STOCK Gt(ROSA)26Sor^{tm1(CAG-Clomeleon*)Kjst}/J

Stock number: 037476
Product name: SuperClomeleon , sClm Lox-P
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

sCLM-floxed KI mice allow Cre-inducible expression of SuperClomeleon, an improved fluorescent fusion protein containing Cerulean and YFP (topaz) that acts as a ratiometric indicator for chloride ions. This strain may be useful for detecting inhibitory synaptic activity in single mouse neurons.

Detailed description

sCLM Lox-P KI knock-in mice use a CAG promoter to direct conditional Cre recombinase-mediated expression of SuperClomeleon. SuperClomeleon is a fusion of yellow fluorescent protein (YFP) containing a Cl^- binding site adjacent to its chromophore and a chloride-insensitive cyan fluorescent protein (Cerulean). (Grimley et al The Journal of Neuroscience, 2013). SuperChlomeleon is modified to provide a higher chloride sensitivity than Clomeleon. In the presence of low chloride ion concentrations, YFP fluoresces as a FRET (Fluorescence Resonance Energy Transfer) acceptor for CFP. As the chloride ion concentration increases, YFP fluorescence decreases and CFP fluorescence increases. Using an appropriate calibration curve, the ratio of YFP and CFP fluorescence peaks indicate [Cl-].

Homozygous sCLM-floxed KI knock-in mice are viable and fertile. Prior to exposure to Cre recombinase SuperClomeleon is not expressed. In the presence of cre, the STOP cassette is excised allowing expression in cre-expressing tissues. This strain may be useful for detecting inhibitory synaptic activity in single mouse neurons.

Development

The sCLM targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAAG), a loxP-flanked STOP/Neo cassette, SuperClomeleon (an improved fluorescent fusion protein containing Cerulean and YFP) and a polyA sequence. SuperClomeleon differs from Clomeleon by the following additions: a short interdomain linker, an S30R point mutation in both domains, intermediate affinity-tuning mutations, Q69T/V163A double mutant in YFP and replacement of the CFP domain with Cerulean. This entire construct was inserted into exon 3 of the Gt(ROSA)26Sor locus via electroporation of 129Sv (GenOway)-derived embryonic stem (ES) cells. Correctly targeted ES cells were selected and chimeric males were bred to 129Sv females (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 129S1/SvImJ oocytes (Stock No. 002448).

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. Many of the 43 markers throughout the genome were segregating with an unknown source, suggesting an incomplete backcross.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-Clomeleon*)Kjst}
Name: targeted mutation 1, Kevin J Staley
MGI accession ID: MGI:7287847
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: sCLM
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: 129/Sv
Molecular note: The targeting vector is designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAAG), a loxP-flanked STOP/Neo cassette, SuperClomeleon (an improved fluorescent fusion protein containing Cerulean and YFP) and a polyA sequence. SuperClomeleon differs from Clomeleon by the following additions: a short interdomain linker, an S30R point mutation in both domains, intermediate affinity-tuning mutations, Q69T/V163A double mutant in YFP and replacement of the CFP domain with Cerulean. It provides a higher choloride sensitivity than Clomeleon.