B6;129-Casq2^tm2.1Kpfe/J

Stock number: 036291
Product name: Casq2RevFlox
Research areas: Cardiovascular Research, Cell Biology Research, Developmental Biology Research, Research Tools

Description

Casq2RevFlox mice contain an inverted promoter and exon 1 that are flanked by loxP66 and loxP71 inward facing sites. Casq2RevFlox mice contain a null allele, the function of which is restored by Cre recombination of the mutant loxP sites. This strain is useful for studying calcium storage in cardiac muscle and polymorphic ventricular tachycardia.

Detailed description

Casq2RevFlox mice have loxP66 and loxP71 inward facing sites flanking the promoter and first exon of the calsequestrin 2 (Casq2) gene (from -561 to +538bp) that are inverted relative to their normal endogenous orientation. This gene encodes a calcium binding protein that stores calcium for muscle function, specifically in the sarcoplasmic reticulum (SR) in cardiac muscle where it plays a role in excitation-contraction coupling in the heart and in regulating the rate of heart beats. Exon 1 includes 3 in-frame ATG translational starts, with only the second ATG site encoding a leader sequence that appropriately targets the nascent CASQ2 peptide to the SR. Mutations in this gene cause stress-induced polymorphic ventricular tachycardia. Homozygous mice are viable and fertile, and retain functional SR Ca2+ storage, yet they exhibit low resting heart rates and stress induced polymorphic ventricular tachycardia. This Casq2RevFlox allele is functionally null but Cre-mediated recombination restores gene function by inverting the 1.1 kb Casq2 gene fragment back to its normal orientation. The recombination is unidirectional because recombination between loxP66 and loxP71 sequences generates one wild type loxP element and one that carries a double point mutation making further recombination between these two substrates inefficient.

Of note, Casq2^floxed mice (Stock No. 036290) contain conventional loxP sites flanking the promoter and exon 1 of the Casq2 gene. Cre recombination renders the Casq2 gene non-functional.

Development

The Casq2RevFlox targeting vector was designed to insert loxP66 and loxP71 inward facing sites flanking the promoter and first exon of the calsequestrin 2 (Casq2) gene (from -561 to +538bp) that are inverted relative to their normal endogenous orientation. A frt-flanked neomycin resistance (neo) cassette was inserted in intron 1, between exons 1 and 2. The construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl⁺-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts and resulting chimeric mice were bred to C57BL/6J mice (Stock No. 000664). Subsequently, offspring were bred to ROSA26::FLPe mice (Stock No. 003946) to remove the neo cassette. Progeny were crossed to remove the FLPe-expressing transgene and resulting Casq2^floxed mice bred to C57BL/6J mice for three additional generations by the donating lab. 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.

Allele

Symbol: Casq2^tm2.1Kpfe
Name: targeted mutation 2.1, Karl Pfeifer
MGI accession ID: MGI:6711695
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed)
Synonyms: Casq2RevFlox; rCasq2
Marker symbol: Casq2
Marker name: calsequestrin 2
Marker synonyms: cardCSQ; cardiac calsequestrin; cCSQ; Csq2; ESTM52; PDIB2
Chromosome: 3
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Molecular note: The targeting vector is designed to insert loxP66 and loxP71 inward facing sites flanking the promoter and first exon of the gene (from -561 to +538bp); they are inverted relative to their normal endogenous orientation. An FRT-flanked neomycin resistance (neo) cassette was inserted in intron 1, between exons 1 and 2. Flp-mediated recombination removed the FRT-flanked neo cassette.