Igs7^{tm93.1(tetO-GCaMP6f)Hze}

The targeting vector was designed with (from 5' to 3') an FRT3 site, two tandem copies of the chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivator protein), a modified Tet response element (TRE or tetO), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the GCaMP6 fast variant calcium indicator (GCaMP6f) coding sequence, a WPRE sequence (to enhance the mRNA transcript stability), a bovine growth hormone polyA signal, two tandem copies of the chicken beta-globin HS4 insulator element, an AttB site, a PGK-5' hygro cassette, an RNA splice donor and an FRT5 site The GCaMP6 fast variant calcium indicator (GCaMP6f) is a detector of single neuronal action potentials with fast response kinetics, and is an improved version of GCaMP5G. GCaMP6f has a chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP, and a rat calmodulin DNA fragment mutation for accelerated kinetics. Embryonic stem cells, previously targeted with FRT5-AttB-Hygro2 cassette-AttP-SV40pA-splice acceptor-PGKpA-FRT3 in the same locus, were re-transfected with the targeting vector and Flp recombinase vector. Genotyping/sequencing revealed that Ai93 had two tandem copies of the pTRE-LSL-GCaMP6f replacement vector inserted during RCME. Chimeric mice were bred with PhiC31 Gt(ROSA)26Sortm3(phiC31*)Sor mice to remove the AttB/AttP-flanked region. The Ai93D allele is therefore TIGRE-frt3-Ins-TRE-LSL-GCaMP6f-WPRE-bGHpA-Ins-AttB-PGK-5'hygro-SD-frt5-frt3-Ins-TRE-LSL-GCaMP6f-WPRE-bGHpA-Ins-AttL. (J:101977, J:219930)