Igs7^{tm86(tetO-CiVS/pHluorin)Hze}

The targeting vector contains (from 5' to 3') an FRT3 site, two copies of chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivators), a modified Tet response element (TRE or tetO), a loxP-STOP-loxP cassette, the voltage-sensitive fluorescent protein Butterfly 1.2 coding sequence (referred to as ArcLight - details below), a WPRE sequence (to enhance mRNA stability), a BGH polyA, two copies of chicken beta-globin HS4 insulator element, an AttB site, a PGK-5'hygro cassette, an RNA splice donor and a FRT5 site inserted into the Igs7 locus (T1 TIGRE - an intergenic region on chromosome 9 [between AB124611 and Carm1]) using recombinase-mediated cassette exchange (RMCE). The voltage-sensitive fluorescent protein ArcLight is a fusion of a CiVS [Ciona intestinalis voltage sensor (S1-S4 transmembrane segment, including the voltage-sensing domain in S4)] and the fluorescent protein super ecliptic pHluorin (an eGFP variant differing from eGFP at 9 positions) with an A227D mutation, and exhibits voltage-dependent Forster resonance energy transfer (FRET) upon membrane depolarization. In this strain, the fluorophore is inserted at position A242 within the CiVS. (J:260362, J:289367)