Generation of a transgenic mouse model with chondrocyte-specific and tamoxifen-inducible expression of Cre recombinase.
ABSTRACT: Postnatal cartilage development and growth are regulated by key growth factors and signaling molecules. To fully understand the function of these regulators, an inducible and chondrocyte-specific gene deletion system needs to be established to circumvent the perinatal lethality. In this report, we have generated a transgenic mouse model (Col2a1-CreER(T2)) in which expression of the Cre recombinase is driven by the chondrocyte-specific col2a1 promoter in a tamoxifen-inducible manner. To determine the specificity and efficiency of the Cre recombination, we have bred Col2a1-CreER(T2) mice with Rosa26R reporter mice. The X-Gal staining showed that the Cre recombination is specifically achieved in cartilage tissues with tamoxifen-induction. In vitro experiments of chondrocyte cell culture also demonstrate the 4-hydroxy tamoxifen-induced Cre recombination. These results demonstrate that Col2a1-CreER(T2) transgenic mice can be used as a valuable tool for an inducible and chondrocyte-specific gene deletion approach.
Project description:We generated transgenic mice in which human CDX2 gene elements control expression of a tamoxifen-regulated Cre protein (CDX2P-CreER(T2)) to allow for inducible gene targeting in intestinal epithelium. After tamoxifen dosing of CDX2P-CreER(T2) mice, Cre activity was detected in the distal ileal, cecal, colonic, and rectal epithelium, with selected crypt base, transit amplifying, and surface cells all capable of activating Cre function. Four weeks after tamoxifen dosing of CDX2P-CreER(T2) mice carrying a Cre-activated fluorescent reporter, single crypts were uniformly fluorescence positive or negative, reflecting Cre activation in crypt stem cells. Biallelic inactivation of the Apc tumor suppressor gene via the CDX2P-CreER(T2) transgene in colon epithelium led to acute alterations in cell proliferation, apoptosis, and morphology, along with mitotic spindle misorientation, ?-catenin nuclear localization, and induction of the intestinal stem cell markers Lgr5 and Musashi-1 and the Sox9 transcription factor. Normal mouse colon epithelium lacks Paneth cells, a key small intestine niche cell type, and Paneth cell differentiation is dependent on Sox9 function. In Apc-deficient colon epithelium, ectopic Paneth-like cells were seen outside the crypt base, such as new crypt budding sites. Our data indicate Apc inactivation via CDX2P-CreER(T2) targeting in mouse colon epithelium is sufficient to induce adenomatous changes and the generation of Paneth-like cells from neoplastic progenitors, with potentially significant roles in colon adenoma development and progression.
Project description:BACKGROUND: The inducible Cre-lox system is a valuable tool to study gene function in a spatial and time restricted fashion in mouse models. This strategy relies on the limited background activity of the modified Cre recombinase (CreER) in the absence of its inducer, the competitive estrogen receptor ligand, tamoxifen. The RIP-CreER mouse (Tg (Ins2-cre/Esr1) 1Dam) is among the few available ?-cell specific CreER mouse lines and thus it has been often used to manipulate gene expression in the insulin-producing cells of the endocrine pancreas. PRINCIPAL FINDINGS: Here, we report the detection of tamoxifen-independent Cre activity as early as 2 months of age in RIP-CreER mice crossed with three distinct reporter strains. SIGNIFICANCE: Evidence of Cre-mediated recombination of floxed alleles even in the absence of tamoxifen administration should warrant cautious use of this mouse for the study of pancreatic ?-cells.
Project description:Pericytes are essential mural cells distinguished by their association with small caliber blood vessels and the presence of a basement membrane shared with endothelial cells. Pericyte interaction with the endothelium plays an important role in angiogenesis; however, very few tools are currently available that allow for the targeting of pericytes in mouse models, limiting our ability to understand their biology. We have generated a novel mouse line expressing tamoxifen-inducible Cre-recombinase under the control of the platelet-derived growth factor receptor ? promoter: PDGFR?-P2A-CreER T2 . We evaluated the expression of the PDGFR?-P2A-CreER T2 line by crossing it with fluorescent reporter lines and analyzed reporter signal in the angiogenic retina and brain at different time points after tamoxifen administration. Reporter lines showed labeling of NG2+, desmin+, PDGFR?+ perivascular cells in the retina and the brain, indicating successful targeting of pericytes; however, signal from reporter lines was also observed in a small subset of glial cells both in the retina and the brain. We also evaluated recombination in tumors and found efficient recombination in perivascular cells associated with tumor vasculature. As a proof of principle, we used our newly generated driver to delete Notch signaling in perivascular cells and observed a loss of smooth muscle cells in retinal arteries, consistent with previously published studies evaluating Notch3 null mice. We conclude that the PDGFR?-P2A-CreER T2 line is a powerful new tool to target pericytes and will aid the field in gaining a deeper understanding of the role of these cells in physiological and pathological settings.
