STOCK Igs7^{tm1(tetO-tdTomato,-Cdkn1a)Rdiez}/AljJ

Stock number: 034777
Product name: Dragon-p21
Research areas: Cell Biology Research, Research Tools

Description

In Dragon-p21 mice, conditional expression of the cyclin-dependent kinase inhibitor 1A (P21) (Cdkn1a) gene is under the control of a tetracycline response element (TRE; tetO) and floxed tdTomato STOP cassette integrated in the Igs7 (TIGRE) locus. Expression of p21 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 are useful for inducible and reversible inhibition of cell cycle progression in specific tissue and at specific times.

Detailed description

Dragon-p21 is a cre recombinase- and tetracycline-controlled transactivator (tTA)-dependent mouse line that conditionally expresses cyclin-dependent kinase inhibitor 1A (P21) (Cdkn1a). Cdkn1a encodes P21, a cyclin-dependent protein kinase (CDK) inhibitor that binds to cyclin-CDK complexes, as well as free CDKs, to inhibit cell cycle progression. This inhibition occurs during cell differentiation and in response to DNA damage and oxidative stress. The Dragon (Doxycycline-controlled and Recombinase Activated Gene OverexpressioN) system requires the intersection of two drivers, Cre and (r)tTA, to achieve p21 expression. Due to its dependence on (r)tTA, the Dragon system is inducible and reversible, allowing the effect of different times and durations of misexpression of p21 to be tested.

For example, when bred to Pitx2-cre mice, expressing cre recombinase asymmetrically in the left lateral plate mesoderm, and also bred to a cartilage-specific Col2a1-rtTA line, containing the reverse tetracycline transactivator under the control of a type II collagen promoter, dox-dependent ectopic p21 expression in resulting Pit-Col-p21 offspring is achieved specifically in non-hypertrophic chondrocytes of the left limb cartilage elements. Due to the asymmetrical expression of cre, cartilage-exclusive expression of tdTomato is seen mainly in right skeletal elements, and p21 expression preferentially in the left limb cartilage. This drives p21 expression in the cartilage region responsible for growth of the long bones, targeting the left limb exclusively and leaving the right limb as an internal control. p21 misexpression during the last gestational week does not lead to left-right limb asymmetry in part due to compensatory proliferation of spared cells within the left cartilage. Moreover, mild growth reduction affects the whole body, contributing to maintenance of limb/body proportions.

Other Dragon line are available:
Dragon-DTA (Stock No. 034778) conditionally expresses a tox176 attenuated version of diphtheria toxin subunit alpha gene (DTA*G128D) and is useful for cell ablation with tissue specificity and temporal control.
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.

Of note, Cdkn1a^SUPER transgenic mice carry a mouse Cdkn1a gene fused to a tdTomato reporter downstream of a floxed-STOP cassette. This strain expresses Cdkn1a with the tdTomato reporter only after cre-mediated excision of the STOP cassette in resulting offspring.

Development

The Dragon-p21 vector 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), cyclin-dependent kinase inhibitor 1A (P21) (Cdkn1a) open reading frame, 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 bred with B6N-Tyr^c-Brd/BrdCrCrl females. Subsequently these mice were backcrossed to Swiss Webster outbred mice 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 the following years 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^{tm1(tetO-tdTomato,-Cdkn1a)Rdiez}
Name: targeted mutation 1, Alberto Rosello Diez
MGI accession ID: MGI:6389245
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible; Inserted expressed sequence
Synonyms: Dragon-p21; Igs7^TRE-LtSL-p21
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 is 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), cyclin-dependent kinase inhibitor 1A (P21) (Cdkn1a) open reading frame, 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).