Stock No. 028861 has the ChAT-IRES-Cre::SV40pA::frt-neo-frt allele (Chattm2(cre)Lowl) on a C57BL/6J genetic background - its generation is described below.
The ChAT-IRES-Cre::SV40pA::frt-neo-frt knock-in allele was designed by Dr. Bradford Lowell (Beth Israel Deaconess Medical Center / Harvard). 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/6J females to rederive our live colony as Stock No. 006410, which was thereafter maintained by breeding mutant mice together.
In 2015, Dr. Melissa R. Warden (Cornell University) purchased some Stock No. 006410 animals (total backcross generation N1 onto C57BL/6, then N1 onto C57BL/6J). They further backcrossed them to C57BL/6J inbred mice for five additional generations. The resulting C57BL/6J-congenic strain, called ChAT-IRES-Cre knock-in (C57BL/6J), were sent to The Jackson Laboratory Repository in 2016 as Stock No. 028861. Upon arrival, males were used to cryopreserve sperm. To establish the living mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred mice (Stock No. 000664).
Of note, Dr. Warden's lab reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).
In 2017, a 154 SNP analysis (with markers covering all 19 chromosomes and the X chromosome) was performed on the first generation of rederived mice at The Jackson Laboratory Repository (heterozygous for the ChAT-IRES-Cre knock-in allele). This showed the genetic background was >99% C57BL/6J allele-type markers. Specifically, 151/154 markers were C57BL/6J allele-type, with the exception of the 3 markers on chromosome 14 closest to the Chat locus were segregating with the ChAT-IRES-Cre knock-in allele (i.e., heterozygous for C57BL/6J;129 allele-type).
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.
