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。     

A novel transgenic technique that allows specific marking of the neural crest cell lineage in mice.

Neural crest cells are embryonic, multipotent stem cells that give rise to various cell/tissue types and thus serve as a good model system for the study of cell specification and mechanisms of cell differentiation. For analysis of neural crest cell lineage, an efficient method has been devised for manipulating the mouse genome through the Cre-loxP system. We generated transgenic mice harboring a Cre gene driven by a promoter of protein 0 (P0). To detect the Cre-mediated DNA recombination, we crossed P0-Cre transgenic mice with CAG-CAT-Z indicator transgenic mice. The CAG-CAT-Z Tg line carries a lacZ gene downstream of a chicken beta-actin promoter and a "stuffer" fragment flanked by two loxP sequences, so that lacZ is expressed only when the stuffer is removed by the action of Cre recombinase. In three different P0-Cre lines crossed with CAG-CAT-Z Tg, embryos carrying both transgenes showed lacZ expression in tissues derived from neural crest cells, such as spinal dorsal root ganglia, sympathetic nervous system, enteric nervous system, and ventral craniofacial mesenchyme at stages later than 9.0 dpc. These findings give some insights into neural crest cell differentiation in mammals. We believe that P0-Cre transgenic mice will facilitate many interesting experiments, including lineage analysis, purification, and genetic manipulation of the mammalian neural crest cells.     

BMP type II receptor regulates positioning of outflow tract and remodeling of atrioventricular cushion during cardiogenesis

Signaling of bone morphogenetic protein (BMP) via type I and type II receptors is involved in multiple processes contributing to cardiogenesis. To investigate the role of the BMP type II receptor (BMPRII) in heart development, the BMPRII gene was deleted throughout the embryo during gastrulation using a Mox2-Cre transgene. BMPRII^flox/−;Mox2-Cre mice exhibited cardiac defects including double-outlet right ventricle, ventricular septal defect (VSD), atrioventricular (AV) cushion defects, and thickened valve leaflets. To characterize the tissue-specific functions of BMPRII in cardiogenesis, a series of Cre transgenes (αMHC-, Tie2-, Wnt1-, and SM22α-Cre) was employed. Interestingly, myocardial development was normal when the BMPRII gene was deleted in myocardial cells using Mox2-Cre, αMHC-Cre, or SM22α-Cre transgenes, suggesting that signaling by other BMP type II receptors may compensate for the absence of BMPRII in the myocardial cells. AV cushion defects including atrial septal defect, membranous VSD, and thickened valve leaflets were found in BMPRII^flox/−;Tie2-Cre mice. Abnormal positioning of the aorta was observed in BMPRII^flox/−;Wnt1-Cre and BMPRII^flox/−;SM22α-Cre mice. Taken together, these results demonstrate that endocardial BMPRII expression is required for septal formation and valvulogenesis. Moreover, mesenchymal BMPRII expression in the outflow tract cushion is required for proper positioning of the aorta.     

Dkk3-Cre BAC transgenic mouse line: a tool for highly efficient gene deletion in retinal progenitor cells

To establish the genetic tools for conditional gene deletion in mouse retinal progenitors, we generated a Dkk3-Cre transgenic mouse line using bacterial artificial chromosome (BAC) transgenesis. Cre recombination efficiency in vivo was assayed by crossing this transgenic line, termed BAC-Dkk3-Cre, with the CAG-CAT-Z reporter line. This BAC-Dkk3-Cre line showed Cre recombinase activity in most retinal progenitors. Cre activity was detectable from embryonic day 10.5 (E10.5) and generally restricted to the retina during embryogenesis. To verify that BAC-Dkk3-Cre mice successfully circumvented lethality, we generated Otx2flox/flox/BAC-Dkk3-Cre+ mice as Otx2 conditional knockout mice. The Otx2flox/flox/BAC-Dkk3-Cre+ mice were viable, and their retina showed loss of mature cell-type markers of photoreceptor cells, bipolar cells, and horizontal cells, in contrast, amacrine-like cells noticeably increased. Thus, the BAC-Dkk3-Cre transgenic mouse line provides a powerful tool for generating conditional knockout mouse lines for studying loss of gene functions in the developing retina.     

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/).     

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.     

Cre-mediated excision of Fgf8 in the Tbx1 expression domain reveals a critical role for Fgf8 in cardiovascular development in the mouse

Tbx1 has been implicated as a candidate gene responsible for defective pharyngeal arch remodeling in DiGeorge/Velocardiofacial syndrome. Tbx1(+/-) mice mimic aspects of the DiGeorge phenotype with variable penetrance, and null mice display severe pharyngeal hypoplasia. Here, we identify enhancer elements in the Tbx1 gene that are conserved through evolution and mediate tissue-specific expression. We describe the generation of transgenic mice that utilize these enhancer elements to direct Cre recombinase expression in endogenous Tbx1 expression domains. We use these Tbx1-Cre mice to fate map Tbx1-expressing precursors and identify broad regions of mesoderm, including early cardiac mesoderm, which are derived from Tbx1-expressing cells. We test the hypothesis that fibroblast growth factor 8 (Fgf8) functions downstream of Tbx1 by performing tissue-specific inactivation of Fgf8 using Tbx1-Cre mice. Resulting newborn mice display DiGeorge-like congenital cardiovascular defects that involve the outflow tract of the heart. Vascular smooth muscle differentiation in the great vessels is disrupted. This data is consistent with a model in which Tbx1 induces Fgf8 expression in the pharyngeal endoderm, which is subsequently required for normal cardiovascular morphogenesis and smooth muscle differentiation in the aorta and pulmonary artery.     