Project description:Recent studies indicate that supporting cells play important roles in inner ear development, function, and regeneration after injury, but the molecular mechanisms underlying these processes remain poorly understood. Inducible cell-specific gene recombination in supporting cells could be a powerful tool to study the roles of specific molecules in these cells. Here we tested the feasibility, effectiveness, and cell specificity of inducible Cre-mediated gene recombination in the postnatal inner ear using mice that express an inducible form of Cre (CreER(T)) under the transcriptional control of the proteolipid protein (PLP) promoter. We assessed the pattern of tamoxifen-induced gene recombination in the inner ear using the ROSA26-LacZ reporter line, in which the beta-galactosidase gene is expressed only after Cre-mediated excision of a loxP-flanked stop cassette. Recombination was detected in cochlear inner phalangeal cells, supporting cells surrounding hair cells in vestibular maculae and cristae. Recombination also occurred in Schwann cells. We also found that this CreER(T) line can be used to increase and decrease the levels of expression of a trophic factor, brain-derived neurotrophic factor, specifically in supporting cells. These results show that PLP/CreER(T) mice are a powerful tool to dissect gene function in inner ear supporting cells.
Project description:Previous observations implicate Indian hedgehog (Ihh) signaling in osteoarthritis (OA) development because it regulates chondrocyte hypertrophy and matrix metallopeptidase 13 (MMP-13) expression. However, there is no direct genetic evidence for the role of Ihh in OA, because mice with cartilage or other tissue-specific deletion of the Ihh gene die shortly after birth. We evaluated the role of Ihh in vivo via a Cre-loxP-mediated approach to circumvent the early death caused by Ihh deficiency.To evaluate the role of Ihh in OA development, Ihh was specifically deleted in murine cartilage using an Ihh conditional deletion construct (Col2a1-CreER(T2); Ihh(fl/fl)). The extent of cartilage degradation and OA progression after Ihh deletion was assessed by histological analysis, immunohistochemistry, real-time PCR and in vivo fluorescence molecular tomography (FMT) 2 months after OA was induced by partial medial meniscectomy. The effect of Ihh signaling on cartilage was compared between Ihh-deleted mice and their control littermates.Only mild OA changes were observed in Ihh-deleted mice, while control mice displayed significantly more cartilage damage. Typical OA markers such as type X collagen and MMP-13 were decreased in Ihh-deleted mice. In vivo FMT demonstrated decreased cathepsins and MMP activity in knee joints of animals with deletion of Ihh.These findings support the protective role of Ihh deletion in surgically induced OA. Thus, our findings suggest the potential to develop new therapeutic strategies that can prevent and treat OA by inhibiting Ihh signaling in chondrocytes.
Project description:To establish a genetic tool for manipulating the neural stem/progenitor cell (NSC) lineage in a temporally controlled manner, we generated a transgenic mouse line carrying an NSC-specific nestin promoter/enhancer expressing a fusion protein encoding Cre recombinase coupled to modified estrogen receptor ligand-binding domain (ER(T2)). In the background of the Cre reporter mouse strain Rosa26(lacZ), we show that the fusion CreER(T2) recombinase is normally silent but can be activated by the estrogen analog tamoxifen both in utero, in infancy, and in adulthood. As assayed by beta-galactosidase activity in embryonic stages, tamoxifen activates Cre recombinase exclusively in neurogenic cells and their progeny. This property persists in adult mice, but Cre activity can also be detected in granule neurons and Bergmann glia at the anterior of the cerebellum, in piriform cortex, optic nerve, and some peripheral ganglia. No obvious Cre activity was observed outside of the nervous system. Thus, the nestin regulated inducible Cre mouse line provides a powerful tool for studying the physiology and lineage of NSCs.
