STOCK Gt(ROSA)26Sor^{tm1(CAG-ECFP*/Venus*)Tpzn}/J

Stock number: 036466
Product name: mt-Cam
Research areas: Cell Biology Research, Metabolism Research, Neurobiology Research, Research Tools

Description

These mt-Cam express a cre-inducible mitochondrial-targeted, Förster resonance energy transfer (FRET)-based Cameleon probe, 4mtD3cpv. These mice are a useful tool to evaluate Ca²⁺ dynamics in the mitochondrial matrix of isolated cells and live tissues.

Detailed description

Mitochondria are involved in the precise regulation of cellular Ca²⁺ homeostasis, and dysfunction in handling mitochondrial Ca²⁺ levels has been implicated in several pathological conditions ranging from cardiovascular diseases to neurological disorders and carcinogenesis.

These mt-Cam mice express a cre-inducible mitochondrial-targeted, Förster resonance energy transfer (FRET)-based Cameleon probe, 4mtD3cpv, sequence under control of a CAG promoter from the endogenous Gt(ROSA)26Sor locus. The FRET-based Cameleon 4mtD3cpv allows ratiometric imaging with reduced sensitivity to motion artifacts and probe expression levels. A floxed STOP cassette (containing a β-geo sequence), prevents mt-Cam expression in the absence of cre recombinase. After removal of the loxP-flanked STOP cassette, mt-Cam is expressed, allowing for labeling cells/tissues where cre is expressed.

Upon ubiquitous Cre expression, Cameleon expression is detected by direct fluorescence in the mitochondrial matrix in all isolated cells and live tissues of the offspring, and allow simple calculations of mitochondrial Ca²⁺ concentrations.

When bred to ChAT-IRES-Cre mice (Stock No. 006410), mt-Cam expression is detected by direct fluorescence in the mitochondrial matrix of cholinergic neurons localized mostly in the midbrain, close to the cerebellum.

Homozygous mt-Cam mice are viable and fertile. Expected coat colors are albino, agouti, and black.

Development

The mt-Cam targeting vector was designed with (from 5' to 3') a ubiquitously expressed CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a loxP-flanked stop cassette (containing a β-geo sequence), and the mt-Cam cDNA. mt-Cam encodes a FRET-based Cameleon calcium sensor (4mtD3cpv) variant containing a codon-optimized "elongated" mitochondria tag, a donor chromophore enhanced cyan fluorescent protein (ECFP), calcium sensor sequences [calmodulin and a calmodulin-binding domain from myosin light-chain kinase (M13)], and the acceptor chromophore circularly permuted Venus (cpV). This targeting vector was inserted into the Gt(ROSA)26Sor locus in opposite orientation to the Gt(ROSA)26Sor promoter. The construct was electroporated into (129S6/SvEvTac x C57BL/6NCr)F1-derived G4 embryonic stem (ES) cells and correctly targeted ES cells were injected into blastocysts. The resulting mt-Cam mice were crossed to C57BL/6J mice for at least 3 generations. 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 (Stock No. 000664).

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 rederived living colony at The Jackson Laboratory Repository. Many of the 43 markers throughout the genome were segregating with 129 as expected. Also, the Tyr recessive mutation associated with albino coat color on Chromosome 1 was found to be segregating from an unknown source. These data suggest the mice distributed by The Jackson Laboratory Repository could be agouti, black, or albino.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-ECFP*/Venus*)Tpzn}
Name: targeted mutation 1, Tullio Pozzan
MGI accession ID: MGI:6728074
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: mt-Cam
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: The targeting vector is designed with (from 5' to 3') a ubiquitously expressed CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a loxP-flanked stop cassette (containing a betageo sequence), and the mt-Cam cDNA. mt-Cam encodes a FRET-based Cameleon calcium sensor (4mtD3cpv) variant containing a codon-optimized elongated mitochondria tag, a donor chromophore enhanced cyan fluorescent protein (ECFP), calcium sensor sequences [calmodulin and a calmodulin-binding domain from myosin light-chain kinase (M13)], and the acceptor chromophore circularly permuted Venus (cpV). This targeting vector is inserted into the Gt(ROSA)26Sor locus in opposite orientation to the Gt(ROSA)26Sor promoter.