B6(Cg)-Tnni3k^em1Hsv/J

Stock number: 034733
Product name: Tnni3k K489R
Research areas: Cardiovascular Research, Metabolism Research

Description

Tnni3k K489R mice carry a lysine to arginine amino acid substitution at position 489 (K489R) resulting in a normally-expressed full-length kinase-dead protein. This strain may be useful when studying regeneration as well as oxidative stress, injury, and remodeling in the postnatal heart.

Detailed description

Tnni3k K489R mice carry a CRISPR/Cas9 generated lysine to arginine amino acid substitution at position 489 (K489R) resulting in a normally-expressed full-length kinase-dead protein. No potential off-target sites are present. Tnni3k is a cardiomyocyte (CMs)-specific enzyme in the MAP kinase kinase kinase (MAPKKK) family. It is thought to play a role in cardiac physiology where it promotes ischemia/reperfusion injury, oxidative stress, and myocyte death.

In the neonatal heart, as CMs are induced to enter cell cycle, Tnni3k influences the percentage of these cells that successfully complete the cell cycle, to remain mononuclear and diploid, or to become polyploid midway through the first postnatal week. Mononuclear diploid CMs are a proliferative and regenerative subpopulation of the postnatal heart.

Mice carrying one Tnni3k K489R allele and one Tnni3k null allele (K489R/-) exhibit significantly increased levels of ventricular mononuclear CMs relative to heterozygous (+/-) mice and equivalent to the level in null (-/-) mice, with no changes in nuclear ploidy seen between groups. Mice that are homozygous for this allele are viable and fertile.

Development

A synthetic guide RNA (5’-AGATGAATTGCATCGTCAGC TGG-3’) was designed to cleave the mouse Tnni3k gene within intron 15. A synthetic 200 nt single strand oligodeoxynucleotide was designed for homology-directed replacement to change the lysine codon at position 489 to arginine (AAA to AGA) in exon 15 of the TNNI3 interacting kinase (Tnni3k) gene. No potential off-target sites are known to be present. Two G-to-T substitutions were also introduced in order to eliminate the Cas9 PAM sequence to prevent retargeting, creating a DraI restriction site at the cleavage site. sgRNA, the Cas9 nuclease, and the oligo replacement sequence were introduced into the cytoplasm of C57BL/6J-derived fertilized eggs with well recognized pronuclei. Correctly targeted embryos were transferred to pseudopregnant females. Tnni3k K489R pups were identified by PCR and DraI restriction digestion and were further bred to C57BL/6J mice to develop the colony (see SNP note below). Some of these mice were bred to mice carrying a Tnni3K null allele on a B6 congenic background. This allele was bred out of the mice being shipped to The Jackson Laboratory. 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. Three of the 5 markers that determine C57BL/6J from C57BL/6N were 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: Tnni3k^em1Hsv
Name: endonuclease-mediated mutation 1, Henry M Sucov
MGI accession ID: MGI:6384879
Generation method: Endonuclease-mediated
Attributes: Not Specified
Synonyms: K489R
Marker symbol: Tnni3k
Marker name: TNNI3 interacting kinase
Marker synonyms: Cark; CCDD; D830019J24Rik
Chromosome: 3
Strain of origin: C57BL/6J
Molecular note: CRISPER/Cas9 genome editing is used to change the lysine codon at position 489 to arginine (AAA to AGA) in exon 15 of the gene. This mutation results in a premature stop codon homologous to one found in exon 16, early in the encoding portion of the kinase domain, in the naked mole-rat species Heterocephalus glaber. Two G-to-T substitutions were also introduced in order to eliminate the Cas9 PAM sequence to prevent retargeting.