STOCK Gt(ROSA)26Sor^{tm5(ACTB-tTA)Luo} Igs7^{tm93.1(tetO-GCaMP6f)Hze}/HzeJ

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

targeted mutation 93

Conditional ready (e.g. floxed); Reporter; Inducible

Igs7

intergenic site 7

9

GCaMP, Genetically encoded calcium indicator

5558086

Expression of fast variant calcium indicator (GCaMP6f) is under the control of tetO in cells/tissues where promoters driving Cre recombinase are expressed.

(129S6/SvEvTac x C57BL/6NCrl)F1

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 siteThe 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.

Wild-type from the colony

When maintaining a live colony, mice heterozygous for both the Ai93D targeted mutation (Igs7tm93.1(tetO-GCaMP6f)Hze) and also for the R26-ZtTA targeted mutation (Gt(ROSA)26Sortm5(ACTB-tTA)Luo) may be bred to wildtype mice from the colony or to C57BL/6J inbred mice. Alternatively, mice heterozygous or homozygous for Ai93D and heterozygous for R26-ZtTA may be bred to wildtype mice from the colony or to C57BL/6J inbred mice.Ai93D;R26-ZtTA mice heterozygous for both targeted mutations are viable and fertile with no reported gross physical or behavioral abnormalities. The R26-ZtTA donating investigator reports that mice heterozygous for R26-ZtTA are more healthy and fertile than mice homozygous for R26-ZtTA. In the Ai93D;R26-ZtTA colony, breeding double heterozygous mice together produces offspring of all genotypes, but results suggest homozygosity of the R26-ZtTA allele may have detrimental effects on viability.

Het Het x Wild Wild (and recip) OR Het Wild x Wild Het (and recip)