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

Stock number: 031570
Product name: Ai170(TIT2L-ASAP2s-Kv-ICL-tTA2)-D (or Ai170D)
Research areas: Neurobiology Research, Research Tools

Description

Ai170(TIT2L-ASAP2s-Kv-ICL-tTA2)-D (or Ai170D) mice are a Cre/Tet-dependent, voltage-sensitive, fluorescent line for neuronal soma and proximal dendrites - created by targeted insertion at the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated). Exposure to Cre recombinase removes both STOP cassettes - resulting in expression of tTA2 and ASAP2s-Kv (highly-sensitive green fluorescent voltage indicator with Kv2.1-tag). ASAP2s-Kv expression may then be diminished by doxycycline. Ai170D mice may be useful for in vivo imaging of voltage dynamics in soma and proximal dendrites.

Detailed description

Ai170(TIT2L-ASAP2s-Kv-ICL-tTA2)-D (also called Ai170D) mice harbor the TIGRE-Ins-TRE2-LSL-ASAP2s-Kv-Ins-CAG-LSL-tTA2 conditional allele, designed with a TRE2 promoter, loxP-flanked STOP cassette and ASAP2s-Kv sequence, and then a CAG promoter, lox2272-flanked STOP cassette and tTA2 sequence.

Prior to Cre recombinase exposure, this allele is designed to have the two floxed-STOP cassettes preventing expression of the downstream genes (ASAP2s-Kv and tTA2).

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) - and depolarization leads to decreased fluorescence. Further administration of tetracycline (or its analog doxycycline [dox]) will directly inhibit tTA2 activity, and therefore is expected to reduce ASAP2s-Kv expression levels.

Specifically, the donating investigator reports Ai170D exhibit no fluorescence in the absence of Cre. To date (January 2018), Ai170D animals have not been characterized regarding dox-inducible attenuation of ASAP2s-Kv expression, nor the Cre-inducible Ai170D expression in tissues other than brain.


For example, when combining Ai170 with Cux2-CreERT2 (see MMRRC_032779-MU), the resulting double transgenic Cux2-CreERT2;Ai170 animals show moderate ASAP2s expression in neocortical layer 2/3/4 excitatory neurons following tamoxifen treatment - and the expression appears enriched in the neuronal soma and proximal dendrites. The same may be expected for Ai170D.


Additionally, the donating investigator reports crossing Ai170 with the pan-excitatory Emx1-IRES-Cre line (Stock No. 005628) or the pan-inhibitory Gad2-IRES-Cre line (Stock No. 010802) may generate offspring with high levels of CAG-driven tTA2 expression early in development - which may result in reduced brain size and neurodegeneration and/or embryonic lethality, respectively. The same may be expected for Ai170D.

The donating investigator reports Ai170D heterozygotes and homozygotes are viable and fertile with no reported gross physical or behavioral abnormalities.

Accelerated sensor of action potential (ASAP)-type sensors have a circularly permutated fluorescent protein linked to the transmembrane helix of a voltage-sensing domain (VSD). Voltage-induced movement of a positively charged VSD is thought to perturb the protonation state of the circularly permuted fluorescent protein - resulting in changes in fluorescence emission. The two-photon max excitation wavelength is ~920 nm.
ASAP2s-Kv is composed of the highly-sensitive green fluorescent voltage indicator ASAP2s (Chamberland et al. 2017 eLife 6:e25690) fused at its C-terminus to a flexible 27 nucleotide linker (GSS*3) and then a rat Kv2.1 potassium channel gene (Kcnb1) C-terminal sequence encoding a 65 amino acid sequence that functions to restrict protein localization to the soma and proximal dendrites. ASAP2s has the VSD 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.

Development

The Ai170(TIT2L-ASAP2s-Kv-ICL-tTA2)-D allele (or Ai170D) has a Tet response element TRE2, loxP-flanked-STOP cassette and green fluorescent voltage indicator protein ASAP2s-Kv sequence followed by a CAG promoter, lox2272-flanked-STOP cassette and tTA2 sequence all 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 pTRE2-LSL-ASAP2s-Kv-CAG-LSL-tTA2 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 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 (tTA2^S), a WPRE, a BGH polyA, 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 replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME). Chimeras were bred with C57BL/6J-congenic PhiC31 mice (see Stock No. 007743) to remove the AttB/AttP-flanked sequences and replace it with the recombined AttB/AttP site (AttL). The Ai170D allele is therefore TIGRE-frt3-Ins-TRE2-LSL-ASAP2s-Kv-WPRE-bGHpA-Ins-CAG-LSL-tTA2-WPRE-bGHpA-AttL. Resulting Ai170D mice were bred to C57BL/6J for several generations (and PhiC31 was removed). In 2018, heterozygous males from generation N5 (black coat color) were sent to The Jackson Laboratory. Upon arrival, sperm is cryopreserved. To establish a live colony, frozen sperm is used to fertilize C57BL/6J oocytes (Stock No. 000664).
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 TRE2 promoter used here is Tet-responsive P_hCMV*-1; containing the Tet response element (seven copies of the 19 bp tet operator sequence [tetO]) just upstream of a minimal cytomegalovirus promoter (P_{min CMV}), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P_hCMV*-1 is silent in the absence of tTA or rtTA binding to tetO.

ASAP2s is a higher-sensitivity variant of the green fluorescent voltage indicator ASAP1 (accelerated sensor of action potentials 1). Designed by Dr. Michael Z. Lin (Stanford), the ASAP2s sequence has a circularly permutated GFP linked to the transmembrane helix of a voltage-sensing domain (VSD) - rendering fluorescence responsive to membrane potential (Chamberland et al. 2017 eLife 6:e25690). ASAP2s-Kv has the C-terminus of the rat Kv2.1 potassium channel fused to the ASAP2s C-terminus via a flexible linker. See strain description for more details on development and characteristics of ASAP2s-Kv.



A 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. One of the 43 markers, on Chromosome 12, was segregating.

Allele

Symbol: Igs7^{tm170.1(tetO-GFP*/TPTE2*,CAG-tTA2)Hze}
Name: targeted mutation 170.1, Hongkui Zeng
MGI accession ID: MGI:6151068
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible; Transactivator
Synonyms: Ai170; Ai170(TIT2L-ASAP2s-Kv-ICL-tTA2)
Marker symbol: Igs7
Marker name: intergenic site 7
Marker synonyms: Iis7; TIGRE
Chromosome: 9
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: TPTE,transmembrane phosphatase with tensin homology,chicken; GFP,Green Fluorescent Protein,; tTA,tetracycline-controlled transactivator,E. coli
Site of expression: 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).
Molecular note: 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 (tTA2^S), 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*-1<; containing the Tet response element (seven copies of the 19 bp tet operator sequence [tetO]) just upstream of a minimal cytomegalovirus promoter (P>min CMV<), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P>hCMV*-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).