B6;129S6-Igs7^{tm140(tetO-EGFP,CAG-tTA2)Hze}/J

Stock number: 034100
Product name: Ai140 [or Ai140(TIT2L-EGFP-ICL-tTA2)]
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Ai140 (also called Ai140(TIT2L-EGFP-ICL-tTA2)) is a Cre-dependent, Tet-controllable fluorescent reporter line, created by targeted insertion at the Igs7 locus (TIGRE). Exposure to Cre recombinase removes both STOP cassettes - resulting in tTA2 expression and robust EGFP fluorescence. EGFP expression may be expected to be diminished/eliminated by doxycycline.

Detailed description

Ai140 mice harbor the TIGRE-Ins-TRE2-LSL-EGFP-Ins-CAG-LSL-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 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 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 and robust EGFP fluorescence. Due to its placement downstream of the TRE2 promoter, subsequent introduction of tetracycline (or its analog doxycycline [dox]) may be expected to diminish/eliminate EGFP expression.

Specifically, the donating investigator reports in the absence of Cre, mice heterozygous for Ai140 are viable and fertile with no reported gross physical or behavioral abnormalities. It has not been attempted to make this strain homozygous to date (April 2021). 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 Ai140 mice are bred with Cre-driver lines to create double transgenic animals (EGFP+tTA2+/Cre+), cells expressing both tTA2 and Cre exhibit robust/bright EGFP fluorescence. For example, they report crosses of Ai140 to several neocortical layer-specific and interneuron Cre-driver lines result in robust native EGFP expression.

Several Cre-driver lines with specific expression patterns have been used with Ai140 - resulting in double mutant offspring with no viability problems (including Tlx3-Cre_PL56, Sst-IRES-Cre and Gnb4-IRES2-CreERT2).
However, they also report that double heterozygous Ai140;Emx1-IRES-Cre mice have reduced brain volume and pronounced neurodegeneration in all hippocampal subfields. In addition, double heterozygous Ai140;Gad2-IRES-Cre mice exhibited embryonic lethality - this is similar to their finding of double heterozygous lethality using Gad2 Cre-driver with the Ai139D, Ai148D or Ai140D alleles. The donating laboratory attributes this to Cre expression during early embryonic development leading to high tTA2 expression levels, which promotes neurodegeneration and cytotoxicity [Daigle et al. 2018 Cell 174:465; PMID:30007418].

The dox-inducible attenuation of EGFP expression has not been characterized to date (April 2021). However, they expect that dox-inducible attenuation of EGFP expression would be variable dependent on which Cre-driver line is utilized - similar to that observed for Ai139 mice (Stock No. 034099).

To date (April 2021), the donating investigator has not evaluated Cre-inducible Ai140 expression in tissues other than brain.

Development

The Ai140 allele (or Ai140(TIT2L-EGFP-ICL-tTA2)) has a Tet response element TRE2, loxP-STOP-loxP cassette and EGFP sequence followed by a CAG promoter, lox2272-STOP-lox2272 cassette and 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-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 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-tTA2 replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME). Chimeric mice were bred with C57BL/6J mice. The Ai140 allele is therefore TIGRE-FRT3-Ins-TRE2-LSL-EGFP-WPRE-bGHpA-Ins-CAG-LSL-tTA2-WPRE-bGHpA-attB-PGK-5'hygro-SD-FRT5-SA-3'hygro-SV40pA-attP. The resulting Ai140 mice were bred with C57BL/6J for several generations. In 2020, males from generation N1 (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^{tm140(tetO-EGFP,CAG-tTA2)Hze}
Name: targeted mutation 140, Hongkui Zeng
MGI accession ID: MGI:6514331
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible; Transactivator
Synonyms: Ai140
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 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-tTA2 replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME).