B6;129S6-Igs7^{tm213(CAG-EGFP,CAG-mOrange2,CAG-mKate2)Tasic}/J

Stock number: 034113
Product name: Ai213 [or Ai213(TICL-EGFP-ICF-mOrange2-ICN-mKate2)]
Research areas: Neurobiology Research, Research Tools

Description

Ai213 (also called Ai213(TICL-EGFP-ICF-mOrange2-ICN-mKate2)) is a triple-recombinase responsive reporter allele designed with a CAG promoter, loxP-STOP-loxP and EGFP coding sequence, followed by a CAG promoter, frt-STOP-frt and mOrange2 coding sequence which is followed by a CAG promoter, nox-STOP-nox and mKate2 coding sequence - all inserted into the Igs7 locus (TIGRE). Exposure to either Cre, FLP or Nigri recombinase results in EGFP fluorescence, mOrange2 fluorescence or mKate2 fluorescence, respectively. In total, Ai213 mice are a Cre::FLP::Nigri intersectional fluorescent reporter line designed to allow EGFP/mOrange2/mKate2 fluorescence in those cells where Cre, FLP and Nigri expression overlaps.
Ai213 may also be useful as dual-recombinase responsive fluorescent reporter (e.g., Cre/FLP-dependent EGFP/mOrange2, Cre/Nigri-dependent EGFP/mKate2 and/or Cre/Nigri-dependent EGFP/mKate2 fluorescence), as well as a single recombinase responsive fluorescent reporter (e.g., Cre-dependent EGFP, FLP-dependent mOrange2 and/or Nigri-dependent mKate2 fluorescence).

Detailed description

Ai213 mice harbor the TIGRE-Ins-CAG-LSL-EGFP-Ins-CAG-FSF-mOrange2-Ins-CAG-NSN-mKate2 conditional allele, designed with a CAG promoter, loxP-STOP-loxP and EGFP coding sequence, followed by a CAG promoter, frt-STOP-frt and mOrange2 coding sequence which is followed by a CAG promoter, nox-STOP-nox and mKate2 coding sequence.

Prior to recombinase exposure, this allele is designed to have the three STOP cassettes preventing expression of their respective downstream genes (EGFP, mOrange2 and mKate2). Some low levels of background fluorescence are observed prior to removal of the STOP cassettes, but those levels are very minimal compared to the fluorescence following recombinase exposure (as described below). Ai213 heterozygotes are viable and fertile with no reported gross physical or behavioral abnormalities. To date (April 2021), it has not been attempted to make this strain homozygous.

Ai213 mice are useful as a triple recombinase responsive reporter line. When bred to mice expressing Cre, FLP and Nigri recombinases, the offspring will have all three STOP cassettes deleted in the cells where Cre, FLP and Nigri expression overlap - resulting in robust/bright EGFP, mOrange2 and mKate2 fluorescence.

Specifically, the donating investigator reports when Ai213 mice are bred with Cre-driver and FLP-driver lines to create quadruple mutant animals, the Ai213+/Cre+/FLP+/Nigri+ cells exhibit bright/robust fluorescence in the expected populations/patterns, including EGFP, mOrange2 and mKate2 fluorescence in any overlapping cell types. As described in Graybuck et al. 2021 Neuron [PMID:33789083], examples of the Cre/FLP/Nigri driver lines used for their Ai213 characterization included Gad2-IRES-Cre, Slc32a1-T2A-FlpO, Snap25-IRES2-Cre, Slc17a7-IRES2-FlpO, etc. Furthermore, PHP.eB serotyped AAVs expressing Cre, FlpO and Nigri delivered into Ai213 also resulted in the expected labeling (for pan-neuronal, GABAergic and excitatory layer 5 types).
To best ensure the Ai213 STOP cassettes remain intact until the desired removal, the donating laboratory suggests that researchers consider first creating their desired triple mutant Cre+/Flp+Nigri+ mice, and then breed them to the Ai213 reporter to generate quadruple mutant Ai213+/Cre+/FLP+/Nigri+ animals for their experiments.

