Igs7^{tm90.1(tetO-COP4*/EGFP)Hze}

The targeting vector was designed with (from 5' to 3') an FRT3 site, two tandem copies of the chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivator protein), a modified Tet response element (TRE or tetO), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the COP4*/EGFP coding sequence, a WPRE sequence (to enhance mRNA transcript stability), a bovine growth hormone polyA signal, two tandem copies of the chicken beta-globin HS4 insulator element, an AttB site, a PGK-5' hygro cassette, an RNA splice donor and an FRT5 site. The COP4*/EGFP fusion protein is composed of a mammalian codon-optimized Stigeoclonium helveticum-derived channelrhodopsin ShChR (Chronos) fused in-frame via a 18 nucleotide linker to the N-terminus of enhanced green fluorescent protein (EGFP). Chronos is a blue-to-green light-driven (~470-530 nm) cation channel that depolarizes the cell and causes action potentials. Chronos has high light sensitivity and faster kinetics than previous channelrhodopsins. Embryonic stem cells, previously targeted with FRT3-AttB-PGK-neoR-polyA::FRT5::splice acceptor::3' hygro cassette::SV40 polyA: AttP in the same locus, were re-transfected with the targeting vector and Flp recombinase vector. Mutant mice were bred with Gt(ROSA)26Sortm3(phiC31*)Sor mice to remove to remove the AttB/AttP-flanked PGK-hygro-SV40pA cassette and replace it with the recombined AttB/AttP site (AttL). The Ai90D allele is therefore TIGRE-frt3-Ins-TRE-LSL-Chronos/EGFP-WPRE-bGHpA-Ins-AttL. (J:101977, J:219930)