B6.Cg-Igs7^{tm133.1(tetO-apx1*)Hze}/J

Stock number: 030213
Product name: Ai133(TITL-ssAPEX2tm)-D (or Ai133D)
Research areas: Neurobiology Research, Research Tools

Description

Ai133(TITL-ssAPEX2tm)-D (also called Ai133D) mice are a Cre/Tet-dependent, substrate-dependent reporter line - created by targeted insertion of floxed-STOP-APEX2 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 APEX2 - an engineered peroxidase with improved catalytic efficiency that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics.

Detailed description

These Ai133(TITL-ssAPEX2tm)-D mice (also called Ai133D) are a Cre/Tet-dependent, substrate-dependent reporter line - created by targeted insertion of floxed-STOP-APEX2 at the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated).

Ai133D mice harbor the TIGRE-Ins-TRE-LSL-ssAPEX2tm conditional allele, designed with a modified Tet response element (TRE or tetO) promoter and loxP-flanked STOP cassette upstream of an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics (APEX2; 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), APEX2 is expressed in cells/tissues where the expression patterns of the individual promoters driving Cre and tTA/rtTA overlap, and expression can be suppressed with tetracycline or its analog doxycycline (dox).

Specifically, the donating investigator reports Ai133D mice have no reported levels of APEX2 expression in the absence of Cre and tTA (APEX2+/Cre-/tTA-)
When Ai133D mice are bred with tTA-driver and Cre-driver lines to create triple transgenic animals (APEX2+/Cre+/tTA+), cells expressing both tTA and Cre exhibit both membrane and cytosolic expression of APEX2 following substrate (diaminobenzidine [DAB]) reactivity in fixed brain sections in several Cre-defined cell types. When using Ai133D with C57BL/6J-congenic driver lines Scnn1a-Tg3-Cre, Camk2a-tTA or ROSA26-ZtTA (see Stock Nos. 009613, 007004, 012266, respectively), the donating investigator observes very efficient reporter function. Additionally, no APEX2 labeling is reported in the absence of substrate (DAB). They have not tested APEX2 expression/function in tissues other than brain. To date (March 2017), APEX2-labeling has not been characterized in Ai133D live cells.

Heterozygous and homozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities.

Ascorbate peroxidase (apx1) is a heme-containing peroxidase family member that catalyzes the H₂O₂-dependent oxidation of a wide range of different biological substrates. Specifically, the apx1 variant used here is the second-generation enzyme APEX2 - an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. APEX2 has improved catalytic efficiency that is suitable for all proteomic applications and electron microscopy applications (Hung et al. 2016 Nat Protoc. 11:456). Unlike horseradish peroxidase, APEX2 lacks disulfides and calcium-binding sites and hence can be expressed in the reducing cytosolic environment of cells without loss of activity. Consequently, APEX2 can be used for intracellular specific protein imaging by electron microscopy (via the substrate diaminobenzidine [DAB]) and spatially resolved proteomic mapping (via the substrate biotin-phenol [BP]). APEX2 activity is undetectable at low expression levels, while high expression causes aggregation of mitochondrial and endoplasmic reticulum membranes. The ssAPEX2tm fusion gene used in Ai133D has APEX2 fused to a T-cell Cd2 antigen signal sequence (ss) and transmembrane targeting sequence (tm). Thus, when expressed, APEX2 is anchored by a transmembrane sequence and faces the cytosol.

Development

The Ai133(TITL-ssAPEX2tm)-D allele (also called Ai133D) has a Tet response element, a floxed-STOP cassette and the APEX2 ascorbate peroxidase 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-ssAPEX2tm 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 ssAPEX2tm 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-ssAPEX2tm 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 Ai133D allele is therefore TIGRE-frt3-Ins-TRE-LSL-ssAPEX2tm-WPRE-bGHpA-Ins-AttL.

The resulting Ai133D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 gene was removed). In 2017, male mice from generation N7 with black coat color were sent to The Jackson Laboratory Repository. Upon arrival, males are used to cryopreserve sperm. To establish the living Ai133D mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (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).

APEX2 is a second-generation variant of soybean Glycine hispida ascorbate peroxidase gene (apx1) that harbors the three APEX mutations (K14D, W41F, E112K) optimized for expression in mice, as well as one additional mutation (A134P) for improved catalytic efficiency. APEX2 has a molecular weight of 27 kDa (~same as GFP), and it is monomeric, free of disulfide bonds and contains a non-covalently bound heme cofactor in its active site. The ssAPEX2tm fusion gene, designed by Dr. Alice Y. Ting (Massachusetts Institute of Technology), has APEX2 fused to a T-cell Cd2 antigen signal sequence (ss) and transmembrane targeting sequence (tm). There is a FLAG-tag domain (DYKDDDDK) between the signal sequence and the APEX2 sequence [Hung et al. 2016 Nat Protoc. 11:456]

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^{tm133.1(tetO-apx1*)Hze}
Name: targeted mutation 1.1, Hongkui Zeng
MGI accession ID: MGI:5903988
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Inducible; Inserted expressed sequence
Synonyms: Ai133; Ai133(TITL-ssAPEX2tm)-D
Marker symbol: Igs7
Marker name: intergenic site 7
Marker synonyms: Iis7; TIGRE
Chromosome: 9
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Site of expression: APEX2 is expressed in cells/tissues where the expression patterns of the individual promoters driving Cre and tTA/rtTA overlap, and expression can be suppressed with tetracycline or its analog doxycycline (dox).
Molecular note: The 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 ssAPEX2tm 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. 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-ssAPEX2tm replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME). PhiC31-mediated recombination removed the AttB/AttP-flanked sequence (PGK-Neo-polyA::frt5::RNA splice acceptor::3'hygro-polyA) and replaced it with the recombined AttB/AttP site (AttL). APEX2 is a second-generation variant of soybean Glycine hispida ascorbate peroxidase gene (apx1) that harbors the three APEX mutations (K14D, W41F, E112K) optimized for expression in mice, as well as one additional mutation (A134P) for improved catalytic efficiency. The ssAPEX2tm fusion gene has APEX2 fused to a T-cell Cd2 antigen signal sequence (ss) and transmembrane targeting sequence (tm). There is a FLAG-tag domain (DYKDDDDK) between the signal sequence and the APEX2 sequence.