The donating investigator reports that these Ai213 mice are also useful as a dual recombinase reporter or as a single recombinase reporter, as outlined below.
Double recombinase reporter: When Ai213 are bred with a double recombinase driver line to create triple mutant animals, the Ai213+/Cre+/FLP+/Nigri- cells had robust EGFP and mOrange2 fluorescence (with only very weak mKate2 expression), the Ai213+/Cre+/Nigri+/FLP- cells had robust EGFP and mKate2 fluorescence (with only very weak mOrange2 expression) and the Ai213+/FLP+/Nigri+/Cre- cells had bright mOrange2 and mKate2 fluorescence (with only very weak EGFP expression) - all in the expected populations (Graybuck et al. 2021 Neuron [PMID:33789083]). To best ensure the Ai213 STOP cassettes remain intact until the desired removal, the donating laboratory suggests that researchers consider first creating their desired double recombinase driver mice, and then breed them to the Ai213 reporter to generate triple mutant animals for their experiments.
Single recombinase reporter: When Ai213 are bred with a single recombinase driver line to create double mutant animals, the Ai213+/Cre+/FLP-/Nigri- cells exhibit robust/bright EGFP fluorescence (with only minimal mOrange2 or mKate2 fluorescence), the Ai213+/FLP+/Cre-/Nigri- cells exhibit robust/bright mOrange2 fluorescence (with only minimal EGFP or mKate2 fluorescence) and the Ai213+/Nigri+/Cre-/FLP- cells exhibit robust/bright mKate2 fluorescence (with only minimal EGFP or mOrange2 fluorescence) - all in the expected populations (Graybuck et al. 2021 Neuron [PMID:33789083]).

To date (April 2021), Ai213 animals have not specifically been characterized for expression in tissues other than brain.

Development

The Ai213 allele (or Ai213(TICL-EGFP-ICF-mOrange2-ICN-mKate2)) has a CAG promoter, loxP-STOP-loxP cassette and EGFP sequence followed by a CAG promoter, frt-STOP-frt cassette and mOrange2 sequence that is then followed by a CAG promoter, nox-STOP-nox cassette and mKate2 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 replacement vector was designed with (from 5' to 3') a Bxb1 attB site, a partial green fluorescent protein sequence (non-functional 500bp fragment to increase the space between the Bxb1 attB site and the insulator sequence to improve stability of the targeting vector), two copies of chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivators), a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG; from pCAGGS), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), a synthetic enhanced green fluorescent protein sequence (EGFP), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA, two copies of chicken beta-globin HS4 insulator element, the CAG promoter, a frt-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), a monomeric orange fluorescent protein sequence (mOrange2; details below) followed by 2xHA tag sequence, a WPRE, a BGH polyA, a PhiC31 attB site, a WPRE, a PGK polyA, two copies of chicken beta-globin HS4 insulator element, the CAG promoter, a nox-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), a sequence encoding the monomeric far-red fluorescent protein mKate2 (details below), a viral 2A oligopeptide that mediates ribosomal skipping (P2A; from porcine teschovirus-1), a WPRE, a BGH polyA, a PhiC31 attB site, a PGK-5'hygro cassette, an RNA splice donor and a Bxb1 attB site.
(129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem cells previously targeted with Bxb1 attP::PhiC31 attB-PGK/gb2-neoR-pA::Bxb1 attP::splice acceptor::3'hygro cassette::SV40pA::PhiC31 attP in TIGRE, were re-transfected with the replacement vector and a mouse codon-optimized Bxb1 integrase recombinase vector for recombinase-mediated cassette exchange (RCME). Chimeric mice were bred with C57BL/6J mice. The Ai213 allele is therefore TIGRE-Bxb1 attL-partial GFP-Ins-CAG-LSL-EGFP-WPRE-BGHpA-Ins-CAG-FSF-mOrange2-2xHA-WPRE-BGHpA-PhiC31 attB-WPRE-PGKpA-Ins-CAG-NSN-mKate2-P2A-WPRE-BGHpA-PhiC31 attB-PGK-5'hygro-SD-Bxb1 attR-SA-3'hygro-SV40pA-PhiC31 attP. The resulting Ai213 mice were bred with C57BL/6J for several generations. In 2020, males from generation N2 (black coat color) were sent to The Jackson Laboratory. Upon arrival, males are used to cryopreserve sperm. To establish our live colony, an aliquot of frozen sperm was 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).

