C.B6N-Rgs4^em1Drue/J

Stock number: 039431
Product name: Rgs4(N128A)
Research areas: Developmental Biology Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Neurobiology Research

Description

Rgs4(N128A) knock-in mice express Rgs4 (regulator of G-protein signaling 4) carrying a conserved amino acid substitution of Asparagine 128 to Alanine (N128A). The Rgs4(N128A) knock-in mice show decreased RGS4 GTPase activating protein activity. This line will be useful for studies of the roles of RGS4 in inflammation and other cellular responses.

 

Detailed description

RGS4 protein, encoded by the regulator of G-protein signaling 4 (Rgs4) gene, is a regulatory molecule that acts as a GTPase activating protein (GAP) for G alpha subunits of heterotrimeric G proteins. Rgs4(N128A) knock-in mice carry a conserved amino acid substitution of Asparagine 128 to Alanine (N128A). The Rgs4(N128A) knock-in mice show decreased RGS4 GTPase activating protein activity.

The donating investigator reported that RGS4 affects asthma severity in part by regulating the airway inflammatory milieu in a G protein-independent manner. Homozygous Rgs4(N128A) knock-in mice exhibited no gross phenotypic abnormalities and had normal body weights. Histological analysis of major organs including brain, heart, and lung revealed no obvious abnormalities. However, Rgs4(N128A) knock-in mice liver sections were vacuolated, suggestive of parenchymal lipid deposits and hepatic steatosis. Blood examination revealed that levels of hemoglobin and various leukocyte numbers were similar in Rgs4(N128A) knock-in mice and WT controls, indicating normal hematopoiesis. Rgs4(N128A) knock-in mice had increased airway hyper-responsiveness (AHR), reduced airway PGE2 levels, and augmented GPCR-induced bronchoconstriction compared with either RGS4 KO mice or WT controls. This line will be useful for studies of the roles of RGS4 for inflammation and other cellular responses.

Development

The Rgs4(N128A) line was generated using the CRISPR-Cas9 method to introduce an AAC>GCT [Chr1:169,571,486-4 (GRCm39)] nucleotide mutation corresponding to an N128A amino acid substitution. An sgRNA designed to cut near the N128 site of mouse Rgs4 genomic DNA and a single-stranded oligonucleotide donor were co-microinjected into the cytoplasm of zygotes collected from C57BL/6N mice together with synthetic Cas9 mRNA. The two-cell stage of embryos were implanted into the oviducts of pseudopregnant surrogate mothers. Of multiple founder mice with the desired amino acid change, one mouse was selected and bred with wild-type C57BL/6N mice to establish the Rgs4(N128A) knock-in mouse line. Rgs4(N128A) mice were subsequently backcrossed to Balb/c mice for five or more generations and maintained on a BALB/c genetic background by the donating laboratory (see SNP below). Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize BALB/cJ oocytes (Stock No. 000651).



In 2024, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. Two of the 43 markers throughout the genome were segregating with C57BL/6, on Chromosomes 1 and 7. The marker on Chromosome 7 is likely linked to the mutant allele.

Allele

Symbol: Rgs4^em1Drue
Name: endonuclease-mediated mutation 1, Kirk M Druey
MGI accession ID: MGI:7705111
Generation method: Endonuclease-mediated
Attributes: Not Applicable
Synonyms: RGS4(N128A)
Marker symbol: Rgs4
Marker name: regulator of G-protein signaling 4
Marker synonyms: ESTM48; ESTM50; RGP4; SCZD9
Chromosome: 1
Strain of origin: C57BL/6N
Molecular note: Using the CRISPR-Cas9 method, AAC to GCT [Chr1:169571486-4 (GRCm39)] mutation corresponding to Rrg4 N128A substitution was introduced.