B6;129S6-Chat^tm2(cre)Lowl/J

Stock number: 006410
Product name: ChAT-IRES-Cre , ChAT-IRES-Cre::frt-neo-frt
Research areas: Neurobiology Research, Research Tools

Description

ChAT-IRES-Cre knock-in mice express Cre recombinase in cholinergic neurons, without disrupting endogenous Chat expression. These mice may be useful in neurobiological research of motor function, learning and memory, Alzheimer's disease, and Down syndrome, as well as obesity and diabetes research.

The Jackson Laboratory Repository has two ChAT-IRES-Cre knock-in alleles from Dr. Bradford Lowell: ChAT-IRES-Cre::SV40pA::frt-neo-frt (Chat^tm2(cre)Lowl; Stock Nos. 006410/018957/028861) and ChAT-IRES-Cre::SV40pA::Δneo (Chat^tm1(cre)Lowl ; Stock No. 031661). Each allele also contains a partial neo fragment between the Cre and SV40 polyA. The donating investigator reports that the presence of the frt-flanked neo cassette may result in ectopic Cre expression, and they have never observed any ectopic expression in their ChAT-IRES-Cre::SV40pA::Δneo mice (January 2018).

Detailed description

The Jackson Laboratory Repository has two ChAT-IRES-Cre knock-in alleles from Dr. Bradford Lowell: ChAT-IRES-Cre::SV40pA::frt-neo-frt (Chat^tm2(cre)Lowl; Stock Nos. 006410/018957/028861) and ChAT-IRES-Cre::SV40pA::Δneo (Chat^tm1(cre)Lowl ; Stock No. 031661). Each allele also contains a partial neo fragment between the Cre and SV40 polyA (see below). The donating investigator reports that the presence of the frt-flanked neo cassette may result in ectopic Cre expression, and they have never observed any ectopic expression in their ChAT-IRES-Cre::SV40pA::Δneo mice (January 2018).

Mice that are homozygous for the ChAT-IRES-Cre::SV40pA::frt-neo-frt knock-in allele are viable and fertile. An "IRES-Cre" sequence is inserted downstream of the stop codon such that cre expression is controlled by the endogenous Chat gene promoter. Chat gene expression, however, is unaffected. Cre recombinase activity is reported in all cholinergic neurons. These mice may be useful for "Cre-lox" technology applications in neurobiology, including studies of motor function, learning and memory, Alzheimer's disease, and Down syndrome, and in obesity and diabetes research.

Characterization performed at The Jackson Laboratory (2019-2020) determined there is an incomplete neo sequence (224 bp) located 310 bp downstream of Cre STOP codon and 70 bp upstream of SV40 polyA sequence. While it is not specifically determined if the 224 bp neo fragment has any effect on transcript levels of Cre or endogenous Chat, the Cre ORF and Cre STOP codon are intact. The 224 bp partial neo fragment includes sequences complimentary to the forward, reverse and probe primers The Jackson Laboratory has used for its generic neo genotyping assay.

View Cre expression characterization for ChAT-IRES-Cre::SV40pA::frt-neo-frt allele (Chat^tm2(cre)Lowl ; Stock No. 006410).

Development

Stock No. 006410 has the ChAT-IRES-Cre::SV40pA::frt-neo-frt allele (Chat^tm2(cre)Lowl) on a mixed C57BL/6;129 genetic background - its generation is described below.

A targeting vector was designed to insert an optimized internal ribosome entry site-linked Cre recombinase gene (followed by an SV40 polyA and frt-flanked neomycin resistance cassette) downstream of the stop codon of the choline acetyltransferase gene (Chat) on chromosome 14. The construct was electroporated into 129S6/SvEvTac-derived W4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts. Chimeric mice were bred to C57BL/6 mice. The resulting mutant offspring (still retaining the frt-flanked neo) were bred to wildtype siblings, and maintained as such prior to sending heterozygous males to The Jackson Laboratory Repository in 2006. Upon arrival, mice were bred once to C57BL/6 females to rederive our living colony. Thereafter, mutant mice were bred together to make this line homozygous.

Characterization performed at The Jackson Laboratory (2019-2020) determined there is an incomplete neo sequence (224 bp) located 310 bp downstream of Cre STOP codon and 70 bp upstream of SV40 polyA sequence. While it is not specifically determined if the 224 bp neo fragment has any effect on transcript levels of Cre or endogenous Chat, the Cre ORF and Cre STOP codon are intact. The 224 bp partial neo fragment includes sequences complimentary to the forward, reverse and probe primers The Jackson Laboratory has used for its generic neo genotyping assay.

Allele

Symbol: Chat^tm2(cre)Lowl
Name: targeted mutation 2, Bradford B Lowell
MGI accession ID: MGI:5475195
Generation method: Targeted
Attributes: Recombinase-expressing
Marker symbol: Chat
Marker name: choline acetyltransferase
Chromosome: 14
Strain of origin: 129S6/SvEvTac
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: Cre recombinase activity is reported in all cholinergic neurons.
Molecular note: An optimized internal ribosome entry sequence (IRES) fused to cre recombinase followed by a neomycin resistance cassette flanked FRT sites was inserted after the ChAT stop codon in BAC (bMQ185k21) using homologous recombination in bacteria. A genomic ChAT fragment with 4kb homology arms on either side of the IRES-Cre insertion site was lifted out from the BAC using reverse recombineering techniques and transformed into ES cells. This allele was used to generate Chat^tm1(cre)Lowl.