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.     

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。     

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.     

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/).     

Characterization of Cre recombinase mouse lines enabling cell type-specific targeting of postnatal intervertebral discs

Cre/loxP technology is an important tool for studying cell type-specific gene functions. Cre recombinase mouse lines, including Agc1-CreER^T2, Col2a1-Cre; Col2a1-CreER^T2, Shh-Cre, Shh-CreER^T2, and Osx-Cre, have been proven to be valuable tools to elucidate the biology of long bones, yet the information for their activity in postnatal intervertebral disc (IVD) tissues was very limited. In this study, we used R26-mTmG fluorescent reporter to systematically analyze cell specificity and targeting efficiency of these six mouse lines in IVD tissues at postnatal growing and adult stages. We found that Agc1-CreER^T2 is effective to direct recombination in all components of IVDs, including annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplate (CEP), upon tamoxifen induction at either 2 weeks or 2 months of ages. Moreover, Col2a1-Cre targets most of the cells in IVDs, except for some cells in the outer AF (OAF) and NP. In contrast, the activity of Col2a1-CreER^T2 is mainly limited to the IAF of IVD tissues at either stage of tamoxifen injection. Similarly, Shh-Cre directs recombination specifically in all NP cells, whereas Shh-CreER^T2 is active only in a few NP cells when tamoxifen is administered at either stage. Finally, Osx-Cre targets cells in the CEP, but not in the NP or AF of IVDs tissues at these two stages. Thus, our data demonstrated that all these Cre lines can direct recombination in IVD tissues at postnatal stages with different cell type specificity and/or targeting efficiency, and can, therefore, serve as valuable tools to dissect cell type-specific gene functions in IVD development and homeostasis.     

血管内皮细胞特异表达Cre重组酶转基因小鼠的建立

血管内皮细胞参与血管形成、血管稳态维持、血栓形成、炎症和血管重建等生理和病理过程。为了便于通过Cre-LoxP系统研究相关基因在血管内皮细胞中的功能,创建了Tie2-Cre转基因小鼠,利用Tie2基因的启动子驱动Cre重组酶基因在血管内皮细胞中表达。经基因组PCR和Southern Blot鉴定有6只小鼠在基因组上整合有Cre基因,整合率为11％。为了验证Cre重组酶的剪切活性和表达组织分布,我们将Tie2-Cre转基因小鼠分别与Smad4条件基因打靶小鼠和报告小鼠ROSA26交配。Tie2-Cre;Smad4^co/＋小鼠的多个组织的基因组DNA的PCR结果显示,Cre重组酶在所有包含血管内皮细胞的组织中表达并能介导LoxP间的重组。Tie2-Cre;ROSA26双转基因胚胎LacZ染色结果显示,Cre重组酶在所有被检测组织的血管内皮细胞中特异性表达。因此．Tie2-Cre转基因小鼠可作血管内皮细胞谱系分析和在血管内皮细胞进行条件基因打靶的理想工具小鼠。     

消化道细胞表达Cre重组酶转基因小鼠的功能鉴定

目的:检测白蛋白启动子介导的Cre重组酶转基因小鼠Alb-Cre-2中Cre重组酶的组织分布及其在体内介导基因重组的作用。方法:将Alb-Cre小鼠与Smad4条件基因打靶小鼠交配,利用PCR对Cre重组酶介导重组的组织特异性进行检测;然后,将Alb-Cre-2转基因小鼠与ROSA26报告小鼠交配,利用LacZ染色对双转基因阳性子代小鼠进行检测。结果:PCR结果显示心、肺、胰、脑及消化道中Cre重组酶介导的Smad4基因发生重组;LacZ染色进一步表明Cre重组酶在肝细胞、胃壁细胞、空肠潘氏细胞、回肠杯状细胞、大肠杯状细胞、大肠柱状细胞及空泡细胞中特异性表达,并介导ROSA位点LoxP序列间的重组。结论:Alb-Cre-2转基因小鼠在消化道中具有一定的组织特异性,只在胃壁细胞、空肠潘氏细胞、回肠杯状细胞、大肠杯状细胞,大肠柱状细胞及空泡细胞等细胞类型中特异性表达,并能在体内成功地介导这些消化道上皮细胞基因组上LoxP位点间的重组,是一种研制在消化道特定细胞中特异性基因剔除小鼠的良好工具小鼠。     

