C57BL/6J-Gjb1^em2Rwb/RwbJ

Stock number: 032526
Product name: Gjb1 R15Q
Strain synonyms: Gjb1^R15Q
Research areas: Cell Biology Research, Mouse/Human Gene Homologs, Neurobiology Research

Description

This R15Q point substitution in the intracellular domain of GJB1 (synonym Gjb1^R15Q) provides a viable delayed-onset model for X-linked Charcot-Marie-Tooth disease.

Detailed description

Gjb1 (Cx32) is an X-linked gene encoding a 4-transmembrane gap junction protein that forms hexameric channels in myelinating glial cells. This protein is expressed in white and gray matter, and is the major connexin expressed in Schwann cells, where it localizes to the nodes of Ranvier. Mutations in GJB1 underlie X-linked Charcot-Marie-Tooth disease; this R15Q point substitution (CGG>CAA) in the short intracellular domain is a model of a human disease-associated allele. GJB1 peptides were found by mass spectroscopy in liver extracts of hemizygous males, and immunocytochemistry of Schwann cells from teased sciatic nerves showed the presence of the mutant GJB1, with some of the mutant protein retained in the cytoplasm, but much of it localized normally to the nodes of Ranvier. At 2 months of age the levels of circulating neurofilament light chain, an indicator of compromised axons, were found to be slightly elevated, then dramatically elevated at 9 months of age. Thinly myelinated axons, demyelinated axons, degenerating axons and regenerating axon bundles were found in peripheral nerves beginning at approximately 5 or 6 weeks of age, which is a later time point than that found in a null allele (Stock No. 032530), and the motor branch of the femoral nerve was more severely affected than the sensory branch, with proximal ventral roots only mildly affected. Nerve conduction velocities of sciatic nerves were found to be reduced by 9 months of age but not 6 months of age, the amplitude of compound muscle action potentials was not reduced, and body weights and triceps surae weights were normal. No deficits were detected in hemizygotes in the accelerated rotarod, latency to fall, or grip strength tests for either forepaws or all four paws. With its later phenotypic onset and intact mutant protein this strain provides a model useful for CMTX treatment development. Homozygous females have not been as rigorously characterized as the hemizygous males, but have a phenotype similar to that of the males.

Development

Cas9 nuclease and plasmids encoding a signal guide RNA designed to introduce the R15Q mutation in exon 2 (arginine to glutamine at amino acid 15) of the X-linked Gjb1 gene were introduced into the cytoplasm of C57BL/6J-derived fertilized eggs. Correctly targeted pups were identified by sequencing and PCR and further bred to C57BL/6J to develop the colony. Sperm was cryopreserved from hemizygous males at generation N2F1.

Allele

Symbol: Gjb1^em2Rwb
Name: endonuclease-mediated mutation 2, Robert W Burgess
MGI accession ID: MGI:8224458
Generation method: Endonuclease-mediated
Attributes: Humanized sequence
Synonyms: R15Q
Marker symbol: Gjb1
Marker name: gap junction protein, beta 1
Marker synonyms: CMTX; CMTX1; connexin 32; connexin-32; CX32; Cxng; Gjb-1
Chromosome: X
Strain of origin: C57BL/6J
Molecular note: This CRISPR/cas9-mediated CGG to CAA mutation results in an R15Q substitution, which replicates a human mutation underlying Charcot-Marie-Tooth disease type 1X.