Expression and activity of osteoblast-targeted Cre recombinase transgenes in murine skeletal tissues

The Cre/loxP recombination system can be used to circumvent many of the limitations of generalized gene ablation in mice. Here we present the development and characterization of transgenic mice in which Cre recombinase has been targeted to cells of the osteoblast lineage with 2.3 kb (Col 2.3-Cre) and 3.6 kb (Col 3.6-Cre) fragments of the rat Col1a1 promoter. Cre mRNA was detected in calvaria and long bone of adult Col 2.3-Cre and Col 3.6-Cre mice, as well as in tendon and skin of Col 3.6-Cre mice. To obtain a historical marking of the temporal and spatial pattern of Cre-mediated gene rearrangement, Col-Cre mice were bred with ROSA26 (R26R) mice in which Cre-mediated excision of a floxed cassette results in LacZ expression. In Col 2.3-Cre;R26R and Col 3.6-Cre;R26R progeny, calvarial and long bone osteoblasts showed intense beta-gal staining at embryonic day 18 and postnatal day 5. The spatial pattern of beta-gal staining was more restricted in bone and in bone marrow stromal cultures established from Col 2.3-Cre;R26R mice. Similar differences in the spatial patterns of expression were seen in transgenic bone carrying Col1a1-GFP visual reporters. Our data suggest that Col 2.3-Cre and Col 3.6-Cre transgenic mice may be useful for conditional gene targeting in vivo or for obtaining osteoblast populations for in vitro culture in which a gene of interest has been inactivated.     

Collagen1alpha1 promoter drives the expression of Cre recombinase in osteoblasts of transgenic mice

Osteoblasts participate in bone formation, bone mineralization, osteoclast differentiation and many pathological processes. To study the function of genes in osteoblasts using Cre-LoxP system, we generated a mouse line expressing the Cre recombinase under the control of the rat Collagen1alpha1 (Col1alpha1) promoter (Col1alpha1-Cre). Two founders were identified by genomic PCR from 16 offsprings, and the integration efficiency is 12.5%. In order to determine the tissue distribution and the activity of Cre recombinase in the transgenic mice, the Col1alpha1-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4(Co/Co)). Multiple tissue PCR of Col1alpha1-Cre;Smad4(Co/+)mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon. LacZ staining in the Col1alpha1-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5. Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice. All these data indicated that the Col1alpha1-Cre transgenic mice could serve as a valuable tool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.     

Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo

Endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme. Here, we created Tie2-Cre transgenic mice, in which expression of Cre recombinase is driven by an endothelial-specific promoter/enhancer. To analyze the lineage of Cre expressing cells, we used CAG-CAT-Z transgenic mice, in which expression of lacZ is activated only after Cre-mediated recombination. We detected pan-endothelial expression of the Cre transgene in Tie2-Cre;CAG-CAT-Z double-transgenic mice. This expression pattern is almost identical to Tie2-lacZ transgenic mice. However, interestingly, we observed strong and uniform lacZ expression in mesenchymal cells of the atrioventricular canal of Tie2-Cre;CAG-CAT-Z double-transgenic mice. We also detected lacZ expression in the mesenchymal cells in part of the proximal cardiac outflow tract, but not in the mesenchymal cells of the distal outflow tract and branchial arch arteries. LacZ staining in Tie2-Cre;CAG-CAT-Z embryos is consistent with endocardial-mesenchymal transformation in the atrioventricular canal and outflow tract regions. Our observations are consistent with previously reported results from Cx43-lacZ, Wnt1-Cre;R26R, and Pax3-Cre;R26R transgenic mice, in which lacZ expression in the cardiac outflow tract identified contributions in part from the cardiac neural crest. Tie2-Cre transgenic mice are a new genetic tool for the analyses of endothelial cell-lineage and endothelial cell-specific gene targeting.     

