B6.Cg-Igs7^{tm62.1(tetO-tdTomato)Hze}/J

Stock number: 022731
Product name: Ai62(TITL-tdT)-D or Ai62D
Research areas: Neurobiology Research, Research Tools

Description

These Ai62(TITL-tdT)-D mice (also called Ai62D) are a fluorescent Cre/Tet 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). 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 tdTomato.

Detailed description

These Ai62(TITL-tdT)-D mice (also called Ai62(TITL-tdT)Δhygro or Ai62D) are a fluorescent Cre/Tet 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). Ai62D mice harbor the TIGRE-Ins-TRE-LSL-tdTomato conditional allele designed with a modified Tet response element (TRE or tetO) and loxP-flanked STOP cassette upstream of the tdTomato fluorescent protein. When bred with other mice expressing Cre recombinase, tetracycline-controlled transactivator protein (tTA) and/or reverse tetracycline-controlled transactivator protein (rtTA), tdTomato expression in cells/tissues where the expression patterns of the individual promoters driving Cre recombinase and tTA/rtTA overlap can be regulated with tetracycline or its analog doxycycline (dox). Heterozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. The donating investigator has not attempted to generate homozygous mice to date (June 2013).
Specifically, the donating investigator reports for triple transgenic mice (Cre+/tTA+/tdTomato+) in the cells expressing both Cre and tTA, tdTomato expression may be detected by direct/native fluorescence and by in situ hybridization (using a tdTomato probe sequence). The donating investigator reports Ai62(TITL-tdT)-D mice have no reported levels of tdTomato expression in absence of tTA. Cre-negative double transgenic mice (Cre-/tTA+/tdTomato+) have no tdTomato expression; although one experimental combination of Cre-/tTA+/tdTomato+ mice were reported to have a few very weakly tdTomato-positive cells in cortex and hippocampus.

For characterization information, see images at the Allen Institute for Brain Science website (Ai62(TITL-tdT) images).

Development

These Ai62(TITL-tdT)-D allele (also called Ai62(TITL-tdT)Δhygro or Ai62D) has a Tet response element, floxed-STOP cassette and tdTomato fluorescent protein, 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 pTRE-LSL-tdTomato replacement vector was designed with (from 5' to 3') an FRT3 site, two tandem copies of the chicken beta-globin HS4 insulator element (to reduce expression of the reporter gene in the absence of transactivator protein), a modified Tet response element (TRE or (tetO); described in detail below), a loxP-flanked STOP cassette (with stop codons in all three reading frames linked to a synthetic pA-hGHpA-PGKpA unit), a sequence encoding the tdTomato fluorescent protein (a non-oligomerizing DsRed fluorescent protein variant with a 12 residue linker fusing two copies of the protein (tandem dimer)), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA signal, two tandem copies of the 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-tdTomato 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 Ai62D allele is therefore TIGRE-frt3-Ins-TRE-LSL-tdTomato-WPRE-bGHpA-Ins-AttL. The resulting Ai62D mice were bred with C57BL/6J wildtype mice for at least three additional generations prior to sending to The Jackson Laboratory Repository in 2013. Upon arrival, mice were bred to C57BL/6J inbred animals (Stock No. 000664) at least once to remove the PhiC31 gene. The Ai62D cryopreserved sperm is the equivalent of six generations backcrossed onto C57BL/6J [June 2014].

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 TetR or rTetR binding to tetO.

Allele

Symbol: Igs7^{tm62.1(tetO-tdTomato)Hze}
Name: targeted mutation 62.1, Hongkui Zeng
MGI accession ID: MGI:5490616
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; RMCE-ready
Marker symbol: Igs7
Marker name: intergenic site 7
Chromosome: 9
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: RFP,Red Fluorescent Protein,coral
Site of expression: Expression of tdTomato is under the control of tetO in cells/tissues where promoters driving Cre recombinase are expressed.
Molecular note: The targeting constructed introduced by recombinase mediated cassette exchange at the existing site inserted anan FRT3 site, two tandem copies of the chicken beta-globin HS4 insulator element (to reduce expression of the reporter gene in the absence of transactivator protein), a modified Tet response element (TRE or (tetO); described in detail below), a loxP-flanked STOP cassette (with stop codons in all three reading frames linked to a synthetic pA-hGHpA-PGKpA unit), a sequence encoding the tdTomato fluorescent protein (a non-oligomerizing DsRed fluorescent protein variant with a 12 residue linker fusing two copies of the protein (tandem dimer)), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA signal, two tandem copies of the chicken beta-globin HS4 insulator element, an AttB site, a PGK-5'hygro cassette, an RNA splice donor and a FRT5 site. PhiC31-mediated recombination removed the hygromycin cassette and replaced it with the recombined AttB/AttP site (AttL). The Ai62D allele is therefore TIGRE-frt3-Ins-TRE-LSL-tdTomato-WPRE-bGHpA-Ins-AttL.
The modified TRE promoter used in these transgenic mice is the Tet-responsive P^tight promoter. This P^tight promoter contains a modified Tet response element (TRE^mod), which consists of seven direct repeats of a 36-bp sequence each containing the 19-bp tet operator sequence (tetO). The TRE^mod is just upstream of a minimal human cytomegalovirus (CMV) promoter (P^minCMVDelta), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P>tight< is silent in the absence of binding of TetR or rTetR to the tetO sequences.