Foxl1-Cre BAC transgenic mice: a new tool for gene ablation in the gastrointestinal mesenchyme

The use of Cre-loxP technology for the purpose of cell type-specific gene ablation has revolutionized developmental biology and biomedicine. Several transgenic mouse lines have been developed for the analysis of gene function in the gastrointestinal tract, but in all of these the expression of Cre is limited to the epithelial cell layer. No Cre- expressing transgenic mouse lines ("Cre lines") exist for the deletion of loxP-flanked genes specifically in gut mesoderm. To address this deficiency, we have derived a bacterial artificial chromosome based transgenic mouse line in which the Cre gene is controlled by the Foxl1 promoter and enhancer elements. X-Gal staining of Foxl1-Cre; Rosa26R bi-transgenic lines confirm that Foxl1-Cre results in recombination specifically in the gastrointestinal mesenchyme. The Foxl1-Cre line will facilitate the dissection of mesenchymal to epithelial signaling that is known to play a major role in the patterning and function of the gastrointestinal tract.     

Essential functions of Alk3 during AV cushion morphogenesis in mouse embryonic hearts

Accumulated evidence has suggested that BMP pathways play critical roles during mammalian cardiogenesis and impairment of BMP signaling may contribute to human congenital heart diseases (CHDs), which are the leading cause of infant morbidity and mortality. Alk3 encodes a BMP specific type I receptor expressed in mouse embryonic hearts. To reveal functions of Alk3 during atrioventricular (AV) cushion morphogenesis and to overcome the early lethality of Alk3(-/-) embryos, we applied a Cre/loxp approach to specifically inactivate Alk3 in the endothelium/endocardium. Our studies showed that endocardial depletion of Alk3 severely impairs epithelium-mesenchymal-transformation (EMT) in the atrioventricular canal (AVC) region; the number of mesenchymal cells formed in Tie1-Cre;Alk3(loxp/loxp) embryos was reduced to only approximately 20% of the normal level from both in vivo section studies and in vitro explant assays. We showed, for the first time, that in addition to its functions on mesenchyme formation, Alk3 is also required for the normal growth/survival of AV cushion mesenchymal cells. Functions of Alk3 are accomplished through regulating expression/activation/subcellular localization of multiple downstream genes including Smads and cell-cycle regulators. Taken together, our study supports the notion that Alk3-mediated BMP signaling in AV endocardial/mesenchymal cells plays a central role during cushion morphogenesis.     

The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field

The vertebrate heart arises from the fusion of bilateral regions of anterior mesoderm to form a linear heart tube. Recent studies in mouse and chick have demonstrated that a second cardiac progenitor population, known as the anterior or secondary heart field, is progressively added to the heart at the time of cardiac looping. While it is clear that this second field contributes to the myocardium, its precise boundaries, other lineages derived from this population, and its contributions to the postnatal heart remain unclear. In this study, we used regulatory elements from the mouse mef2c gene to direct the expression of Cre recombinase exclusively in the anterior heart field and its derivatives in transgenic mice. By crossing these mice, termed mef2c-AHF-Cre, to Cre-dependent lacZ reporter mice, we generated a fate map of the embryonic, fetal, and postnatal heart. These studies show that the endothelial and myocardial components of the outflow tract, right ventricle, and ventricular septum are derivatives of mef2c-AHF-Cre expressing cells within the anterior heart field and its derivatives. These studies also show that the atria, epicardium, coronary vessels, and the majority of outflow tract smooth muscle are not derived from this anterior heart field population. Furthermore, a transgene marker specific for the anterior heart field is expressed in the common ventricular chamber in mef2c mutant mice, suggesting that the cardiac looping defect in these mice is not due to a failure in anterior heart field addition to the heart. Finally, the Cre transgenic mice described here will be a crucial tool for conditional gene inactivation exclusively in the anterior heart field and its derivatives.     

