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Transgenic Mice Expressing a Ligand-Inducible Cre Recombinase in Osteoblasts and Odontoblasts

2004; Elsevier BV; Volume: 165; Issue: 6 Linguagem: Inglês

10.1016/s0002-9440(10)63240-3

1525-2191

Jung‐Eun Kim, Kazuhisa Nakashima, Benoît De Crombrugghe,

Bone and Dental Protein Studies

The skeleton supports body structures in vertebrates and helps maintain calcium homeostasis throughout life. Disruption of genes involved in mammalian bone formation has often led to embryonic lethality, hence preventing study of these genes' role in adult animals. To develop a usable tool for such study, we generated transgenic mice in which a 2.3-kb mouse Col1a1 proximal promoter, which is active in all osteoblasts, drives a transgene coding for a polypeptide consisting of Cre recombinase fused to a mutated ligand-binding domain of the estrogen receptor. In this Col1a1-CreERT2 mouse line, expression patterns of the transgene and of the resulting Cre-mediated DNA recombination are analyzed by crossing with ROSA26 reporter mice and by measurement of β-galactosidase activity and X-gal staining. Exposure to 4-hydroxytamoxifen induced Cre-mediated recombination in osteoblasts in virtually all bones and in odontoblasts in teeth of both embryos and postnatal mice. The generation of these transgenic mice provides a new and important tool with which to study the function of specific genes in bone and tooth physiology and diseases in intact animals after birth. The skeleton supports body structures in vertebrates and helps maintain calcium homeostasis throughout life. Disruption of genes involved in mammalian bone formation has often led to embryonic lethality, hence preventing study of these genes' role in adult animals. To develop a usable tool for such study, we generated transgenic mice in which a 2.3-kb mouse Col1a1 proximal promoter, which is active in all osteoblasts, drives a transgene coding for a polypeptide consisting of Cre recombinase fused to a mutated ligand-binding domain of the estrogen receptor. In this Col1a1-CreERT2 mouse line, expression patterns of the transgene and of the resulting Cre-mediated DNA recombination are analyzed by crossing with ROSA26 reporter mice and by measurement of β-galactosidase activity and X-gal staining. Exposure to 4-hydroxytamoxifen induced Cre-mediated recombination in osteoblasts in virtually all bones and in odontoblasts in teeth of both embryos and postnatal mice. The generation of these transgenic mice provides a new and important tool with which to study the function of specific genes in bone and tooth physiology and diseases in intact animals after birth. Composed of cartilage and bone, the vertebrate skeleton not only supports body structures but also helps maintain calcium homeostasis throughout life. Manipulating mammalian genomes by disrupting them by homologous recombination1Aszodi A Bateman JF Gustafsson E Boot-Handford R Fassler R Mammalian skeletogenesis and extracellular matrix: what can we learn from knockout mice?.Cell Struct Funct. 2000; 25: 71-82Crossref Scopus (77) Google Scholar, 2Mundlos S Olsen BR Heritable diseases of the skeleton. Part I: molecular insights into skeletal development-transcription factors and signaling pathways.FASEB J. 1997; 11: 125-132Crossref PubMed Scopus (158) Google Scholar, 3Mundlos S Olsen BR Heritable diseases of the skeleton. Part II: molecular insights into skeletal development-matrix components and their homeostasis.FASEB J. 1997; 11: 227-233Crossref PubMed Scopus (126) Google Scholar, 4Capecchi MR The new mouse genetics: altering the genome by gene targeting.Trends Genet. 1989; 5: 70-76Abstract Full Text PDF PubMed Scopus (455) Google Scholar in embryonic stem cells has provided insights into gene functions in skeletal development and bone-related diseases. However, many questions related to the structure of genetic pathways that regulate cellular differentiation and function remain to be clarified. In particular, dissection of genetic pathways that control embryonic skeletal development is often hampered by the disruption of some critical genes that function in early embryonic decisions, thereby leading to embryonic or perinatal lethality and preventing study of the role of these genes in postnatal bone development and physiology. To overcome this difficulty, gene-targeting methods using site- and time-specific recombination based on the Cre/loxP system have been used to delete particular genes in specific tissues and stages of development.5Rossant J McMahon A “Cre”-ating mouse mutants—a meeting review on conditional mouse genetics.Genes Dev. 1999; 13: 142-145Crossref PubMed Scopus (95) Google Scholar, 6Rajewsky K Gu H Kuhn R Betz UA Muller W Roes J Schwenk F Conditional gene targeting.J Clin Invest. 