These Cart-IRES2-Cre-D mice (also called Cart-IRES2-Cre-Δ or Cart-IRES2-Cre-Δneo) have an IRES2 sequence and a Cre recombinase gene inserted immediately downstream of the translational STOP codon of the CART prepropeptide gene (Cartpt) on chromosome 13. The specific details are below.
The targeting vector contained, from 5' to 3',
a partial Cartpt sequence spanning exon 1 through intron 2,
an frt3 site within Cartpt intron 2,
a partial Cartpt exon 3 sequence up to and including the endogenous stop codon,
an internal ribosome entry site 2 (IRES2) sequence (allows translation initiation in the middle of an mRNA sequence),
a Cre recombinase gene,
a bovine growth hormone polyA sequence,
an AttB site,
a PGK promoter-Neomycin resistance gene-PGK polyA cassette,
an frt5 site,
an mRNA splice acceptor sequence,
the 3' portion of the hygromycin gene (Hygro2) with SV40 late polyA signal,
and an AttP site.
This construct was electroporated into (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. Chimeric mice were bred to PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) to remove the AttB/AttP-flanked sequences (PGK-Neo-polyA::frt5::RNA splice acceptor::3'hygro-polyA) and replace it with the recombined AttB/AttP site (AttL).
The resulting Cart-IRES2-Cre-D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 transgene was removed). In 2016, black male mice from generation N4 were sent to The Jackson Laboratory Repository. Upon arrival, males are used to cryopreserve sperm. To establish the living Cartpt-IRES2-Cre-D mouse colony, an aliquot of the frozen sperm is used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
Of note, the donating investigator 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).
