B6.Cg-Igs7^{tm92.1(tetO-ECFP*/Venus*)Hze}/J

Stock number: 026262
Product name: Ai92(TITL-YCX2.60)-D or Ai92D
Research areas: Neurobiology Research, Research Tools

Description

Ai92(TITL-YCX2.60)-D (also called Ai92D) mice are a Cre/Tet-dependent, fluorescent calcium indicator line, created by targeted insertion of floxed-STOP-YCX2.60 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 cassettes by Cre recombinase, they may be used to generate Tet-Off/Tet-On mutant animals with conditional (inducible/reversible) expression of a FRET-based Ca2+ sensor with expanded dynamic range (YCX2.60). Following subsequent calcium binding (such as neuronal activation), robust yellow fluorescence and diminished cyan fluorescence is observed.

Detailed description

These Ai92(TITL-YCX2.60)-D mice (also called Ai92D) are a Cre/Tet-dependent, fluorescent calcium indicator line, created by targeted insertion of floxed-STOP-YCX2.60 at the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated). Ai92D mice harbor the TIGRE-Ins-TRE-LSL-YCX2.60 conditional allele, designed with a modified Tet response element (TRE or tetO) promoter and loxP-flanked STOP cassette upstream of the FRET-based Ca2+ sensor YCX2.60 (see detailed description below). When bred with mice expressing Cre recombinase and either the tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), YCX2.60 is expressed in cells/tissues where the expression patterns of the individual promoters driving Cre and tTA/rtTA overlap, and expression can be regulated with tetracycline or its analog doxycycline (dox).

Specifically, the donating investigator reports Ai92D mice have no reported levels of YCX2.60 expression (CFP or YFP fluorescence) in the absence of Cre and tTA (YCX2.60+/tTA-/Cre-), in Cre-negative double transgenic mice (YCX2.60+/tTA+/Cre-) or in tTA-negative double transgenic mice (YCX2.60+/tTA-/Cre+).
When Ai92D mice are bred with tTA-driver and Cre-driver lines to create triple transgenic animals (YCX2.60+/tTA+/Cre+), cells expressing both tTA and Cre exhibit cyan fluorescence (~480 nm) and minimal yellow fluorescence (~535 nm) before calcium transients. Following calcium binding (such as neuronal activation), robust bright yellow fluorescence and diminished cyan fluorescence is observed in these cells. The Ai92D reporter has high sensitivity; allowing observation of spontaneous and sensory-evoked neuronal activity. The donating investigator has not tested YCX2.60 expression/function in cells other than brain.

Heterozygous Ai92D mice are viable and fertile with no reported gross physical or behavioral abnormalities. The donating investigator has not attempted to generate homozygous Ai92D mice to date (May 2015).

For characterization information, see images at the Allen Institute for Brain Science website (Ai92(TITL-YCX2.60) images).

The FRET-based Ca2+ sensor YCX2.60 contains a Ca2+ sensor domain (CaM-GG-M13) fused between a FRET chromophore pair (yellow fluorescent protein donor [cp173 Venus YFP] / cyan fluorescent protein acceptor [ECFPΔC11]). YCX2.60 is an improved version of YC2.60 with expanded dynamic range and 2-3-fold greater dissociation constant. Upon Ca2+ binding (such as neuronal activation), a conformational change in the sensor domain alters YFP-CFP proximity and results in increased FRET (strong yellow fluorescence and diminished cyan fluorescence is observed). YCX2.60 shows strong calcium-evoked expression in excitatory neurons and inhibitory interneurons, and can be used to measure spontaneous and sensory-evoked neuronal activity with high sensitivity. YCX2.60 has single-photon and two-photon fluorescence excitation with ~440 nm and ~840 nm light, respectively, with emissions collected at ~480 nm for CFP and ~535 nm for YFP.

Development

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.

Allele

Symbol: Igs7^{tm92.1(tetO-ECFP*/Venus*)Hze}
Name: targeted mutation 92.1, Hongkui Zeng
MGI accession ID: MGI:5645699
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible
Marker symbol: Igs7
Marker name: intergenic site 7
Chromosome: 9
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: CFP,Cyan Fluorescent Protein,jellyfish; YFP,Yellow Fluorescent Protein,jellyfish
Site of expression: Expression of yellow fluorescent protein and cyan fluorescent protein is under the control of tetO in cells/tissues where promoters driving Cre recombinase are expressed.
Molecular note: The vector was designed with (from 5' to 3') an FRT3 site, two copies of chicken beta-globin HS4 insulator element, a modified Tet response element promoter, a floxed-STOP cassette, the YCX2.60 coding sequence (details below), a WPRE sequence, 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. 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 fusion gene is an enhanced cyan fluorescent protein with C-terminal 11 amino acids deleted, 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). PhiC31-mediated recombination removed the AttB/AttP-flanked PGK-hygromycin-SV40polyA cassette and replaced it with the recombined AttB/AttP site (AttL).