B6.Cg-Chat^{tm1.1(flpo)Rmcld}/J

Stock number: 036281
Product name: Chat^FlpO
Research areas: Research Tools

Description

Chat^FlpO knock-in mice express optimized FLP recombinase (FLPo) specifically in ChAT+ neuron populations in the forebrain. These mice are useful when using intersectional genetics to study cholinergic neurons throughout the brain.

Detailed description

Choline O-acetyltransferase (Chat) encodes a major neurotransmitter in both the central and peripheral nervous systems. Mutations in ChAT are associated with Alzheimer's disease and congenital myasthenic syndrome. Chat^FlpO knock-in mice express an optimized FLP recombinase (FLPo) from the endogenous Chat promoter in cholinergic neurons. The insertion of an IRES allows for co-expression of flpo and Chat. Homozygous mice are viable and fertile. When Chat^FlpO mice are bred with mice containing frt-flanked sequences, Flpo-mediated recombination will result in deletion of the frt-flanked sequences in Chat-expressing cells in the offspring.

For studying cholinergic projections within the cortex, these Chat^FlpO mice were bred with Ai65 intersectional reporter mice that are both Cre- and Flp-dependent (Stock No. 021875), and offspring were subsequently injected with an axon terminal-specific CAV-2::Cre virus. This results in selectively labelled cholinergic neurons with axons projecting to specific cortical layers. The fluorescent reporter from Ai65 fills the entire axonal arborization of labeled cholinergic neurons and thus allows for determination of the extent of arborization of individual cells across laminae.

Of note, a transgenic line expressing Flpo from a mouse Chat promoter from a BAC is also available as Stock No. 033850.

Development

The Chat^FlpO targeting vector containing an internal ribosome entry site (IRES) fused to a FlpO recombinase sequence and a polyadenylation site was inserted into the 3' UTR of the choline acetyltransferase (Chat) gene. The construct also included a loxP-flanked neomycin resistance cassette. The construct was electroporated into B6.Cg-Thy1^a-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and resulting chimeric mice were bred with CMV-cre mice (Stock No. 006054) to remove the neo cassette. Resulting Chat^FlpO mice were bred to C57BL/6NJ mice (Stock No. 005304) to remove the Cre expressing transgene, and heterozygous mice were subsequently bred with each other to establish a homozygous Chat^FlpO colony. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6NJ 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. One of the 43 markers throughout the genome was foudn to be segregating with an unknown source.

Allele

Symbol: Chat^{tm1.1(flpo)Rmcld}
Name: targeted mutation 1.1, Robert Machold
MGI accession ID: MGI:6512860
Generation method: Targeted
Attributes: Recombinase-expressing
Marker symbol: Chat
Marker name: choline acetyltransferase
Chromosome: 14
Strain of origin: B6.Cg-Thy1^a
Molecular note: The targeting vector containing an internal ribosome entry site (IRES) fused to a FlpO recombinase sequence and a polyadenylation site was inserted into the 3' UTR of the choline acetyltransferase (Chat) gene. The construct also included a loxP-flanked neomycin resistance cassette. Chimeric mice were bred with CMV-cre mice to remove the neo cassette.