Ai92(TITL-YCX2.60)-D allele (also called Ai92D) has a Tet response element, a floxed-STOP cassette and the YCX2.60 FRET-based Ca2+ indicator inserted into the T1 TIGRE locus (an intergenic region on chromosome 9 [between AB124611 and Carm1] determined to have low basal transcriptional activity and allow high Cre and/or Tet inducibility).

Specifically, the pTRE-LSL-YCX2.60 replacement vector was designed with (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) promoter, a floxed-STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the YCX2.60 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.
(129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem cells previously targeted with FRT3::AttB::PGK-neoR-polyA::FRT5::splice acceptor::3’hygro cassette::SV40 polyA:AttP in TIGRE, were re-transfected with the pTRE-LSL-YCX2.60 replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME). Chimeric mice were bred with C57BL/6J-congenic PhiC31 mice (see Stock No. 007743) to remove the AttB/AttP-flanked PGK-hygromycin-SV40polyA cassette and replace it with the recombined AttB/AttP site (AttL). The Ai92D allele is therefore TIGRE-frt3-Ins-TRE-LSL-YCX2.60-WPRE-bGHpA-Ins-AttL. The resulting Ai92D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 gene was removed). In 2016, male mice from generation N7F1 with black coat color were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Of note, the donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).

The fluorescence resonance energy transfer (FRET)-based calcium sensor Yellow Cameleon variant X 2.60 (YCX2.60) is an improved version of YC2.60 with expanded dynamic range and 2-3-fold greater dissociation constant. The YCX2.60 fusion gene was designed by Dr. Atsushi Miyawaki (Brain Science institute, Riken) with an enhanced cyan fluorescent protein with C-terminal 11 amino acids deleted (ECFPΔC11 with R27K and K213N mutations, a rat calmodulin DNA fragment (CaM; aa 5-148 with N60D and D78Y mutations [to increase the fluorescence change for small calcium transients] and E127V mutation), a GG linker, a chicken smooth muscle M13 fragment of myosin light chain kinase (with I-to-S mutation in the 20th aa of the sequence), and a circularly permutated Venus enhanced yellow fluorescent protein (cp173 Venus; aa 174-239, a 15 bp linker and aa 1-173).

The TRE promoter used here is Tet-responsive P_tight; containing a modified Tet response element (TRE_mod) composed of seven direct 36 bp repeats (each containing the 19 bp tet operator sequence [tetO]). TRE_mod is just upstream of a minimal human cytomegalovirus promoter (P_minCMVΔ), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P_tight is silent in the absence of tTA or rtTA binding to tetO.

• Wild-type from the colony
• 000664 C57BL/6J

Additional Information

• Considerations for Choosing Controls

• Madisen L; Garner AR; Shimaoka D; Chuong AS; Klapoetke NC; Li L; van der Bourg A; Niino Y; Egolf L; Monetti C; Gu H; Mills M; Cheng A; Tasic B; Nguyen TN; Sunkin SM; Benucci A; Nagy A; Miyawaki A; Helmchen F; Empson RM; Knopfel T; Boyden ES; Reid RC; Carandini M; Zeng H. 2015. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85(5):942-58PubMed: 25741722MGI: J:219930