A targeting vector was used to insert a synthetic modified tetracycline-regulated transactivator (tTA2S) gene and 3' UTR region of the somatostatin exon 2 into the ATG transcriptional start site of the somatostatin gene (Sst). Specifically, the targeting vector was designed with (from 5' to 3') an frt3 site, a Cre recombinase gene, a bovine growth hormone polyA sequence, a second frt3 site, a tTA2S gene, the 3' UTR region from somatostatin exon 2, an AttB site, PGK-Neo-polyA cassette, an frt5 site, an RNA splice acceptor, the 3' portion of the hygromycin gene with SV40 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, and chimeric animals were bred to C57BL/6J mice to generate the SST-Cre-pA-tTA2 mutant colony. To remove the frt-flanked Cre recombinase and polyA sequence, the mice were bred to ROSA26::FLPe deleter mice (129S4 genetic background; Stock No. 003946). The SST-tTA2 offspring were then bred to PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) to replace the PGK-Neo and hygromycin-polyA cassettes with the recombined AttB/AttP site (AttL). The resulting SST-tTA2-D (or SST-tTA2-Δ) mice have a single frt3 site, tTA2S, the 3' UTR from somatostatin exon 2, and AttL site targeted into the somatostatin ATG site. The SST-tTA2-Δ mice were subsequently backcrossed to C57BL/6J wildtype mice for at least five additional generations (and the FLP-expressing mutation and PhiC31 transgene was removed) prior to sending to The Jackson Laboratory Repository. Upon arrival, mice were bred with C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the living colony.