Development of mice with osteoblast-specific connexin43 gene deletion

Genetic deficiency of Cx43 in vivo causes skeletal developmental defects, osteoblast dysfunction and perinatal lethality. To determine the role of Cx43 in the adult skeleton, we developed two models of osteoblast-specific Cx43 gene deletion using Cre mediated replacement of a "floxed" Cx43 allele with a LacZ reporter gene. Cre recombinase expression in osteoblasts was driven by either the osteocalcin OG2 promoter or the 2.3 kb fragment of the Colalpha1(I) promoter. Homozygous Cx43(fl/fl) mice, in which the Cx43 coding region is flanked by two loxP sites, were crossed with Cre expressing mice in a heterozygous Cx43-null background [Cx43(+/-); Colalpha1(I)-Cre or Cx43(+/-); OG2-Cre]. Cx43 gene ablation was demonstrated in tissues by selective X-gal staining of cells lining the endosteal surface, and in cultured osteoblastic cells from calvaria using different approaches. Although no LacZ expression was observed in proliferating calvaria cells, before osteoblast differentiation begins, post-proliferative cells isolated from conditional knockout mice [Cx43(fl/-); Colalpha1(I)-Cre or Cx43(fl/-); OG2-Cre] developed strong LacZ expression as they differentiated, in parallel to a progressive disappearance of Cx43 mRNA and protein abundance relative to controls. Selective Cre mediated Cx43 gene inactivation in bone forming cells will be useful to determine the role of Cx43 in adult skeletal homeostasis and overcome the perinatal lethality of the conventional null model.     

International Society for Biological and Environmental Repositories

Genetically altered mice are an important tool for biomedical research. Several transgenic mice have been created in which activation of the transgene results in production of β-galactosidase that can be detected by histological means. While preservation and subsequent visualization of enzyme activity in soft tissues can be complicated, it is particularly difficult in bone specimens, especially those that have been decalcified. For these studies, we examined the bones of parathyroid hormone-related peptide (PTHrP) knock-in mice in which expression of PTHrP resulted in β-galactosidase production. During the past decade, several studies have demonstrated the importance of PTHrP in bone. Thus, it is important to preserve and detect β-galactosidase enzymatic activity in bone for these studies. We demonstrate here that β-galactosidase was visualized better in slides with bone sections taken from PTHrP knock-in mice when bones were frozen and sectioned compared to bones that were embedded in plastic and sectioned using a microtome. Importantly, we were able to visualize β-galactosidase in plastic embedded bones when specimens were fixed, stained (X-gal), embedded in plastic, and then sectioned rather than being fixed, embedded in plastic, sectioned, then stained.     

Development of Mice with Osteoblast-Specific Connexin43 Gene Deletion

Genetic deficiency of Cx43 in vivo causes skeletal developmental defects, osteoblast dysfunction and perinatal lethality. To determine the role of Cx43 in the adult skeleton, we developed two models of osteoblast-specific Cx43 gene deletion using Cre mediated replacement of a “floxed” Cx43 allele with a LacZ reporter gene. Cre recombinase expression in osteoblasts was driven by either the osteocalcin OG2 promoter or the 2.3 kb fragment of the Colα1(I) promoter. Homozygous Cx43^fl/flmice, in which the Cx43 coding region is flanked by two loxP sites, were crossed with Cre expressing mice in a heterozygous Cx43-null background [Cx43^±; Colα1(I)-Cre or Cx43^±; OG2-Cre]. Cx43 gene ablation was demonstrated in tissues by selective X-gal staining of cells lining the endosteal surface, and in cultured osteoblastic cells from calvaria using different approaches. Although no LacZ expression was observed in proliferating calvaria cells, before osteoblast differentiation begins, post-proliferative cells isolated from conditional knockout mice [Cx43^fl/?; Colα1(I)-Cre or Cx43^fl/?; OG2-Cre] developed strong LacZ expression as they differentiated, in parallel to a progressive disappearance of Cx43 mRNA and protein abundance relative to controls. Selective Cre mediated Cx43 gene inactivation in bone forming cells will be useful to determine the role of Cx43 in adult skeletal homeostasis and overcome the perinatal lethality of the conventional null model.     

