explore NMDA Receptors Functions
Attention for Chapter 11: Gene Targeted Mice with Conditional Knock-In (-Out) of NMDAR Mutations
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Chapter title
Gene Targeted Mice with Conditional Knock-In (-Out) of NMDAR Mutations
Chapter number 11
Book title
NMDA Receptors
Published in
Methods in molecular biology, January 2017
DOI 10.1007/978-1-4939-7321-7_11
Pubmed ID
Book ISBNs
978-1-4939-7320-0, 978-1-4939-7321-7
Authors

Rolf Sprengel, Ahmed Eltokhi, Frank N. Single

Abstract

For the genetic alterations of NMDA receptor (NMDAR) properties like Ca(2+)-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.

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Mendeley readers

The data shown below were compiled from readership statistics for 6 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 6 100%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 1 17%
Student > Bachelor 1 17%
Researcher 1 17%
Student > Doctoral Student 1 17%
Unknown 2 33%
Readers by discipline Count As %
Nursing and Health Professions 2 33%
Psychology 1 17%
Unknown 3 50%