Selective expression of the Cre recombinase in late-stage thymocytes using the distal promoter of the Lck gene

Transgenic mouse lines were generated that express the Cre recombinase under the control of the distal promoter of the mouse Lck gene. Cre recombination in four of these lines of transgenic mice was characterized at the single cell level using ROSA26-regulated loxP-Stop-loxP-betageo and loxP-Stop-loxP-YFP reporter mouse lines. Two of the lines showed T cell-restricted Cre recombination, whereas the other two also expressed Cre in B cells, NK cells, and monocytes. Cre recombination began at a late stage of T cell development (at or after up-regulation of the TCR during positive selection) in the two T cell-restricted lines. Lines of mice that express the Cre recombinase at late stages of thymocyte development are of value for determining the impact of mutations on T cell function in the absence of complicating effects on early thymocyte selection.     

Mid-facial developmental defects caused by the widely used LacZ reporter gene when expressed in neural crest-derived cells

Reporter genes play important roles in transgenic research. LacZ is a widely used reporter gene that encodes Escherichia coli β-galactosidase, an enzyme that is well known for its ability to hydrolyze X-gal into a blue product. It is unknown whether transgenic LacZ has any adverse effects. R26R reporter mice, containing a LacZ reporter gene, were generated to monitor the in vivo recombination activity of various transgenic Cre recombinase via X-gal staining. P0-Cre is expressed in neural crest-derived cells, which give rise to the majority of the craniofacial bones. Herein, we report that 12% of the R26R reporter mice harboring P0-Cre had unexpected mid-facial developmental defects manifested by the asymmetrical growth of some facial bones, thus resulting in tilted mid-facial structure, shorter skull length, and malocclusion. Histological examination showed a disorganization of the frontomaxillary suture, which may at least partly explain the morphological defect in affected transgenic mice. Our data calls for the consideration of the potential in vivo adverse effects caused by transgenic β-galactosidase.     

Novel reporter and deleter mouse strains generated using VCre/VloxP and SCre/SloxP systems,and their system specificity in mice

DNA site-specific recombination by Cre/loxP is a powerful tool for gene manipulation in experimental animals. VCre/VloxP and SCre/SloxP are novel site-specific recombination systems, consisting of a recombinase and its specific recognition sequences, which function in a manner similar to Cre/loxP. Previous reports using Escherichia coli and Oryzias latipes demonstrated the existence of stringent specificity between each recombinase and its target sites; VCre/VloxP, SCre/SloxP, and Cre/loxP have no cross-reactivity with each other. In this study, we established four novel knock-in (KI) mouse strains in which VloxP-EGFP, SloxP-tdTomato, CAG-VCre, and CAG-SCre genes were inserted into the ROSA26 locus. VloxP-EGFP and SloxP-tdTomato KI mice were reporter mice carrying EGFP or tdTomato genes posterior to the stop codon, which was floxed by VloxP or SloxP fragments, respectively. CAG-VCre and CAG-SCre KI mice carried VCre or SCre genes that were expressed ubiquitously. These two reporter mice were crossed with three different deleter mice, CAG-VCre KI, CAG-SCre KI, and Cre-expressing transgenic mice. Through these matings, we found that VCre/VloxP and SCre/SloxP systems were functional in mice similar to Cre/loxP, and that the recombinases showed tight specificity for their recognition sequences. Our results suggest that these novel recombination systems allow highly sophisticated genome manipulations and will be useful for tracing the fates of multiple cell lineages or elucidating complex spatiotemporal regulations of gene expression.     

