Pvalb-T2A-Dre-D (Pvalb-2A-Dre-D) mice were created in the laboratory of Dr. Hongkui Zeng (Allen Institute for Brain Science) to have a T2A sequence and a mammalian-optimized D6 site-specific DNA recombinase (DreO recombinase) inserted in-frame at the 3' end of the parvalbumin (Pvalb) coding region on chromosome 15. The specific details are below.
(129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells, already targeted with a T2A-Cre vector inserted in-frame at the 3' end of the Pvalb coding region (see Stock No. 012358), were re-targeted with a "T2A-DreO" vector and a FLP-expressing plasmid to facilitate recombination. Correctly targeted ES cells had (from 5' to 3') a three amino acid linker sequence, an frt3 site within Pvalb intron 3, a partial Pvalb exon 4 sequence up to (but not including) the endogenous stop codon, a viral 2A oligopeptide (T2A; mediates ribosomal skipping) that is in-frame with the Pvalb coding sequence, a D6 site-specific DNA recombinase optimized for mammalian cell expression (DreO recombinase), the Pvalb 3' UTR sequence from exon 4, an attB site, a PGK-hygromycin-SV40polyA cassette (with an RNA splice donor-frt5 site-RNA splice acceptor in the hygromycin gene), and an attP site.
Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were bred to PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) to delete the attB/attP-flanked PGK-hygromycin-polyA cassette and replace it with the recombined attB/attP site (attL). The resulting Pvalb-T2A-Dre-D mice were bred with C57BL/6J wildtype mice for at least five additional generations (and the PhiC31 transgene was removed) prior to sending to The Jackson Laboratory Repository in 2013.
Upon arrival, males were used to cryopreserve sperm. To establish the living Pvalb-T2A-Dre-D mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
