B6.Cg-Igs7^{tm139.1(tetO-EGFP,CAG-tdTomato,-tTA2)Hze}/J

Stock number: 030219
Product name: Ai139(TIT2L-GFP-ICL-TPT)-D (or Ai139D)
Research areas: Neurobiology Research, Research Tools

Description

Ai139(TIT2L-GFP-ICL-TPT)-D (also called Ai139D) mice are a Cre-dependent, Tet-controllable dual fluorescent reporter 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). Exposure to Cre recombinase removes both STOP cassettes - resulting in EGFP fluorescence, tdTomato fluorescence and tTA2 expression - and that EGFP expression may be diminished/eliminated by doxycycline.

Detailed description

Ai139D mice harbor the TIGRE-Ins-TRE2-LSL-EGFP-Ins-CAG-LSL-tdT-P2A-tTA2 conditional allele, designed with a TRE2 promoter, loxP-flanked STOP cassette and EGFP sequence, and then a CAG promoter, lox2272-flanked STOP cassette and bicistronic tdTomato-P2A-tTA2 sequence. Prior to Cre recombinase exposure, this allele is designed to have the two floxed-STOP cassettes preventing expression of the downstream genes (EGFP, tdTomato and tTA2). When bred to mice expressing Cre recombinase, the resulting offspring will have both floxed-STOP cassettes deleted in the cre-expressing cells/tissues - resulting in EGFP fluorescence, tdTomato fluorescence and tTA2 expression. Due to its placement downstream of the TRE2 promoter, subsequent introduction of tetracycline (or its analog doxycycline [dox]) will diminish/eliminate EGFP expression.

Specifically, the donating investigator reports in the absence of Cre, mice heterozygous or homozygous for Ai139D exhibit no fluorescence, and are viable and fertile with no reported gross physical or behavioral abnormalities. Anti-GFP immunostaining reveals some background expression in cortex and hippocampus, but this is several fold less than the levels observed following exposure to Cre recombinase. When Ai139D mice are bred with Cre-driver lines to create double transgenic animals (EGFP+tdT+tTA2+/Cre+), cells expressing both tTA2 and Cre exhibit robust/bright fluorescence (both EGFP and tdTomato) in living and fixed brain sections. The dox-inducible attenuation of EGFP expression has been characterized - EGFP fluorescence is completely suppressed after two weeks of dox treatment. Their experience is the extent of two-color fluorescence (EGFP and tdTomato co-expression) within a given cell type is variable dependent on which Cre-driver line is utilized.
Several Cre-driver lines with specific expression patterns have been used with Ai139D - resulting in double mutant offspring with no viability problems (including C57BL/6J-congenic Nr5a1-Cre and Sst-IRES-Cre (see Stock Nos. 006364 and 013044). In addition, their experience is double heterozygous Gad2-Cre;Ai139D mice exhibited embryonic lethality (see Stock No. 019022) - this is similar to their finding of double heterozygous lethality using the same Gad2-Cre driver with the with the Ai140D or Ai148D allele (Stock Nos. 030220 or 030228).

To date (March 2017), the donating investigator has not evaluated Cre-inducible Ai139D expression in tissues other than brain.

Development

The Ai139(TIT2L-GFP-ICL-TPT)-D allele (or Ai139D) has a Tet response element TRE2, loxP-flanked-STOP cassette and EGFP sequence followed by a CAG promoter, lox2272-flanked-STOP cassette, tdTomato-P2A-tTA2 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 pTRE2-LSL-EGFP-CAG-LSL-tdT-P2A-tTA2 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 Tet response element/promoter (TRE2; details below), 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, a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG; from pCAGGS), a lox2272-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), a tdTomato sequence (tdT; encoding the non-oligomerizing DsRed fluorescent protein variant with a 12 residue linker fusing two copies of the protein (tandem dimer)), a viral 2A oligopeptide that mediates ribosomal skipping (P2A; from porcine teschovirus-1), a synthetic modified tetracycline-regulated transactivator gene (tTA2^S), a WPRE, a BGH polyA, 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 pTRE2-LSL-EGFP-CAG-LSL-tdT-P2A-tTA2 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-hygro-SV40polyA cassette and replace it with the recombined AttB/AttP site (AttL). The Ai139D allele is therefore TIGRE-frt3-Ins-TRE2-LSL-EGFP-WPRE-bGHpA-Ins-CAG-LSL-tdT-P2A-tTA2-WPRE-bGHpA-AttL. The resulting Ai139D mice were bred with C57BL/6J for several generations (and the PhiC31 gene was removed). In 2017, males from generation N6 (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).

The TRE2 promoter used here is Tet-responsive P_hCMV*-1; containing the Tet response element (seven copies of the 19 bp tet operator sequence [tetO]) just upstream of a minimal cytomegalovirus promoter (P_{min CMV}), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P_hCMV*-1 is silent in the absence of tTA or rtTA binding to tetO.

Allele

Symbol: Igs7^{tm139.1(tetO-EGFP,CAG-tdTomato,-tTA2)Hze}
Name: targeted mutation 139.1, Hongkui Zeng
MGI accession ID: MGI:5903998
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible; Transactivator
Synonyms: Ai139(TIT2L-GFP-ICL-TPT)-D; Ai139D
Marker symbol: Igs7
Marker name: intergenic site 7
Marker synonyms: Iis7; TIGRE
Chromosome: 9
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: GFP,Green Fluorescent Protein,; RFP,Red Fluorescent Protein,coral; tTA,tetracycline-controlled transactivator,E. coli
Site of expression: When bred to mice expressing Cre recombinase, the resulting offspring will express EGFP fluorescence, tdTomato fluorescence and tTA2 expression in the cre-expressing cells/tissues.
Molecular note: The replacement vector is designed with (from 5' to 3') an FRT3 site, two copies of chicken beta-globin HS4 insulator element, a Tet response element/promoter (TRE2), a loxP-flanked STOP cassette, 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, a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG; from pCAGGS), a lox2272-flanked STOP cassette, a tdTomato sequence, a viral 2A oligopeptide that mediates ribosomal skipping, a synthetic modified tetracycline-regulated transactivator gene (tTA2S), a WPRE, a BGH polyA, 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 pTRE2-LSL-EGFP-CAG-LSL-tdT-P2A-tTA2 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).