B6.NOD-Ins2^em1Kplr/ArvnJ

Stock number: 037293
Product name: Ins2-R(B22)E
Research areas: Cell Biology Research, Diabetes and Obesity Research, Research Tools

Description

Ins2-R(B22)E mice carry a CRISPR/Cas9 generated CGT to a GAG mutation, resulting in an arginine to glutamic acid substitution at position 46 (R46E) in the insulin B chain (B22) of the Ins2 gene [R(B22)E]. These mice may be useful for studying the effects of proinsulin misfolding on the development of diet-induced diabetes.

Detailed description

Ins2-R(B22)E mice carry a CRISPR/Cas9 generated CGT to a GAG mutation, resulting in an arginine to glutamic acid substitution at position 46 (R46E) in the insulin B chain (B22) of the insulin II (Ins2) gene [R(B22)E]. Ins2 encodes one of the two insulin genes in the mouse. Ins2 is a peptide hormone involved in the regulation of carbohydrate and lipid metabolism. These Ins2-R(B22)E mice possess a mutation of the Arginine residue at position B22 that has been associated with proinsulin misfolding, resulting in impaired insulin processing and secretion.

Neither heterozygous male or female Ins2-R(B22)E mice develop diabetes at any age while on normal chow diet, yet increased proinsulin misfolding in the endoplasmic reticulum is evident in isolated islets. Males have indications of glucose intolerance and develop diabetes after 6 weeks on a high-fat diet (60% fat). Heterozygous mice have a much milder phenotype when compared to mice carrying the spontaneous Ins2^Akita mutation (Stock No. 003548).

Homozygous males and females develop spontaneous diabetes by 5 weeks of age. The development of disease parallels a progressive heterogeneity of β-cells with increasing fractions of proinsulin-rich/insulin-poor cells as well as glucagon-positive cells. Homozygous mice display a more severe phenotype than Ins2^Akita mice. Due to the severity and early onset of diabetes in homozygous males and females, the donating investigator did not attempt to make a homozygous colony. It is unknown if homozygous mice are fertile.

Development

Guide RNA and a mutant ssDNA oligonucleotide template were designed to create a CGT to a GAG mutation, resulting in an arginine to glutamic acid substitution at position 46 (R46E) in the insulin B chain (B22) of the insulin II (Ins2) gene [R(B22)E]. A silent mutation was also added, creating an Alu I restriction site. sgRNA, ssDNA, and the Cas9 nuclease mRNA were introduced into the cytoplasm of zygotes derived from NOD mice with well recognized pronuclei. Correctly targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by PCR, restriction digest, and sequencing, and mice were further backcrossed to C57BL/6J mice (Stock No. 000664) for at least 9 generations to remove off-target CRISPR changes (see SNP note below). Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

In 2022, 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. One of the 5 markers that determine C57BL/6J from C57BL/6N was found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.

Allele

Symbol: Ins2^em1Kplr
Name: endonuclease-mediated mutation 1, John Kappler
MGI accession ID: MGI:7275270
Generation method: Endonuclease-mediated
Attributes: Not Applicable
Synonyms: Ins2-proinsulin-R(B22)E
Marker symbol: Ins2
Marker name: insulin II
Marker synonyms: CP-II; IDDM; IDDM1; IDDM2; ILPR; Ins-2; InsII; IRDN; Mody; MODY10; Mody4; PNDM4
Chromosome: 7
Strain of origin: NOD
Molecular note: Guide RNA and a mutant ssDNA oligonucleotide template are designed to create a CGT to a GAG mutation, resulting in an arginine to glutamic acid substitution at position 46 (R46E) in the insulin B chain (B22) of the insulin II (Ins2) gene [R(B22)E]. A silent mutation was also added, creating an Alu I restriction site.