1996; 98: 600-603Crossref PubMed Scopus (251) Google Scholar, 7Le Y Sauer B Conditional gene knockout using Cre recombinase.Methods Mol Biol. 2000; 136: 477-485PubMed Google Scholar Thus, the generation and characterization of transgenic mice expressing Cre recombinase under the control of a tissue- and stage-specific promoter is a prerequisite to studying skeletogenesis. Type I collagen, a major constituent of many mammalian tissues,8Rossert J de Crombrugghe B Type I collagen: structures, synthesis, and regulation.in: Bilezikian J Raisz L Rodan G Principles of Bone Biology. Academic Press, San Diego1996: 127-142Google Scholar is synthesized by osteoblasts and odontoblasts and by various types of fibroblasts and mesenchymal cells. Previous reports identified an osteoblast-specific enhancer element in the mouse Col1a1 gene.9Ducy P Karsenty G Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene.Mol Cell Biol. 1995; 15: 1858-1869Crossref PubMed Scopus (529) Google Scholar, 10Rossert J Eberspaecher H de Crombrugghe B Separate cis-acting DNA elements of the mouse pro-alpha-1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice.J Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (214) Google Scholar Indeed, transgenic mice harboring a 2.3-kb proximal fragment of mouse or rat Col1a1 promoter showed high activity of the transgene in bone-forming cells, osteoblasts, and in the dentin-forming cells, odontoblasts in teeth, very low activity in tendons, and no activity in other tissues.10Rossert J Eberspaecher H de Crombrugghe B Separate cis-acting DNA elements of the mouse pro-alpha-1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice.J Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (214) Google Scholar, 11Dacquin R Starbuck M Schinke T Karsenty G Mouse alpha-1(I)-collagen promoter is the best known promoter to drive efficient Cre recombinase expression in osteoblast.Dev Dyn. 2002; 224: 245-251Crossref PubMed Scopus (267) Google Scholar, 12Braut A Kalajzic I Kalajzic Z Rowe DW Kollar EJ Mina M Col1a1-GFP transgene expression in developing incisors.Connect Tissue Res. 2002; 43: 216-219Crossref PubMed Scopus (28) Google Scholar, 13Rossert JA Chen SS Eberspaecher H Smith CN de Crombrugghe B Identification of a minimal sequence of the mouse pro-alpha-1(I) collagen promoter that confers high-level osteoblast expression in transgenic mice and that binds a protein selectively present in osteoblasts.Proc Natl Acad Sci USA. 1996; 93: 1027-1031Crossref PubMed Scopus (95) Google Scholar, 14Marijanovic I Jiang X Kronenberg MS Stover ML Erceg I Lichtler AC Rowe DW Dual reporter transgene driven by 2.3Col1a1 promoter is active in differentiated osteoblasts.Croat Med J. 2003; 44: 412-417PubMed Google Scholar In the present study, we generated transgenic mouse lines in which a 2.3-kb Col1a1 osteoblast- and odontoblast-specific promoter drives a transgene coding for a polypeptide consisting of Cre recombinase fused to a mutated ligand-binding domain (LBD) of the estrogen receptor (ER). This CreER fusion polypeptide becomes active only after administration of the synthetic estrogen antagonist 4-hydroxytamoxifen (4-OHT). The Cre recombinase is inactive in the absence of 4-OHT. To generate these mice, we have used the CreERT2 recombinase, which contains a G400V/M543A/L544A triple mutation in the ER LBD and is more sensitive to 4-OHT than is the mutant ER LBD with a single G521R substitution.15Feil R Wagner J Metzger D Chambon P Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains.Biochem Biophys Res Commun. 1997; 237: 752-757Crossref PubMed Scopus (749) Google Scholar, 16Indra AK Warot X Brocard J Bornert JM Xiao JH Chambon P Metzger D Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases.Nucleic Acids Res. 1999; 27: 4324-4327Crossref PubMed Scopus (588) Google Scholar These transgenic mice also harbor the ubiquitously active ROSA26 locus, in which the LacZ gene is preceded by a transcriptional stop cassette flanked by loxP sites. By measuring β-galactosidase (β-gal) activity and assessing X-gal staining, we found that in this transgenic mouse line, referred to as Col1a1-CreERT2, recombination of loxP sites could be induced by 4-OHT in both osteoblasts and odontoblasts, and that osteoblast- and odontoblast-specific gene deletion could be achieved both during embryonic development and after birth. Use of these mice provides a novel experimental approach to studying the role of specific genes in the physiology and disease of bone and tooth in intact animals after birth. pGS-CreERT2 plasmid DNA15Feil R Wagner J Metzger D Chambon P Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains.Biochem Biophys Res Commun. 1997; 237: 752-757Crossref PubMed Scopus (749) Google Scholar containing CreERT2 cDNA and pJ2300LacZ plasmid DNA10Rossert J Eberspaecher H de Crombrugghe B Separate cis-acting DNA elements of the mouse pro-alpha-1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice.J Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (214) Google Scholar containing a 2.3-kb promoter fragment of the gene for the α1 chain of mouse type I collagen (Col1a1) were used to construct the Col1a1-CreERT2 transgene. A 2.9-kb AvrII/SalI fragment with CreERT2 cDNA from pGS-CreERT2 and a 2.3-kb NotI/XbaI Col1a1 promoter fragment from pJ2300LacZ were subcloned into the NotI/SalI site of the pBluscript II KS(+) vector (Stratagene, La Jolla, CA). To remove vector sequences for pronuclear injection, a 5.2-kb fragment of the Col1a1-CreERT2 transgene was digested with NotI and SalI. The Col1a1-CreERT2 fragment excised from the vector backbone was microinjected into the pronuclei of fertilized B6D2 F1 oocytes. Founder mice were genotyped using tail genomic DNA by polymerase chain reaction (PCR) with the Cre-specific primers, 5′-ATCCGAAAAGAAAACGTTGA-3′ and 5′-ATCCAGGTTACGGATATAGT-3′. The size of the amplified product was ∼700 bp for the Col1a1-CreERT2. Total RNA from limbs of embryos at 13.5 days post coitum (dpc), 16.5 dpc, and 18.5 dpc, and from limbs of 3-day-old pups was prepared using TRIzol reagent (Invitrogen Inc., Carlsbad, CA) according to the manufacturer's instructions. Briefly, each limb was homogenized in 1 ml of TRIzol reagent and incubated for 5 minutes at room temperature. After adding 0.2 ml of chloroform, the mixtures were separated by centrifugation into a phenol-chloroform phase, an interphase, and an upper aqueous phase. RNA was precipitated from the aqueous phase by mixing it with 0.5 ml of isopropanol. For RT-PCR analysis, cDNA was synthesized from 1 μg of total RNA by using the First Strand cDNA Synthesis kit for RT-PCR (Roche Applied Science, Indianapolis, IN). PCR was performed according to standard procedure by using the Cre-specific primers. Control PCR was performed with the mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers, 5′-TGAAGGTCGGTGTGAACGGATTTGGC-3′ and 5′-CATGTAGGCCATGAGGTCCACCAC-3′. After crossing Col1a1-CreERT2 male transgenic mice with female ROSA26 reporter (R26R) mice,17Soriano P Generalized lacZ expression with the ROSA26 Cre reporter strain.Nat Genet. 1999; 21: 70-71Crossref PubMed Scopus (4197) Google Scholar offspring were genotyped from the yolk sac or pup tail genomic DNA by PCR using Cre-specific and LacZ-specific primers for CreERT2 and R26R alleles, respectively. LacZ PCR was performed with the LacZ-specific primers, 5′-GCATCGAGCTGGGTAATAAGGGTTGGCAAT-3′ and 5′-GACACCAGACCAACT-GGTAATGGTAGCGAC-3′. 4-OHT (Sigma-Aldrich, St. Louis, MO) used for testing the activity and ligand dependency of the Col1a1-CreERT2 transgene was prepared as suspensions of 2.5, 5.0, 7.5, or 10.0 mg/ml 4-OHT in autoclaved sunflower oil (Sigma-Aldrich). For analysis of embryonic activity of Cre recombinase, pregnant female mice were injected intraperitoneally with 1 mg of 4-OHT for 3 consecutive days starting at 12.5, 13.5, or 14.5 dpc. Injected mice were sacrificed 48 hours after the final injection, and the embryos were processed for whole-mount X-gal (5-bromo-4-chloro-3-indoyl β-d-galactopyranoside; Brinkmann, Westbury, NY) staining. Control embryos whose dams were treated with sunflower oil instead of 4-OHT were used for comparison to confirm the activity and ligand dependency of Cre recombinase. To analyze the postnatal activity of the conditional Cre recombinase, pups were injected intraperitoneally with 0.25, 0.5, 0.75, or 1.0 mg of 4-OHT for 5 consecutive days starting when they were 12 days old. Forty-eight hours after the final injection, mice were killed and various tissue samples were processed for measurement of β-galactosidase (β-gal) activity and X-gal staining. Tissue β-gal activity was assessed in 18-day-old pups by using the Galacto-Light Plus systems (Applied Biosystems, Bedford, MA). Dissected tissues from tail, skin, limb, liver, and calvaria were homogenized in 1 ml of extraction buffer (0.1 mol/L potassium phosphate, 1 mol/L dithiothreitol, and 10% Triton X-100). After centrifuging the samples at 13,000 rpm for 10 minutes, 10-μl extracts of each sample were used for the assay, following the instructions of the manufacturer. Protein concentrations were determined with the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). β-Gal activity was normalized to the protein concentration to determine the number of relative light units per microgram of protein. X-Gal staining in whole-mount embryos at various stages of development or in various tissue samples from postnatal mice was performed as described previously.10Rossert J Eberspaecher H de Crombrugghe B Separate cis-acting DNA elements of the mouse pro-alpha-1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice.J Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (214) Google Scholar Briefly, whole embryos or dissected tissue fragments from pups were fixed for 45 to 60 minutes in 0.1 mol/L phosphate buffer (pH 7.5) with 5 mmol/L EGTA (pH 8.0), 2 mmol/L MgCl2, 0.2% glutaraldehyde, and 0.8% formaldehyde, and rinsed three times for 30 minutes each with 0.1 mol/L phosphate buffer (pH 7.3) containing 2 mmol/L MgCl2, 0.2% Nonidet P-40, and 0.1% sodium deoxycholate. Samples were then stained with the same rinsing solution supplemented with 1 mg/ml of X-gal, 5 mmol/L potassium ferrocyanide, and 5 mmol/L potassium ferricyanide. For embryos older than 16.5 dpc or postnatal pups, skin was removed for fixation and stained. After staining, the embryos or pup tissues were washed in 1× phosphate-buffered saline (PBS, pH 7.8) with 10 mmol/L EDTA (pH 8.0) and were photographed. For histological analysis, X-gal stained samples were fixed again in 4% formaldehyde/1× PBS, pH 7.8, at 4°C overnight and bone samples were decalcified in 0.25 mol/L EDTA/0.1 mol/L NaPB, pH 6.5, for 2 to 3 weeks. Then, all embryo and pup samples were dehydrated, embedded in paraffin, and sections of 12 μm were cut and counterstained with nuclear fast red. Founders of two types of transgenic mice were successfully generated. In the first type, the transgene consisted of the 2.3-kb mouse Col1a1 promoter linked to CreER DNA with a G521R mutation in the LBD of the ER. The second type harbored the CreERT2 transgene (Figure 1A), which consisted of the same promoter linked to CreER with a triple mutation G400V, M543A, and L544A in the ER LBD. Although in both types of mice recombinase activity was specifically induced in osteoblasts and odontoblasts by 4-OHT, this study focuses on the characterization of transgenic mice harboring the CreERT2 transgene because the CreERT2 recombinase achieves higher activity with 4-OHT than the CreERT recombinase. As assessed using RT-PCR, Cre mRNA expression in 13.5, 16.5, and 18.5-dpc embryos, and in 3-day-old pups was consistent with activation of the Col1a1 promoter at the time of osteoblasts differentiation and bone formation (Figure 1B). To determine the ability of Cre to induce recombination specifically in bone, male Col1a1-CreERT2 transgenic mice were crossed with female mice of the ROSA26 reporter strain (R26R).17Soriano P Generalized lacZ expression with the ROSA26 Cre reporter strain.Nat Genet. 1999; 21: 70-71Crossref PubMed Scopus (4197) Google Scholar In latter strain, the LacZ gene, which is inserted in the ubiquitously expressed ROSA locus, is preceded by a transcriptional stop cassette flanked by loxP sites. Thus, in Col1a1-CreERT2;R26R double-transgenic mice, β-galactosidase (β-gal) should be expressed in bones when Cre recombinase is activated by administration of 4-OHT. In 18-day-old offspring that had been injected with 4-OHT, we detected high β-gal activity in the tail, limb, and calvaria (Figure 2). The level of β-gal activity strongly depended on the concentration of 4-OHT injected and was higher after five injections than after three injections (data not shown). No β-gal activity was detected in the liver and only a low level of activity in the skin of the double-transgenic mice regardless of treatment with 4-OHT. In other control mice containing only the Col1a1-CreERT2 or only the R26R allele allele, little or no β-gal activity was detected. In some tissues, low-level endogenous galactosidase activity could be present. X-Gal staining was also examined in the same tissues that showed high levels of β-gal activity (ie, tail, limb, and calvaria). Strong X-gal staining occurred in all of the long bones of the forelimb and hindlimb (data not shown) and in the ribs, vertebrae, and calvaria (Figure 3; A1 to A4). In contrast, uninjected 18-day-old double-transgenic control pups showed weak staining, mainly at sites corresponding to endogenous galactosidase activity (Figure 3; B1 to B4). Other tissues (eg, skin, liver, and kidney), whether exposed to 4-OHT or not, revealed no X-gal staining (data not shown). To analyze patterns of Cre recombinase activity during embryonic development, we repeated the crossbreeding of male Col1a1-CreERT2 mice and female R26R mice. The pregnant mice were then given intraperitoneal injections of 1 mg of 4-OHT starting at 12.5, 13.5, or 14.5 dpc for 3 consecutive days. Injected female mice were sacrificed at 48 hours after the final injection. The pattern of X-gal staining of skeletal elements in the Col1a1-CreERT2;R26R double-transgenic embryos whose dams were treated with 4-OHT was similar at different stages of embryonic development (data not shown). X-Gal staining was very strong in all bones of these embryos, but no staining was observed in other tissues except the intestine (Figure 3C, top). Magnified images of the skull, forelimb, vertebra, and ribs, which stained strongly, indicated the presence of ligand-inducible Cre recombinase in all bones of these embryos (Figure 3; D1 to D5). In contrast, in control embryos whose dams were treated with sunflower oil instead of 4-OHT, no X-gal staining was observed in bone or any other tissues except the intestine [Figure 3, C (bottom row) and E1 to E5]. The staining of the intestine was present in all embryos regardless of 4-OHT treatment because of endogenous galactosidase activity. We then performed histological analysis of different bones of the Col1a1-CreERT2;R26R double-transgenic embryos and postnatal mice after administration of 4-OHT. For embryos whose dams had been treated with 4-OHT, coronal sections of the skull revealed strong X-gal staining in osteoblasts of the calvaria and the jaw (Figure 4, A1 and A2), and sections of long bones showed strong staining in osteoblasts but not chondrocytes (Figure 4, A3 and A4). At 18.5 dpc after 4-OHT administration, ∼61% of the osteoblasts stained with X-gal indicating gene excision at the ROSA locus by Cre recombinase. In contrast, no staining of osteoblasts was detectable in oil-treated control embryos (Figure 4B). No X-gal staining occurred in other organs such as heart, lung, liver, and kidney of embryos whose dams were treated with 4-OHT (Figure 4C). In histological analysis of bones in postnatal mice, the staining pattern was similar to that seen in the embryos. X-Gal staining was clearly seen in the long bones (Figure 4D), whereas none was visible in osteoblasts of control mice (Figure 4E) or in other organs such as skin, liver, and kidney of pups treated with 4-OHT (Figure 4F). There was also no X-gal staining in brain, eyes, intestine, bladder, blood vessels, muscle, cartilage, and hematopoietic cells (data not shown). These results indicated that Cre recombination activated in a ligand-dependent manner occurred in osteoblasts under the control of the 2.3-kb Col1a1 promoter. X-Gal-staining cells were also detected in teeth of 4-OHT-treated Col1a1-CreERT2;R26R double-transgenic embryos and pups. Odontoblasts in molars and incisors showed strong X-gal staining in 18.5 dpc Col1a1-CreERT2;R26R double-transgenic embryos whose dams had been treated with 4-OHT (Figure 5A) but not in control embryos (Figure 5B). X-Gal staining of odontoblasts also occurred in 18-day-old pups treated with 4-OHT (Figure 5C) but not in control pups (Figure 5D). Considerable progress has recently been made in understanding the role of transcription factors, signaling molecules, and extracellular matrix components in the formation of bones during embryonic development.18Nakashima K de Crombrugghe B Transcriptional mechanisms in osteoblast differentiation and bone formation.Trends Genet. 2003; 19: 458-466Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar These studies have relied in large part on the identification of genes mutated in specific human skeletal diseases19Prockop DJ Kivirikko KI Collagens: molecular biology, diseases, and potentials for therapy.Annu Rev Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1399) Google Scholar, 20McKenna MJ Kristiansen AG Bartley ML Rogus JJ Haines JL Association of COL1A1 and otosclerosis: evidence for a shared genetic etiology with mild osteogenesis imperfecta.Am J Otol. 1998; 19: 604-610PubMed Google Scholar and on the generation of mouse embryos and mice with targeted mutations in genes with major roles in bone formation.21Komori T Yagi H Nomura S Yamaguchi A Sasaki K Deguchi K Shimizu Y Bronson RT Go YH Inada M Sato M Okamoto R Kitamura Y Yoshiki S Kishimoto T Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts.Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3716) Google Scholar, 22Otto F Thornell AP Crompton T Denzel A Gilmour KC Rosewell IR Stamp GW Beddington RS Mundlos S Olsen BR Selby PB Owen MJ Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development.Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2450) Google Scholar However, our understanding of the role of these factors in the physiology and homeostasis of bones after birth and in adult animals is much less complete. Often, mice with a gene that is essential for bone formation but that has been inactivated die during embryonic development or perinatally. Thus, embryonic or perinatal lethality because of disruption of these genes has prevented our studying their role in bone physiology in adult animals. Gene-targeting technology based on the Cre/loxP system has been used as a tool to delete genes in specific tissues or at specific stages of development.5Rossant J McMahon A “Cre”-ating mouse mutants—a meeting review on conditional mouse genetics.Genes Dev. 1999; 13: 142-145Crossref PubMed Scopus (95) Google Scholar, 6Rajewsky K Gu H Kuhn R Betz UA Muller W Roes J Schwenk F Conditional gene targeting.J Clin Invest. 1996; 98: 600-603Crossref PubMed Scopus (251) Google Scholar, 7Le Y Sauer B Conditional gene knockout using Cre recombinase.Methods Mol Biol. 2000; 136: 477-485PubMed Google Scholar In this system, the bacteriophage P1 Cre recombinase excises specific DNA fragments by recognizing loxP sites.23Sternberg N Hamilton D Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites.J Mol Biol. 1981; 150: 467-486Crossref PubMed Scopus (523) Google Scholar Therefore, the generation and characterization of mouse strains expressing Cre recombinase in specific cell types have provided an essential tool for studying the function of specific genes in specific tissues.5Rossant J McMahon A “Cre”-ating mouse mutants—a meeting review on conditional mouse genetics.Genes Dev. 1999; 13: 142-145Crossref PubMed Scopus (95) Google Scholar A transcriptional enhancer previously identified in the mouse and rat Col1a1 gene directs expression of reporter genes selectively in osteoblasts and odontoblasts of transgenic mice.10Rossert J Eberspaecher H de Crombrugghe B Separate cis-acting DNA elements of the mouse pro-alpha-1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice.J Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (214) Google Scholar, 11Dacquin R Starbuck M Schinke T Karsenty G Mouse alpha-1(I)-collagen promoter is the best known promoter to drive efficient Cre recombinase expression in osteoblast.Dev Dyn. 2002; 224: 245-251Crossref PubMed Scopus (267) Google Scholar, 12Braut A Kalajzic I Kalajzic Z Rowe DW Kollar EJ Mina M Col1a1-GFP transgene expression in developing incisors.Connect Tissue Res. 2002; 43: 216-219Crossref PubMed Scopus (28) Google Scholar, 13Rossert JA Chen SS Eberspaecher H Smith CN de Crombrugghe B Identification of a minimal sequence of the mouse pro-alpha-1(I) collagen promoter that confers high-level osteoblast expression in transgenic mice and that binds a