Igs7^{tm136.1(tetO-COP3*/COP4*/EYFP)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 (TRE/tetO), a floxed-STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the ReaChR/EYFP sequence (details below), a WPRE, 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-ReaChR/EYFP 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). The ReaChR/EYFP fusion protein has the red-activatable channelrhodopsin variant ReaChR fused in-frame at its C-terminus (via a G-S-G linker) to an enhanced yellow fluorescent protein (EYFP). ReaChR is a chimeric channelrhodopsin composed of transmembrane domains A-E and G of VChR1 (Volvox carterichannelrhodopsin 1 [cop3]), transmembrane domain F of VChR2 (V. carterichannelrhodopsin 2 [cop4]) and the ChEF/ChIEF variant N-terminal extracellular sequences (derived fromChlamydomonas reinhardtiichannelrhodopsin 1 [cop3]). The ChEF/ChIEF, VChR1 and VChR2 sequences are mammalian-codon optimized, and transmembrane domain C harbors two point mutations encoding I170V (to accelerate rate of channel closure while retaining reduced inactivation) and L171I (to enhance activation by red light [~590 nm]). (J:101977, J:260362)