mOrange2 is a mammalian-codon optimized, photostable, fast-bleaching, monomeric orange fluorescent protein that was designed by Dr. Roger Y. Tsien (University of California at San Diego, HHMI; Shaner et al. 2008 Nat Methods 5:545). mOrange2 is an improved version of mOrange (a derivative of DsRed). Compared to its predecessor, mOrange2 has substantially enhanced photostability (Q64H, F99Y, E160K and G196D mutations) and also exhibits slightly shifted excitation and emission peaks (549nm and 565nm, respectively).

mKate2 is a monomeric far-red fluorescent protein optimized for high-brightness, photostability, pH resistance and low-toxicity (Shcherbo et al. 2009 Biochem J 418:567). mKate2 has excitation and emission peaks of 588nm and 633nm, respectively. Compared with its predecessor, the fluorescence of mKate2 is far more pH-stable, thus increasing its brightness at physiological pH and making mKate2 a more reliable reporter for imaging in acidic organelles. For applications in combination with channelrhodopsin/EYFP fusion proteins, the more red-shifted mKate2 may be desirable to tdTomato.

Allele

Symbol: Igs7^{tm213(CAG-EGFP,CAG-mOrange2,CAG-mKate2)Tasic}
Name: targeted mutation 213, Bosiljka Tasic
MGI accession ID: MGI:6514387
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: Ai213; Igs7^{tm213(CAG-EGFP,CAG-mOrange2,CAG-mKate2)Hze}
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 vector contains (from 5' to 3') a Bxb1 attB site, a partial green fluorescent protein sequence (non-functional 500bp fragment to increase the space between the Bxb1 attB site and the insulator sequence to improve stability of the targeting vector), two copies of chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivators), a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter, a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), a synthetic enhanced green fluorescent protein sequence (EGFP), a woodchuck hepatitis virus post-transcriptional regulatory element, a BGH polyA, two copies of chicken beta-globin HS4 insulator element, the CAG promoter, an FRT-flanked STOP cassette, a monomeric orange fluorescent protein sequence (mOrange2; details below) followed by 2xHA tag sequence, a WPRE, a BGH polyA, a PhiC31 attB site, a WPRE, a PGK polyA, two copies of chicken beta-globin HS4 insulator element, the CAG promoter, a nox-flanked STOP cassette, a sequence encoding the monomeric far-red fluorescent protein mKate2 (details below), a viral 2A oligopeptide that mediates ribosomal skipping, a WPRE, a BGH polyA, a PhiC31 attB site, a PGK-5'hygro cassette, an RNA splice donor, Bxb1 attR-SA-3'hygro-SV40pA-PhiC31 attP. mOrange2 is a mammalian-codon optimized, photostable, fast-bleaching, monomeric orange fluorescent protein. Compared to its predecessor, mOrange2 has substantially enhanced photostability (Q64H, F99Y, E160K and G196D mutations) and also exhibits slightly shifted excitation and emission peaks (549nm and 565nm, respectively). Kate2 is a monomeric far-red fluorescent protein optimized for high-brightness, photostability, pH resistance and low-toxicity. Compared with its predecessor, the fluorescence of mKate2 is far more pH-stable, thus increasing its brightness at physiological pH and making mKate2 a more reliable reporter for imaging in acidic organelles.