protein selectively present in osteoblasts.Proc Natl Acad Sci USA. 1996; 93: 1027-1031Crossref PubMed Scopus (95) Google Scholar This promoter has recently been used to generate mice expressing Cre recombinase in osteoblasts.11Dacquin R Starbuck M Schinke T Karsenty G Mouse alpha-1(I)-collagen promoter is the best known promoter to drive efficient Cre recombinase expression in osteoblast.Dev Dyn. 2002; 224: 245-251Crossref PubMed Scopus (267) Google Scholar Because this promoter is activated during embryonic development when osteoblast differentiation starts, inactivation of specific genes will occur during embryonic development and eventually affect bone formation and development.11Dacquin R Starbuck M Schinke T Karsenty G Mouse alpha-1(I)-collagen promoter is the best known promoter to drive efficient Cre recombinase expression in osteoblast.Dev Dyn. 2002; 224: 245-251Crossref PubMed Scopus (267) Google Scholar In this study, to examine the function of specific genes on bone and tooth physiology and homeostasis after birth, we have generated transgenic mice that express an inducible Cre recombinase (CreERT2) in osteoblasts and odontoblasts using the same 2.3-kb mouse Col1a1 promoter. Our results showed that the Cre-ERT2 gene was expressed specifically in osteoblasts and odontoblasts and that the Cre recombinase was active only after systemic administration of 4-OHT. On the basis of our results with the R26R reporter mouse strain, we estimate Cre-ERT2 gene was active in most osteoblasts and odontoblasts of 18.5 dpc embryos. This appears also to be the case 3 weeks after birth although the penetration of X-gal into bone and tooth tissues may be less complete at that time. Furthermore, our results are consistent with a previous report that the 2.3-kb fragment of Col1a1 promoter directs strong expression of reporter genes in osteoblasts and odontoblasts.10Rossert J Eberspaecher H de Crombrugghe B Separate cis-acting DNA elements of the mouse pro-alpha-1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice.J Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (214) Google Scholar The mouse strain we generated will provide opportunities to examine the consequences of the ablation of a number of specific genes after birth. For instance, null mutations in Runx221Komori T Yagi H Nomura S Yamaguchi A Sasaki K Deguchi K Shimizu Y Bronson RT Go YH Inada M Sato M Okamoto R Kitamura Y Yoshiki S Kishimoto T Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts.Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3716) Google Scholar, 22Otto F Thornell AP Crompton T Denzel A Gilmour KC Rosewell IR Stamp GW Beddington RS Mundlos S Olsen BR Selby PB Owen MJ Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development.Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2450) Google Scholar or Osterix,24Nakashima K Zhou X Kunkel G Zhang Z Deng JM Behringer RR de Crombrugghe B The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation.Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2896) Google Scholar which are transcription factors required for osteoblast differentiation, cause lethality in the immediate perinatal period. When conditional alleles for the genes of these proteins become available, their postnatal inactivation should generate essential information about their role after embryonic development is completed and should provide a proof of principle about their function during steady-state bone remodeling. Similar experiments with genes for components of signaling pathways (eg, the Wnt/β-catenin, the bone morphogenetic protein, and the TGF-β pathway) known to affect bone formation will also be possible. The same approach might also be used with genes for bone extracellular matrix components. Furthermore, this approach may be important to understand better the chronic diseases of bone (eg, osteoporosis) that characteristically occur late in life. Thus, we believe that the Col1a1-CreERT2 transgenic mice we have generated will become an important tool with which to study bone and tooth physiology and diseases. We thank Mr. Chad Smith and Dr. Jan Parker-Thornburg of the University of Texas M.D. Anderson's Genetically Engineered Mouse Facility for performing the pronuclear injections; and Dr. John Clifford of M.D. Anderson Cancer Center for providing plasmid pSG-CreERT2.