STOCK Igs7^{tm86(tetO-CiVS/pHluorin)Hze}/VpiJ

Stock number: 034694
Product name: Ai86(TITL-ArcLight)
Research areas: Neurobiology Research, Research Tools

Description

Ai86(TITL-ArcLight) mice are a Cre/Tet-dependent, voltage-sensitive, fluorescent line, created by targeted insertion at the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated). After removal of the floxed-STOP cassette by Cre recombinase, they may be used to generate Tet-Off/Tet-On mutant animals with conditional (inducible/reversible) expression of the voltage-sensitive fluorescent protein ArcLight. ArcLight exhibits voltage-dependent FRET (Förster resonance energy transfer pairs) upon membrane depolarization, and enables optical recordings at high spatial resolution with intrinsic assignment of recorded signals to targeted cell populations.

Detailed description

These Ai86(TITL-ArcLight) mice are a Cre/Tet-dependent, voltage-sensitive, fluorescent line, created by targeted insertion at the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated). Ai86 mice harbor the TIGRE-Ins-TRE-LSL-Arclight conditional allele, designed with a modified Tet response element (TRE or tetO) and loxP-flanked STOP cassette upstream of the voltage-sensitive fluorescent protein ArcLight (see detailed description below). When bred with other mice expressing Cre recombinase, tetracycline-controlled transactivator protein (tTA) and/or reverse tetracycline-controlled transactivator protein (rtTA), ArcLight expression in cells/tissues where the expression patterns of the individual promoters driving Cre and tTA/rtTA overlap can be regulated with tetracycline or its analog doxycycline (dox).

The voltage-sensitive fluorescent protein ArcLight is a fusion of a CiVS (Ciona intestinalis voltage sensor) and the fluorescent protein super ecliptic pHluorin (an eGFP variant differing from eGFP at 9 positions) with an A227D mutation, and exhibits voltage-dependent FRET upon membrane depolarization, reporting in vivo signals at a high signal-to-noise ratio using 1- and 2-photon imaging (Jin et al. 2012).

Heterozygous Ai86 mice are viable and fertile with no reported gross physical or behavioral abnormalities. The donating investigator has not attempted to generate homozygous Ai86 mice to date (May 2020).

Development

The Ai86(TITL-ArcLight)-A242 allele (also called Ai86) has a Tet response element, floxed-STOP cassette and voltage-sensitive fluorescent protein ArcLight 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-ArcLight 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), a floxed-STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), ), the voltage-sensitive fluorescent protein ArcLight_A242 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-ArcLight replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME), thereby replacing the neo cassette. Chimeric mice were bred with the F1 progeny of a cross between Emx1-cre (Stock No. 005628) and Camk2a-tTA (Stock No. 007004) for germline transmission. The resulting Ai86D mice were bred with C57BL/6J wildtype mice for several generations prior to sending males to The Jackson Laboratory Repository in 2020. Upon arrival, sperm was cryopreserved. To establish our live colony, frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

The voltage-sensitive fluorescent protein ArcLight is a fusion of a CiVS [Ciona intestinalis voltage sensor (S1-S4 transmembrane segment, including the voltage-sensing domain in S4)] and the fluorescent protein super ecliptic pHluorin (an eGFP variant differing from eGFP at 9 positions) with an A227D mutation, and exhibits voltage-dependent Forster resonance energy transfer (FRET) upon membrane depolarization. In this strain, the fluorophore is inserted at position A242 within the CiVS (See Jin et al. 2012 for details).

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 binding of TetR or rTetR to the tetO sequences.

Allele

Symbol: Igs7^{tm86(tetO-CiVS/pHluorin)Hze}
Name: targeted mutation 86, Hongkui Zeng
MGI accession ID: MGI:6436677
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible
Synonyms: Ai86; Ai86(TITL-ArcLight)
Marker symbol: Igs7
Marker name: intergenic site 7
Marker synonyms: Iis7; TIGRE
Chromosome: 9
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: The targeting vector contains (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), a loxP-STOP-loxP cassette, the voltage-sensitive fluorescent protein Butterfly 1.2 coding sequence (referred to as ArcLight - 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 inserted into the Igs7 locus (T1 TIGRE - an intergenic region on chromosome 9 [between AB124611 and Carm1]) using recombinase-mediated cassette exchange (RMCE). The voltage-sensitive fluorescent protein ArcLight is a fusion of a CiVS [Ciona intestinalis voltage sensor (S1-S4 transmembrane segment, including the voltage-sensing domain in S4)] and the fluorescent protein super ecliptic pHluorin (an EGFP variant differing from EGFP at 9 positions) with an A227D mutation, and exhibits voltage-dependent Forster resonance energy transfer (FRET) upon membrane depolarization. In this strain, the fluorophore is inserted at position A242 within the CiVS.