Igs7^{tm85.1(tetO-gltI/GFP*)Hze}

The pTRE-LSL-iGluSnFr replacement vector was designed with (from 5' to 3') an FRT3 site, two copies of chicken beta-globin HS4 insulator element, a modified Tet response element promoter, a floxed-STOP cassette, the iGluSnFr coding sequence (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. The iGluSnFr fusion gene is constructed from the Escherichia coli gltI gene and circularly permutated green fluorescent protein (cpGFP). Specifically, iGluSnFr has a 21 aa IgG K-leader sequence, the gltI aa 1-253, the LILV linker, the cpGFP (aa 148-239, an 18 bp flexible linker and aa 1-147), the L2NP linker, the gltI aa 254-279, an 11 aa c-Myc epitope and a 49 aa PDGR sequence (PDGR aa 513-561). iGluSnFr is an extremely fast and sensitive glutamate indicator with large signal-to-noise ratio, and kinetics appropriate for in vivo imaging. PhiC31-mediated recombination removed the AttB/AttP-flanked PGK-hygromycin-SV40polyA cassette and replaced it with the recombined AttB/AttP site (AttL). (J:219930)