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 Gt(ROSA)26Sor locus via electroporation of (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells (clone Ai32) were selected. Chimeric mice were bred with C57BL/6 wildtype mice and/or PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) - with some resulting offspring retaining the attB/attP-flanked cassette (more detail below). Mice with the Ai32 allele (Rosa-CAG-LSL-ChR2(H134R)-EYFP-WPRE-BGHpA-attB-PGK-FRT-Neo-pA-attP) were then bred with C57BL/6J wildtype mice for at least one additional generation prior to sending to The Jackson Laboratory Repository in 2010 as Stock No. 012569. Upon arrival, some Ai32 mice were backcrossed with C57BL/6J inbred mice (Stock No. 000664) for five additional generations to establish this C57BL/6-congenic strain as Stock No. 024109. This colony shows 99.5%-100% of 183 SNP markers throughout the genome are C57BL/6 allele-type.
All of the mice originally sent to The Jackson Laboratory Repository, as well as several generations of breeding Ai32 mutant mice with C57BL/6J inbred mice, have resulted in mice that genotype as completely co-segregating for the neo cassette and YFP-WPRE portion of the Ai32 construct. As such, these mice harbor the Ai32 mutation (retaining the attB/attP-flanked PGK-FRT-Neo-polyA cassette) rather than the Ai32Δneo mutation.