Project description:Conditional gene targeting has been extensively used for in vivo analysis of gene function in adipocyte cell biology but often with debate over the tissue specificity and the efficacy of inactivation. To directly compare the specificity and efficacy of different Cre lines in mediating adipocyte specific recombination, transgenic Cre lines driven by the adipocyte protein 2 (aP2) and adiponectin (Adipoq) gene promoters, as well as a tamoxifen-inducible Cre driven by the aP2 gene promoter (iaP2), were bred to the Rosa26R (R26R) reporter. All three Cre lines demonstrated recombination in the brown and white fat pads. Using different floxed loci, the individual Cre lines displayed a range of efficacy to Cre-mediated recombination that ranged from no observable recombination to complete recombination within the fat. The Adipoq-Cre exhibited no observable recombination in any other tissues examined, whereas both aP2-Cre lines resulted in recombination in endothelial cells of the heart and nonendothelial, nonmyocyte cells in the skeletal muscle. In addition, the aP2-Cre line can lead to germline recombination of floxed alleles in ~2% of spermatozoa. Thus, different "adipocyte-specific" Cre lines display different degrees of efficiency and specificity, illustrating important differences that must be taken into account in their use for studying adipose biology.
Project description:To generate new mouse lines that facilitate inducible gene activation in the murine trabecular meshwork in vivo.Two expression cassettes were knocked into the 3'-UTR of the Myocilin (Myoc) locus, an abundantly expressed extracellular matrix protein produced by cells of the trabecular meshwork. The first cassette directs expression of an inducible form of Cre recombinase, CreER(T2), which is activated by tamoxifen administration under the control of endogenous Myoc regulatory elements. The second cassette contains a reverse tetracycline transactivator, rtTA(M2), which directs the expression of tetracycline-operator transgenes on exposure of animals to doxycycline (Dox). These lines were crossed to GFP and lacZ reporter mice to assay for tamoxifen or Dox-induced transgene expression.Both the Myoc-CreER(T2) and the Myoc-rtTA(M2) lines were capable of directing efficient and inducible expression of transgenes in the murine trabecular meshwork in vivo. In addition, activation of transgenes by Myoc-rtTA(M2) was reversible with loss of transgene expression after Dox withdrawal. Examination of multiple tissues demonstrates efficient transgene activation in the trabecular meshwork, with additional sites of transgene activation including cells in the retina, sclera, lung, kidney, and abundant activation in the neocortex and hippocampus.Two new mouse lines have been generated that allow for efficient and inducible transgene activation in the murine trabecular meshwork in vivo.
Project description:Tamoxifen (Tm)-inducible Cre recombinases are widely used to perform gene inactivation and lineage tracing studies in mice. Although the efficiency of inducible Cre-loxP recombination can be easily evaluated with reporter strains, the precise length of time that Tm induces nuclear translocation of CreER(Tm) and subsequent recombination of a target allele is not well defined, and difficult to assess. To better understand the timeline of Tm activity in vivo, we developed a bioassay in which pancreatic islets with a Tm-inducible reporter (from Pdx1(PB)-CreER(Tm);R26R(lacZ) mice) were transplanted beneath the renal capsule of adult mice previously treated with three doses of 1 mg Tm, 8 mg Tm, or corn oil vehicle. Surprisingly, recombination in islet grafts, as assessed by expression of the ?-galactosidase (?-gal) reporter, was observed days or weeks after Tm treatment, in a dose-dependent manner. Substantial recombination occurred in islet grafts long after administration of 3×8 mg Tm: in grafts transplanted 48 hours after the last Tm injection, 77.9±0.4% of ?-cells were ?-gal+; in ?-cells placed after 1 week, 46.2±5.0% were ?-gal+; after 2 weeks, 26.3±7.0% were ?-gal+; and after 4 weeks, 1.9±0.9% were ?-gal+. Islet grafts from mice given 3×1 mg Tm showed lower, but notable, recombination 48 hours (4.9±1.7%) and 1 week (4.5±1.9%) after Tm administration. These results show that Tm doses commonly used to induce Cre-loxP recombination may continue to label significant numbers of cells for weeks after Tm treatment, possibly confounding the interpretation of time-sensitive studies using Tm-dependent models. Therefore, investigators developing experimental approaches using Tm-inducible systems should consider both maximal recombination efficiency and the length of time that Tm-induced Cre-loxP recombination occurs.