B6;129S6-Igs7^{tm139(tetO-EGFP,CAG-tdTomato,-tTA2)Hze}/J

Stock number: 034099
Product name: Ai139 [or Ai139(TIT2L-EGFP-ICL-TPT)]
Research areas: Neurobiology Research, Research Tools

Description

Ai139 (also called Ai139(TIT2L-EGFP-ICL-TPT)) is a Cre-dependent, Tet-controllable dual fluorescent reporter line, created by targeted insertion at the Igs7 locus (TIGRE). Exposure to Cre recombinase removes both STOP cassettes resulting in tTA2 expression, robust EGFP fluorescence and robust tdTomato fluorescence. EGFP expression may be diminished/eliminated by doxycycline.

Detailed description

Ai139 mice harbor the TIGRE-Ins-TRE2-LSL-EGFP-Ins-CAG-LSL-tdT-P2A-tTA2 conditional allele, designed with a TRE2 promoter, loxP-STOP-loxP cassette and EGFP sequence, and then a CAG promoter, lox2272-STOP-lox2272 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 tTA2 expression, robust EGFP fluorescence and robust tdTomato fluorescence. Due to its placement downstream of the TRE2 promoter, subsequent introduction of tetracycline (or its analog doxycycline [dox]) is expected to diminish/eliminate EGFP expression.

Specifically, the donating investigator reports in the absence of Cre, mice heterozygous or homozygous for Ai139 and are viable and fertile with no reported gross physical or behavioral abnormalities. Weak GFP fluorescence is observed throughout the cortex and in the hippocampus, but this is much weaker than the levels observed following exposure to Cre recombinase.

When Ai139 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.

Their experience is that 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. For example, crossing Ai139 to Sst-IRES-Cre, Pvalb-T2A-CreERT2 or Ntsr1-Cre yields robust and bright GFP and tdTomato expression (see Daigle et al. 2018 Cell 174:465 [PMID:30007418]). Meanwhile, crossing Ai139 to Calb1-IRES2-Cre gives robust GFP and tdTomato expression, though some cells express only one or the other.

Similarly they report that dox-inducible attenuation of EGFP expression is variable dependent on which Cre-driver line is utilized. For example, EGFP is completely repressed in Ai139;Ntsr1-Cre cortical layer 6 neurons after 14 days of dox chow, but EGFP is only partially repressed in Ai139;Calb1-IRES2-Cre cortical layers 2/3-4 neurons.

In addition, they do not recommend crossing Ai139 to pan-excitatory or pan-inhibitory driver lines because the resulting double mutant Cre expression during early embryonic development leads to high tTA2 expression levels, which promotes neurodegeneration and cytotoxicity (as shown for their other similarly designed reporters in Daigle et al. 2018 Cell 174:465 [PMID:30007418]).

To date (April 2021), the donating investigator has not evaluated Cre-inducible Ai139 expression in tissues other than brain.

Development

The Ai139 allele (or Ai139(TIT2L-EGFP-ICL-TPT)) has a Tet response element TRE2, loxP-STOP-loxP and EGFP sequence followed by a CAG promoter, lox2272-STOP-lox2272 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::SV40pA::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 mice.
The Ai139 allele is therefore TIGRE-frt3-Ins-TRE2-LSL-EGFP-WPRE-bGHpA-Ins-CAG-LSL-tdT-P2A-tTA2-WPRE-bGHpA-AttB-PGK-5'hygro-SD-frt5-SA-3'hygro-SV40pA-AttP.
The resulting Ai139 mice were bred with C57BL/6J for several generations. In 2020, males from generation N3 (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(tetO-EGFP,CAG-tdTomato,-tTA2)Hze}
Name: targeted mutation 139, Hongkui Zeng
MGI accession ID: MGI:6514329
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible; Transactivator
Synonyms: Ai139
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 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).