Gt(ROSA)26Sor^{tm32(CAG-COP4*H134R/EYFP)Hze}

The Rosa-CAG-LSL-ChR2(H134R)-EYFP-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 ChR2(H134R)-EYFP 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 ChR2(H134R)-EYFP fusion gene, a cDNA sequence encoding the first 315 amino acids of channelrhodopsin-2 (derived from the green alga Chlamydomonas reinhardtii) was modified with a gain-of-function H134R substitution (CAC to CGC) designed to cause larger stationary photocurrents. This ChR2(H134R) sequence was fused in-frame to the amino terminus of an enhanced yellow fluorescent protein sequence, resulting in the final ChR2(H134R)-EYFP fusion protein sequence. This entire construct was inserted between exons 1 and 2 of the locus. (J:182204)