STOCK Igs7^{tm2(tetO-tdTomato,-DTA*G128D)Rdiez}/AljJ

Stock number: 034778
Product name: Dragon-DTA
Research areas: Research Tools

Description

In Dragon-DTA mice, conditional expression of the tox176 attenuated DTA Gene (DTA*G128D) is under the control of a tetracycline response element (TRE; tetO) and floxed tdTomato STOP cassette integrated in the Igs7 (TIGRE) locus. Expression of DTA*G128D requires the expression of (r)tTA, and induction of the tetracycline analog, doxycycline (dox), as well as cre recombinase-mediated excision of the floxed tdTomato STOP cassette. These mice enable cell ablation with tissue specificity and temporal control.

Detailed description

Dragon-DTA is a cre recombinase- and tetracycline-controlled transactivator (tTA)-dependent mouse line that conditionally expresses a tox176 attenuated version of diphtheria toxin subunit alpha gene (DTA*G128D). The Dragon (Doxycycline-controlled and Recombinase Activated Gene OverexpressioN) system requires the intersection of two drivers, Cre and (r)tTA, to generate DTA*G128D. Due to its dependence on (r)tTA, the Dragon system is inducible and reversible, providing the ability to manipulate time and duration of DTA*G128D expression and subsequent ablation of DTA-expressing cells. DTA*G128D exhibits diminished enzyme activity/toxicity compared to diphtheria toxin, but retains substantial toxicity levels for ablation of mammalian cells.

For example, when bred to Atoh1-tTA mice and Atoh1-Cre mice (Stock No. 011104), induction with dox restricts DTA*G128D expression to excitatory cerebellar nuclei (eCN), which house the main output neurons of the cerebellum. The resulting mice with DTA expression exhibit ectopic cell death specifically in the Nuclear Transitory Zone (NTZ), where the precursors of the eCN migrate from the rhombic lip.

Other Dragon line are available:
Dragon-p21 (Stock No. 034777) conditionally expresses P21 and is useful for inhibiting cell cycle progression in specific tissue and at specific times.
Dragon-Ctgf (Stock No. 034779) conditionally expresses cellular communication network factor 2 (Ccn2) gene, and is useful for the enhancing or blocking of cell proliferation in specific tissue and at specific times.

Development

The Dragon-DTA 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 the absence of transactivators), a Tet-response element/promoter (TRE tight, composed of seven TetO repeats and a minimal CMV promoter), a loxP-flanked red fluorescent protein variant (tdTomato) acting as a STOP cassette with 2 polyadenylation sequences (synthetic pA and hGHpA), a tox176 attenuated version of diphtheria toxin subunit alpha gene, a woodchuck virus RNA stabilizing sequence (WPRE), 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 (Igs7), were transfected with the replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME). Correctly targeted ES cells were injected into recipient blastocysts from albino C57BL/6J mice (Stock No. 000058) and the resulting chimeric males were outbred to a Swiss Webster background for two generations before crossing heterozygous non-siblings to establish a homozygous line. The process of backcrossing to Swiss Webster background and breeding back to homozygosity was repeated twice in order to maintain high fertility. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Igs7^{tm2(tetO-tdTomato,-DTA*G128D)Rdiez}
Name: targeted mutation 2, Alberto Rosello Diez
MGI accession ID: MGI:6389249
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible; Inserted expressed sequence
Synonyms: Dragon-DTA; Igs7^TRE-LtSL-DTA
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 targeting 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 the absence of transactivators), a Tet-response element/promoter (TRE tight, composed of seven TetO repeats and a minimal CMV promoter), a loxP-flanked red fluorescent protein variant (tdTomato) acting as a STOP cassette with 2 polyadenylation sequences (synthetic pA and hGHpA), a tox176 attenuated version of diphtheria toxin subunit alpha gene, a BGH polyA, a woodchuck virus RNA stabilizing sequence (WPRE), 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. G4 embryonic stem cells previously targeted with FRT3::AttB::PGK-neoR-polyA::FRT5::splice acceptor::3'hygro cassette::SV40 polyA:AttP in Igs7, were transfected with the replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME).