Igs7^{tm94.1(tetO-GCaMP6s)Hze}

The replacement vector was designed with (from 5' to 3') an FRT3 site, two copies of chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivators), a modified Tet response element (TRE or tetO), a floxed-STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the GCaMP6 slow variant calcium indicator coding sequence, a WPRE sequence (to enhance mRNA stability), 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. The GCaMP6 slow variant calcium indicator (GCaMP6s) is an ultrasensitive detector of single neuronal action potentials with slow response kinetics and is an improved version of GCaMP5G. 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-GCaMP6s replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME). Chimeric mice were bred with PhiC31 Gt(ROSA)26Sortm3(phiC31*)Sor mice to remove the AttB/AttP-flanked PGK-hygromycin-SV40polyA cassette and replace it with the recombined AttB/AttP site (AttL). (J:101977)