Gt(ROSA)26Sor^{tm39.1(CAG-hop/EYFP)Hze}

he Rosa-CAG-LSL-eNpHR3.0-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), an eNpHR3.0-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. The NpHR-EYFP fusion protein was first designed with the halorhodopsin from the halophilic bacterium Natronomas pharaonis (NpHR) fused in-frame with an enhanced yellow fluorescent protein (EYFP). To create the eNpHR3.0-EYFP fusion protein, the NpHR-EYFP fusion protein was modified via addition of a membrane trafficking signal from the potassium channel Kir2.1 gene (amino acids KSRITSEGEYIPLDQIDINV) between NpHR and EYFP, as well as addition of an endoplasmic reticulum (ER) exporting sequence from the potassium channel Kir2.1 gene (amino acids FCYENEV) at the C-terminal of the EYFP. These modifications result in optimized expression in mammalian systems by preventing ER aggregation/enhancing membrane translocation, reducing bleb formation, and enhancing inhibitory capacity. The entire Rosa-CAG-LSL-eNpHR3.0-EYFP-WPRE targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. PhiC31-mediated recombination replace the neo cassette with the recombined attB/attP site (attL). (J:172634)