An optimized protocol for detection of <Emphasis Type="Italic">E. coli</Emphasis> β-galactosidase in lung tissue following gene transfer

Staining by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) typically detects activity of E. coli β-galactosidase (β-gal) in transduced tissues that express the LacZ reporter gene. In lung tissue from mice that received β-galactosidase-expressing adeno-associated virus (AAV) vectors via intranasal inhalation, we observed only a low frequency of positive cells after X-gal staining in contrast to other reporter genes, such as alkaline phosphatase or green fluorescent protein. In this study, we systematically tested a number of parameters to improve the sensitivity of X-gal staining in lungs transduced with β-galactosidase-expressing AAV2/5 vectors. We observed that the use of nuclear-targeted LacZ instead of cytoplasmic LacZ as the reporter gene substantially increases the number of positive cells after X-gal staining. The pH of the staining solution determines staining sensitivity and background staining with pH 7.0 resulting in high sensitivity and no background levels. Glutaraldehyde at 0.2% or 0.5% in PBS as fixative provides optimal results for X-gal staining. The alternative substrate, Bluo-gal, showed no improvement compared with X-gal but instead caused nonspecific background staining. We further stained intact fixed lungs with X-gal and processed them for paraffin embedding or cryosectioning, resulting in equal staining intensities. However, en bloc staining of intact tissues resulted in the absence of positive cells within deeper-located lung areas.     

逆转录病毒载体介导的LacZ基因在NIH-3T3细胞中的稳定表达

精原干细胞(SSCs)介导的转基因技术很可能成为制作转基因动物及治疗雄性不育的一条新途径。为了研究逆转录病毒载体介导法转染体外培养SSCs的可行性,用脂质体介导法将携带LacZ基因的重组逆转录病毒载体pLNCL导入包装细胞PA317,用含G418的培养液筛选得到5株稳定转染的产毒细胞。收集这些克隆的产毒上清,过滤后进行倍比稀释,用NIH-3T3细胞通过X-gal染色测定其浓缩前病毒滴度。结果显示,PA3173培养上清中病毒的浓缩前滴度最高,达1.1×103CFU/mL。再将筛选到的稳定转染的NIH-3T3细胞培养至单层,进行X-gal染色检测β-半乳糖苷酶的表达。结果显示,大多数稳定转染的NIH-3T3细胞均为X-gal ,表明这些细胞成功表达了目的基因LacZ。本研究结果为后期工作中用该载体感染体外培养SSCs奠定了基础。     

Generation of the tamoxifen-inducible DBH-Cre transgenic mouse line DBH-CT

We generated transgenic mice bearing a tamoxifen-dependent Cre recombinase expressed under the control of the dopamine-β-hydroxylase promoter. By crossing to the ROSA26 reporter mice we show that tamoxifen-induced Cre recombinase in adult mice specifically activates β-galactosidase expression in differentiated noradrenergic neurons of the central and peripheral nervous system. Tamoxifen application in adult mice did not induce β-galactosidase activity in parasympathetic neurons that transiently express DBH during development. Thus, this transgenic mouse line represents a valuable tool to study gene function in mature noradrenergic neurons by conditional inactivation.     

Transgenic mice that express Cre recombinase in hypertrophic chondrocytes

In order to investigate the physiological control of hypertrophic chondrocytes which present the terminally differentiated form of chondrocytes, we generated a mouse line expressing the Cre recombinase under the control of the mouse type X collagen (Col10a1) promoter. In situ hybridization analysis demonstrated the expression of Col10a1-Cre transgene in hypertrophic chondrocytes of femur at postnatal day 2 (P2). In order to test the excision activity of the Cre recombinase, the Col10a1-Cre transgenic line was crossed with the mouse strain carrying the Smad4 conditional alleles (Smad4co/co) and the reporter line ROSA26. Multiple tissue PCR of Col10a1-Cre;Smad4co/+ mice revealed the restricted Cre activity in tissues containing hypertrophic chondrocytes. LacZ staining revealed that the Cre activity was observed in the cartilage primordia of ribs at E14.5 and only detected in the lower hypertrophic region of ribs at P1. These data suggest that the Col10a1-Cre mouse line described here could be used to achieve conditional gene targeting in hypertrophic chondrocytes.     

