B6.Cg-Igs7^{tm170.1(tetO-GFP*/TPTE2*, CAG-tTA2)Hze}/J

Igs7^{tm170.1(tetO-GFP*/TPTE2*,CAG-tTA2)Hze}

targeted mutation 170.1

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

Igs7

intergenic site 7

9

TPTE, transmembrane phosphatase with tensin homology, chicken; GFP, Green Fluorescent Protein; tTA, tetracycline-controlled transactivator, E. coli

6151068

When bred to mice expressing Cre recombinase, the offspring will have both floxed-STOP cassettes deleted in the Cre-expressing cells/tissues - resulting in ASAP2s-Kv expression and tTA2 expression. When ASAP2s-Kv is expressed, robust GFP fluorescence is observed with the expected localization (membrane-localized in neuronal soma and proximal dendrites).

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

The vector is 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 Tet response element/promoter (TRE2; details below), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), a green fluorescent voltage indicator protein ASAP2s sequence with soma and proximal dendrite targeting sequences (ASAP2s-Kv; details below), a WPRE (to enhance the mRNA transcript stability), a BGH polyA, two copies of chicken beta-globin HS4 insulator element, a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG; from pCAGGS), a lox2272-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), a synthetic modified tetracycline-regulated transactivator gene (tTA2S), a WPRE, a BGH polyA, an AttB site, a PGK-5'hygro cassette, an RNA splice donor and a FRT5 site.The TRE2 promoter used here is Tet-responsive P>hCMV*-1min CMVhCMV*-1< is silent in the absence of tTA or rtTA binding to tetO.GFP*/TPTE2* has VSD (voltage sensor domwin) sequences (GgVSD) from the Gallus gallus voltage-sensitive phosphatase TPTE2, including the R154Q mutation for desired voltage response. A circularly permuted superfolder GFP 1-10 OPT variant (cpsfGFP-OPT) is inserted into the GgVSD extracellular loop between the third and fourth transmembrane segments (S3-S4). The GgVSD includes the R154Q mutation for desired voltage response (equivalent to R153Q in original descriptions). The GFP 1-10 OPT includes the substitutions from folding reporter GFP (F99S, M153T, V163A, F64L, S65T) and superfolder GFP (S30R, Y145F, I171V, A206V), and also has seven additional substitutions (N39I, T105K, E111V, I128T, K166T, I167V, S205T). Together, these mutations result in improved brightness and dynamic range, while maintaining efficient expression at the membrane. In addition, this voltage sensor is tagged with a 65-amino-acid cytosolic segment of a rat Kv2.1 to restrict protein localization to the soma and proximal dendrites. PhiC31-mediated recombination removed the AttB/AttP-flanked sequence and replaced it with the recombined AttB/AttP site (AttL).

C57BL/6J

The donating investigator reports Ai170D heterozygotes and homozygotes are viable and fertile with no reported gross physical or behavioral abnormalities. When maintaining a live colony, heterozygous mice may be bred together, to wildtype mice from the colony or to C57BL/6J inbred mice. Alternatively, this strain may be maintained as homozygous.

Wild-type x Heterozygote; Heterozygote x Wild-type