Cre recombinase-mediated gene targeting of mesenchymal cells

Loss-of-function approaches by the Cre/loxP technology have provided powerful tools for functional analyses of genes of interest expressed preferentially in a particular tissue. Here we describe the generation of transgenic mouse lines expressing Cre recombinase under the control of the promoter/enhancer unit of the gene for the alpha2 chain of collagen type I (Col1alpha2). As an expression vector, we used a P1-derived artificial chromosome (PAC), which harbors approximately 100 kb carrying the col1alpha2 gene. The improved coding sequence of the Cre recombinase was introduced to replace the first exon of col1alpha2. Cre expression was determined by immunohistochemistry and Cre-mediated onset of beta-galactosidase expression in ROSA26R-Cre reporter mice. In four analyzed transgenic lines, Cre recombinase was efficiently expressed during embryogenesis and in adult animals in cells of mesenchymal origin, such as dermal fibroblasts, mesenchymal cells of blood vessel walls, and cells in fibrous connective tissues surrounding internal organs.     

Rearrangement of a conditional allele regardless of inheritance of a Cre recombinase transgene

To generate conditional gene knockouts in osteoblasts, we previously developed transgenic mice in which Cre recombinase cDNA was cloned downstream of a 3.6 or 2.3 kb fragment of the rat Col1a1 promoter (Col3.6-Cre and Col2.3-Cre, respectively). Col-Cre mice were bred with mice in which exon 4 of the Igf1 gene is flanked by loxP sites. Mating units were arranged such that either the male or the female breeder transmitted the Col-Cre transgenes. Progeny were evaluated for Cre-mediated Igf1 gene rearrangement. We found that the loxP-flanked Igf1 locus was rearranged in the absence of inheritance of the Cre transgene. The incidence was 50 and 28% with Col2.3-Cre and Col3.6-Cre females, respectively, and 15 and 18% with Col2.3-Cre and Col3.6-Cre males, respectively.     

Tamoxifen‐inducible podocyte‐specific iCre recombinase transgenic mouse provides a simple approach for modulation of podocytes in vivo

We report the generation and initial characterization of a mouse line expressing tamoxifen‐inducible improved Cre (iCre) recombinase (iCre‐ER^T2) under the regulation of NPHS2 (podocin) gene promoter. The resulting transgenic mouse line was named podocin‐iCreER^T2 mice. The efficiency of iCre activity was confirmed by crossing podocin‐iCreER^T2 with the ROSA26 reporter mouse. By using the floxed ROSA reporter mice, we found that tamoxifen specifically induced recombination in the kidneys. In the absence of tamoxifen, recombination was undetectable in podocin‐iCreER^T2;ROSA26 mice. However, following intraperitoneal injection of tamoxifen, selective recombination was observed in the podocytes of adult animals. We further examined the efficiency of recombination by assessing various tamoxifen exposure regimens in adult mice. These results suggest that podocin‐iCre‐ER^T2 mouse provides an excellent genetic tool to examine the function of candidate genes in podocytes in a spatially and temporally‐restricted manner. genesis 48:446–451, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Atp4b promoter directs the expression of Cre recombinase in gastric parietal cells of transgenic mice

Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid.To study the function of gastric parietal cells during gastric epithelium homeostasis,we generated a transgenie mouse line,namely,Atp4b-Cre,in which the expression of Cre recombinase was controlled by a 1.0 kb promoter of mouse β-subunit of H+-,K+-ATPase gene(Atp4b).In order to test the tissue distribution and excision activity of Cre recombinase in vivo,the Atp4b-Cre transgenic mice were bred with the reporter strain ROSA26 and a mouse strain that carries Smad4 conditional alleles(Smad4Co/Co).Multiple-tissue PCR of Atp4b-Cre;Smad4Co/+mice revealed that the recombination only happened in the stomach.As indicated by LacZ staining,ROSA26;Atp4b-Cre double transgenic mice showed efficient expression of Cre recombinase within the gastric parietal cells.These results showed that this Atp4b-Cre mouse line could be used as a powerful tool to achieve conditional gene knockout in gastric parietal cells.     

Collagenl α1 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 Collagenlal (Coilal) promoter(Coilatl-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 rccombinase in the transgenic mice,the Collal-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4co/co).Multiple tissue PCR of Collal-Cre;Smad4co/ mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon.LacZ staining in the Coilal-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 Collal-Cre transgenic mice could Serve as a valuabletool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.