These Slc17a7-IRES2-Cre-D mice (also called Slc17a7-IRES2-Cre-Δ or Slc17a7-IRES2-Cre-Δneo/hygro) have an IRES2 sequence and a Cre recombinase gene inserted immediately downstream of the translational STOP codon of the vesicular glutamate transporter 1 gene (Slc17a7 or Vglut1). The specific details are below.
The targeting vector contained, from 5' to 3',
a partial Slc17a7 sequence spanning exon 1 through intron 9,
an frt3 site within Slc17a7 intron 9,
a partial Slc17a7 exon 10 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,
the 3' portion of the hygromycin gene 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 Slc17a7-IRES2-Cre-D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 transgene was removed). In 2013, males from generation N3F1 were sent to The Jackson Laboratory Repository. Upon arrival, males were used to cryopreserve sperm. To establish the living Slc17a7-IRES2-Cre-D mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
