Gt(ROSA)26Sor^{tm27.1(CAG-COP4*H134R/tdTomato)Hze}

The Rosa-CAG-LSL-hChR2(H134R)-tdTomato-WPRE targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), a mammalianized ChR2(H134R)-tdTomato fusion gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette. To create the mammalianized ChR2(H134R)-tdTomato fusion gene, a cDNA sequence encoding the first 315 amino acids of channelrhodopsin-2 (derived from the green alga Chlamydomonas reinhardtii) was modified to harbor condons optimized for mammalian expression and a gain-of-function H134R substitution (CAC to CGC) designed to cause larger stationary photocurrents. This mhChR2 sequence (also called hChR2(H134R) or hChR2-H134R) was fused in-frame to the amino terminus of a tdTomato sequence (a non-oligomerizing DsRed fluorescent protein variant with a 12 residue linker fusing two copies of the protein (tandem dimer)), resulting in the final hChR2(H134R)-tdTomato fusion protein sequence. The entire Rosa-CAG-LSL-hChR2(H134R)-tdTomato-WPRE targeting vector was inserted between exons 1 and 2 of the locus. PhiC31 mediated recombination removed the PGK-FRT-Neo-polyA cassette. (J:182204)