肥大软骨细胞特异性表达Cre重组酶转基因小鼠的建立

肥大软骨细胞是软骨细胞的终末分化形式,在软骨内成骨过程中发挥十分关键的作用。为了研究肥大软骨细胞在骨骼发育过程中的功能,我们构建了在8.2 kb小鼠X型胶原基因（Col10a1）启动子控制下表达Cre重组酶的转基因小鼠品系（Col10a1-8.2-Cre）。采用显微注射法将11.5 kb的转基因片段引入小鼠基因组,共注射受精卵328枚,获得子代鼠51只,经PCR基因型鉴定有3只在基因组上整合有Cre重组酶基因。PCR检测发现Col10a1-8.2-Cre转基因在含有肥大软骨细胞的组织中表达。为了检测Cre重组酶表达的强度和组织特异性,转基因小鼠与ROSA26报告小鼠交配。子代ROSA26;Col10a1-8.2-Cre双转基因小鼠LacZ染色检测的结果显示,Cre重组酶在所有的肥大软骨细胞中表达。原位杂交的结果验证Col10a1-8.2-Cre转基因表达在肥大区的上端。以上结果表明,我们建立的肥大软骨细胞特异性表达Cre重组酶的转基因小鼠品系可以作为一种遗传学工具,介导目的基因在肥大软骨细胞中的敲除。     

BAC constructs in transgenic reporter mouse lines control efficient and specific LacZ expression in hypertrophic chondrocytes under the complete Col10a1 promoter

During endochondral ossification hypertrophic chondrocytes in the growth plate of fetal long bones, ribs and vertebrae play a key role in preparing growth plate cartilage for replacement by bone. In order to establish a reporter gene mouse to facilitate functional analysis of genes expressed in hypertrophic chondrocytes in this process, Col10a1- BAC reporter gene mouse lines were established expressing LacZ specifically in hypertrophic cartilage under the control of the complete Col10a1 gene. For this purpose, a bacterial artificial chromosome (BAC RP23-192A7) containing the entire murine Col10a1 gene together with 200 kb flanking sequences was modified by inserting a LacZ-Neo cassette into the second exon of Col10a1 by homologous recombination in E. coli. Transgenic mice containing between one and seven transgene copies were generated by injection of the purified BAC-Col10a1- lLacZ DNA. X-gal staining of newborns and embryos revealed strong and robust LacZ activity exclusively in hypertrophic cartilage of the fetal and neonatal skeleton of the transgenic offspring. This indicates that expression of the reporter gene in its proper genomic context in the BAC Col10a1 environment is independent of the integration site and reflects authentic Col10a1 expression in vivo. The Col10a1 specific BAC recombination vector described here will enable the specific analysis of effector gene functions in hypertrophic cartilage during skeletal development, endochondral ossification, and fracture callus healing. Sonja Gebhard and Takako Hattori equally contributed to this work.     

Identification and Transgenic Analysis of a Murine Promoter that Targets Cholinergic Neuron Expression

Abstract : Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell type-specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized a promoter region of the mouse ChAT gene in transgenic mice, using β-galactosidase ( LacZ ) as a reporter gene. A 3,402-bp segment from the 5'-untranslated region of the mouse ChAT gene (from -3,356 to +46, +1 being the translation initiation site) was sufficient to direct the expression of LacZ to selected neurons of the nervous system ; however, it did not provide complete cholinergic specificity. A larger fragment (6,417 bp, from -6,371 to +46) of this region contains the requisite regulatory elements that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4-kb DNA fragment encompasses 633 bp of the 5'-flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.     

Transgenic mice that express Cre recombinase in osteoclasts

To study the physiological control of osteoclasts, the bone resorbing cells, we generated transgenic mice carrying the Cre recombinase gene driven by either the tartrate-resistant acid phosphatase (TRAP) or cathepsin K (Ctsk) promoters. TRAP-Cre and Ctsk-Cre transgenic mouse lines were characterized by breeding with LacZ ROSA 26 (R26R) reporter mice and immunohistochemistry for Cre recombinase. The Cre transgene was functional in all lines, with Cre-mediated recombination occurring primarily in the long bones, vertebrae, ribs, and calvaria. Histological analyses of the bones demonstrated that functional Cre protein was present in 1) osteoclasts (Ctsk-Cre); 2) osteoclasts, columnar proliferating, and hypertrophic chondrocytes (TRAP-Cre line 4); and 3) round proliferating chondrocytes (TRAP-Cre line 3). In conclusion, we generated transgenic mouse lines that will enable the deletion of floxed target genes in osteoclasts, which will be valuable tools for studying the regulation of osteoclast function.     

