B6;129S-Igs7^{tm85.1(tetO-gltI/GFP*)Hze}/J

Stock number: 026260
Product name: Ai85D
Strain synonyms: Ai85(TITL-iGluSnFr)-D
Research areas: Neurobiology Research, Research Tools

Description

Ai85(TITL-iGluSnFr)-D (also called Ai85D) mice are a Cre/Tet-dependent, fluorescent glutamate indicator line, created by targeted insertion of floxed-STOP-iGluSnFr 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 iGluSnFr, an intensity-based glutamate-sensing fluorescent reporter that is fast and sensitive. Increased GFP fluorescence is observed following subsequent glutamate binding (such as excitatory synaptic activity).

Detailed description

These Ai85(TITL-iGluSnFr)-D mice (also called Ai85D) are a Cre/Tet-dependent, fluorescent glutamate indicator line, created by targeted insertion of floxed-STOP-iGluSnFr at the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated). Ai85D mice harbor the TIGRE-Ins-TRE-LSL-iGluSnFr conditional allele, designed with a modified Tet response element (TRE or tetO) promoter and loxP-flanked STOP cassette upstream of the intensity-based glutamate-sensing fluorescent reporter (iGluSnFr; 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), iGluSnFr 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 Ai85D mice have no reported levels of iGluSnFr expression (GFP fluorescence) in the absence of Cre and tTA (iGluSnFr+/tTA-/Cre-), in Cre-negative double transgenic mice (iGluSnFr+/tTA+/Cre-) or in tTA-negative double transgenic mice (iGluSnFr+/tTA-/Cre+).
When Ai85D mice are bred with tTA-driver and Cre-driver lines to create triple transgenic animals (iGluSnFr+/tTA+/Cre+), cells expressing both tTA and Cre exhibit low GFP fluorescence in the absence of glutamate binding. Following glutamate binding (such as excitatory synaptic activity), robust GFP fluorescence is observed in these cells. The donating investigator has not tested iGluSnFr expression/function in cells other than brain.

Heterozygous Ai85D mice are viable and fertile with no reported gross physical or behavioral abnormalities. In 2017, breeding homozygous mice together at The Jackson Laboratory successfully identified that homozygous Ai85D mice are viable and fertile.

For characterization information, see images at the Allen Institute for Brain Science website (Ai85(TITL-iGluSnFr) images).

The intensity-based glutamate-sensing fluorescent reporter iGluSnFr optimized for in vivo imaging with fast kinetics, single-wavelength glutamate sensitivity and maximum fluorescence response (Marvin et al. 2013 Nat Methods 10:162). Designed by Dr. Loren Looger (Janelia Farm, HHMI), the iGluSnFr fusion gene is constructed from the Escherichia coli gltI gene (YbeJ) and circularly permutated green fluorescent protein (cpGFP). The iGluSnFr fusion gene has an IgG K-leader sequence (to direct the protein to the secretory pathway) at the N-terminal end, and a c-Myc epitope tag and a 49 aa PDGR sequence (to anchor the protein to the plasma membrane [displaying it on the extracellular side]) at the C-terminal end. Because iGluSnFr can directly access the synaptic cleft, it allows extremely rapid detection of glutamate transients with high spatial resolution; enabling direct visualization of excitatory synaptic release (as opposed to calcium imaging). iGluSnFr is sensitive to glutamate peaks just prior to calcium peaks and action potential. iGluSnFr has single-photon and two-photon fluorescence excitation with ~470-485 nm and ~910-920 nm light, respectively. The pK_a of iGluSnFr is 6.5 in the glutamate-bound and 7.0 in the ligand-free state. The purified iGluSnFr protein has no affinity to a panel of other amino acids or other neurotransmitters, excepting that some in vitro affinity to aspartate is reported. In the absence of glutamate binding, dim GFP fluorescence is expected (presumably due to distortion of the cpGFP β-barrel). Upon glutamate binding, the correct conformation is restored; resulting in bright and photostable fluorescence.

Development

The Ai85(TITL-iGluSnFr)-D allele (also called Ai85D) has a Tet response element, a floxed-STOP cassette and the iGluSnFr glutamate 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-iGluSnFr 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 iGluSnFr 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-iGluSnFr 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 Ai85D allele is therefore TIGRE-frt3-Ins-TRE-LSL-iGluSnFr-WPRE-bGHpA-Ins-AttL.

The resulting Ai85D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 gene was removed) prior to sending black males from generation N2 to The Jackson Laboratory Repository in 2016. Upon arrival, sperm was cryopreserved. To establish our live colony, frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

The intensity-based glutamate-sensing fluorescent reporter iGluSnFr (also called GltI253-cpGFP.L1LV/L2NP) is an extremely fast and sensitive glutamate indicator with large signal-to-noise ratio, and kinetics appropriate for in vivo imaging (Marvin et al. 2013 Nat Methods 10:162). Designed by Dr. Loren Looger (Janelia Farm, HHMI), the iGluSnFr fusion gene is constructed from the Escherichia coli gltI gene (YbeJ; encoding the periplasmic component of the ABC transporter complex for glutamate and aspartate) and circularly permutated green fluorescent protein (cpGFP). Specifically, iGluSnFr has a 21 aa IgG K-leader sequence, the gltI aa 1-253, the LILV linker, the cpGFP (aa 148-239, an 18 bp flexible linker and aa 1-147), the L2NP linker, the gltI aa 254-279, an 11 aa c-Myc epitope and a 49 aa PDGR sequence (PDGR aa 513-561).

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^{tm85.1(tetO-gltI/GFP*)Hze}
Name: targeted mutation 85.1, Hongkui Zeng
MGI accession ID: MGI:5645698
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: GFP,Green Fluorescent Protein,; myc tag,myc tag,
Site of expression: Expression of GFP is under the control of tetO in cells/tissues where promoters driving Cre recombinase are expressed.
Molecular note: The pTRE-LSL-iGluSnFr replacement 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 iGluSnFr 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. The iGluSnFr fusion gene is constructed from the Escherichia coli gltI gene and circularly permutated green fluorescent protein (cpGFP). Specifically, iGluSnFr has a 21 aa IgG K-leader sequence, the gltI aa 1-253, the LILV linker, the cpGFP (aa 148-239, an 18 bp flexible linker and aa 1-147), the L2NP linker, the gltI aa 254-279, an 11 aa c-Myc epitope and a 49 aa PDGR sequence (PDGR aa 513-561). iGluSnFr is an extremely fast and sensitive glutamate indicator with large signal-to-noise ratio, and kinetics appropriate for in vivo imaging. PhiC31-mediated recombination removed the AttB/AttP-flanked PGK-hygromycin-SV40polyA cassette and replaced it with the recombined AttB/AttP site (AttL).