B6.FVB-Mir486^em1Mtm/MsalxJ

Stock number: 036767
Product name: miR-486 KO
Research areas: Cardiovascular Research, Cell Biology Research, Neurobiology Research, Reproductive Biology Research

Description

miR-486 knock-out mice have 85 base pairs deleted from the Mir486 (microRNA 486) locus. Mice homozygous for the mutation exhibit cardiomyopathy and skeletal muscle weakness and slightly decreased fertility in females. These mice be useful in studies of dystrophic skeletal muscles and cardiac muscle pathologies as well as Ago2-dependent RNA silencing in the hematopoietic system.

Please note: Mir486 KO mice on the FVB/N background are available as FVB/N-Mir486^em1Mtm/MsalxJ Stock No. 036901.

Detailed description

Mir486 (microRNA 486) encodes a muscle specific short, noncoding regulatory RNA (myomiR) that acts as negative regulator of gene expression by binding target mRNAs. Specifically, miR486 is markedly downregulated/reduced in the muscles of Duchenne muscular dystrophy (DMD) patients and dystrophin-deficient mice (e.g., Dmd^mdx-5Cv mice). miR-486 knock-out mice have 85 base pairs deleted from the loop of the Mir486 locus, removing the mir-486-5p region.

Mice homozygous for the mutation exhibit early cardiomyopathy and skeletal muscle weakness beginning at around 4-6 months or age. Specifically, tibialis anterior (TA) muscles from mir-486 KO mice develop a decreased myofiber cross-sectional area, increased centralized myonuclei, and increased fibrosis. The phenotype is exacerbated when combined with the Dmd^mdx-5Cv mutation (B6Ros.Cg-Dmd^mdx-5Cv/J Stock No. 002379). In the heart, KO mice develop increased fibrosis, decreased fractional shortening and ejection fraction. The donating investigator indicates that no phenotypic differences (gross or skeletal/cardiac muscle) have been observed between the C57BL/6J background and the FVB/N background.

On the FVB/N background, homozygotes develop impaired homeostasis under stress and erythroid differentiation defects resulting in a reduction in mature red blood cells and an accumulation of immature reticulocytes. The hematopoietic phenotype has not been verified on the C57BL/6 background. On the C57BL/6 background, homozygous female mice exhibit slightly decreased fertility at 6 months of age, and male KO mice are viable and fertile.

Please note: Mir486 KO mice on the FVB/N background are available as FVB/N-Mir486^em1Mtm/MsalxJ Stock No. 036901.

Development

The miR-486-KO allele was created by Dr. Michael McManus (University of California, San Francisco). CRISPR/cas9 endonuclease-mediated genome editing was used to create an 85 bp deletion in the loop of the Mir486 sequence on chromosome 8. Of note, Mir486 is embedded within the Ank1 (ankyrin 1) locus. The mutation removes mature mir-486-5p. Single guide RNA and cas9 endonuclease were introduced into FVB/N-derived fertilized eggs by pronuclear injection. Embryos were transferred to pseudopregnant females, and correctly targeted pups were bred to FVB/N mice for germline transmission. Mice were maintained on the FVB/N background. Northern blot analysis confirms the absence of Mir486 RNA in TA (tibialis anterior) muscle and whole heart. Some of these mice were further backcrossed to C57BL/6J mice for more than 6 generations in the laboratory of Dr. Matthew Alexander (University of Alabama at Birmingham). Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J (Stock No. 000664) oocytes.

In 2021, 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 43 markers throughout the genome was segregating with FVB on Chromosome 8.

Allele

Symbol: Mir486^em1Mtm
Name: endonuclease-mediated mutation 1, Micheal McManus
MGI accession ID: MGI:6147598
Generation method: Endonuclease-mediated
Attributes: Null/Knockout
Marker symbol: Mir486
Marker name: microRNA 486
Chromosome: 8
Strain of origin: FVB
Molecular note: CRISPR-targeting produced an 85 bp deletion. Northern blot analysis confirmed the absence of RNA production.