Novel ROSA26 Cre-reporter Knock-in C57BL/6N Mice Exhibiting Green Emission before and Red Emission after Cre-mediated Recombination

The Cre/loxP system is a strategy for controlling temporal and/or spatial gene expression through genome alteration in mice. As successful Cre/loxP genome alteration depends on Cre-driver mice, Cre-reporter mice are essential for validation of Cre gene expression in vivo. In most Cre-reporter mouse strains, although the presence of reporter product indicates the expression of Cre recombinase, it has remained unclear whether a lack of reporter signal indicates either no Cre recombinase expression or insufficient reporter gene promoter activity. We produced a novel ROSA26 knock-in Cre-reporter C57BL/6N strain exhibiting green emission before and red after Cre-mediated recombination, designated as strain R26GRR. Ubiquitous green fluorescence and no red fluorescence were observed in R26GRR mice. To investigate the activation of tdsRed, EGFP-excised R26GRR, R26RR, mice were produced through the crossing of C57BL/6N mice with R26GRR/Ayu1-Cre F₁ mice. R26RR mice showed extraordinarily strong red fluorescence in almost all tissues examined, suggesting ubiquitous activation of the second reporter in all tissues after Cre/loxP recombination. Moreover, endothelial cell lineage and pancreatic islet-specific expression of red fluorescence were detected in R26GRR/Tie2-Cre F₁ mice and R26GRR /Ins1-Cre F₁ mice, respectively. These results indicated that R26GRR mice are a useful novel Cre-reporter mouse strain. In addition, R26GRR mice with a pure C57BL/6N background represent a valuable source of green-to-red photoconvertible cells following Cre/loxP recombination for application in transplantation studies. The R26GRR mouse strain will be available from RIKEN BioResource Center (http://www.brc.riken.jp/lab/animal/en/).     

Primary Spermatocyte-Specific Cre Recombinase Activity in Transgenic Mice

We have evaluated the specificity of Cre recombinase activity in transgenic mice expressing Cre under the control of the synatonemal complex protein 1 (Sycp1) gene promoter. Sycp1Cre mice were crossed with the ROSA26 reporter line R26R, to monitor the male germ cell stage-specificity of Cre activity as well as to verify that Cre was not active previously during development of other tissues. X-gal staining detected Cre-mediated recombination only in testis. Detailed histological examination indicated that weak Cre-mediated recombination occurred as early as in zygotene spermatocytes at stage XI of the cycle of the seminiferous epithelium. Robust expression of X-gal was detected in early to mid-late spermatocytes at stages V-VIII. We conclude that this transgenic line is a powerful tool for deleting genes of interest specifically during male meiosis.     

Conditional expression of Wnt4 during chondrogenesis leads to dwarfism in mice

Wnts are expressed in the forming long bones, suggesting roles in skeletogenesis. To examine the action of Wnts in skeleton formation, we developed a genetic system to conditionally express Wnt4 in chondrogenic tissues of the mouse. A mouse Wnt4 cDNA was introduced into the ubiquitously expressed Rosa26 (R26) locus by gene targeting in embryonic stem (ES) cells. The expression of Wnt4 from the R26 locus was blocked by a neomycin selection cassette flanked by loxP sites (floxneo) that was positioned between the Rosa26 promoter and the Wnt4 cDNA, creating the allele designated R26(floxneoWnt4). Wnt4 expression was activated during chondrogenesis using Col2a1-Cre transgenic mice that express Cre recombinase in differentiating chondrocytes. R26(floxneoWnt4); Col2a1-Cre double heterozygous mice exhibited a growth deficiency, beginning approximately 7 to 10 days after birth, that resulted in dwarfism. In addition, they also had craniofacial abnormalities, and delayed ossification of the lumbar vertebrae and pelvic bones. Histological analysis revealed a disruption in the organization of the growth plates and a delay in the onset of the primary and secondary ossification centers. Molecular studies showed that Wnt4 overexpression caused decreased proliferation and altered maturation of chondrocytes. In addition, R26(floxneoWnt4); Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF). These studies demonstrate that Wnt4 overexpression leads to dwarfism in mice. The data indicate that Wnt4 levels must be regulated in chondrocytes for normal growth plate development and skeletogenesis. Decreased VEGF expression suggests that defects in vascularization may contribute to the dwarf phenotype.