Igs7^{tm133.1(tetO-apx1*)Hze}

The replacement vector was designed with (from 5' to 3') an FRT3 site, two copies of chicken beta-globin HS4 insulator element, a modified Tet response element promoter, a floxed-STOP cassette, the ssAPEX2tm coding sequence (details below), a WPRE sequence, a BGH polyA, two copies of chicken beta-globin HS4 insulator element, an AttB site, a PGK-5'hygro cassette, an RNA splice donor and a FRT5 site. Embryonic stem cells previously targeted with FRT3::AttB::PGK-neoR-polyA::FRT5::splice acceptor::3'hygro cassette::SV40 polyA:AttP in TIGRE, were re-transfected with the pTRE-LSL-ssAPEX2tm replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME). PhiC31-mediated recombination removed the AttB/AttP-flanked sequence (PGK-Neo-polyA::frt5::RNA splice acceptor::3'hygro-polyA) and replaced it with the recombined AttB/AttP site (AttL). APEX2 is a second-generation variant of soybean Glycine hispida ascorbate peroxidase gene (apx1) that harbors the three APEX mutations (K14D, W41F, E112K) optimized for expression in mice, as well as one additional mutation (A134P) for improved catalytic efficiency. The ssAPEX2tm fusion gene has APEX2 fused to a T-cell Cd2 antigen signal sequence (ss) and transmembrane targeting sequence (tm). There is a FLAG-tag domain (DYKDDDDK) between the signal sequence and the APEX2 sequence. (J:101977, J:260362)