Nkx-2.5 gene induction in mice is mediated by a Smad consensus regulatory region

In the forming vertebrate heart, bone morphogenetic protein signaling induces expression of the early cardiac regulatory gene nkx-2.5. A similar regulatory interaction has been defined in Drosophila embryos where Dpp signaling mediated by the Smad homologues Mad and Medea directly regulates early cardiac expression of tinman. A conserved cluster of Smad consensus binding sequences was identified in early cardiac regulatory sequences of the mouse nkx-2.5 gene. The importance of the nkx-2.5 Smad consensus region in early cardiac gene expression was examined in transgenic mice and in cultured mouse embryos. In transgenic mice, deletion of the Smad consensus region delays induction of embryonic DeltaSmadnkx-2.5/lacZ gene expression during early heart formation. Induction of DeltaSmadnkx-2.5/lacZ expression is also delayed in the outflow tract myocardium and visceral mesoderm. Targeted mutation of the three Smad consensus sequences inhibited nkx-2.5/lacZ expression in the cardiac crescent, demonstrating a specific requirement for the Smad consensus sites in early cardiac gene induction. Cultured DeltaSmadnkx-2.5/lacZ transgenic mouse embryos also exhibit delayed induction of transgene expression. In the four-chambered heart, deletion of the Smad consensus region resulted in expanded DeltaSmadnkx-2.5/lacZ transgene expression. Thus, the nkx-2.5 Smad consensus region can have positive or negative regulatory function, depending on the developmental context and cellular environment.     

Proline-rich-protein promoters direct LacZ expression to the granular convoluted tubular cells of the submandibular gland in adult transgenic mice

The ability of two mouse PRP gene promoters to direct the expression of the bacterial lacZ reporter gene was tested in transgenic mice. Transgenes A1-lacZ and C1-lacZ consisted of 8.2 kb A1 and 7.8 kb C1 PRP promoters respectively fused to the lacZ coding sequence. A1 and C1 are two A-type PRP genes isolated from the inbred SWR mice, which show the same gene structure and similar sequence to the closely related MP2 and M14 PRP genes previously cloned from outbred CD-1 mice. We here show that both A1-lacZ and C1-lacZ transgenes have very similar expression patterns: (1) they expressed the lacZ gene in all 14 established transgenic lines under normal (non-stimulated) conditions; (2) the expression was restricted to the granular convoluted tubular cells of the submandibular glands; (3) the expression was developmentally regulated beginning at sexual maturation and lasting to at least 1.5 years of age; and (4) expression in some lines was probably influenced by sex hormones, since higher expression was found in males than in females. A1-lacZ and C1- lacZ are the first transgenes derived from the PRP/GRP (glutamine/glutamic acid-rich protein) gene superfamily to be expressed in the granular convoluted tubular cells (with known endocrine functions), rather than in the acinar cells (with mainly exocrine functions) of the submandibular glands     

Analysis of Fibroblast growth factor 15 cis-elements reveals two conserved enhancers which are closely related to cardiac outflow tract development

Fibroblast growth factor 15 (Fgf15) is expressed in the developing mouse central nervous system and pharyngeal arches. Fgf15 mutant mice showed defects of the cardiac outflow tract probably because of aberrant behavior of the cardiac neural crest cells. In this study, we examined cis-elements of the Fgf15 gene by transient transgenic analysis using lacZ as a reporter. We identified two enhancers: one directed lacZ expression in the hindbrain/spinal cord and the other in the posterior midbrain (pmb), rhombomere1 (r1) and pharyngeal epithelia. Interestingly, human genomic regions which are highly homologous to these two mouse enhancers showed almost the same enhancer activities as those of mice in transgenic mouse embryos, indicating that the two enhancers are conserved between humans and mice. We also showed that the mouse and human pmb/r1 enhancer can regulate lacZ expression in chick embryos in almost the same way as in mouse embryos. We found that the lacZ expression domain with this enhancer was expanded by ectopic Fgf8b expression, suggesting that this enhancer is regulated by Fgf8 signaling. Moreover, over-expression of Fgf15 resulted in up-regulation of Fgf8 expression in the isthmus/r1. These findings suggest that a reciprocal positive regulation exists between Fgf15 and Fgf8 in the isthmus/r1. Together with cardiac outflow tract defects in Fgf15 mutants, the conservation of enhancers in the hindbrain/spinal cord and pharyngeal epithelia suggests that human FGF19 (ortholog of Fgf15) is involved in early development and the distribution of cardiac neural crest cells and is one of the candidate genes for congenital heart defects.     

Efficient Cre-mediated deletion in cardiac progenitor cells conferred by a 3'UTR-ires-Cre allele of the homeobox gene Nkx2-5

Conditional gene targeting and transgenic strategies utilizing Cre recombinase have been successfully applied to the analysis of development in mouse embryos. To create a conditional system applicable to heart progenitor cells, a Cre recombinase gene linked at its 5' end to an internal ribosome entry site (IRES) was inserted into the 3' untranslated region of the cardiac homeobox gene Nkx2-5 using gene targeting. Nkx2-5IRESCre mice were fully viable as homozygotes. We evaluated the efficacy of Cre-mediated deletion by crossing Nkx2-5IRESCre mice with the Cre-dependent R26R and Z/AP reporter strains. Efficient deletion was observed in the cardiac crescent and heart tube in both strains. However, the Z/AP locus showed transient resistance to deletion in caudal heart progenitors. Such resistance was not evident at the R26R locus, suggesting that Cre-mediated deletion in myocardium may be locus-dependent. From cardiac crescent stages, deletion was seen not only in myocardium, but also endocardium, dorsal mesocardium and pericardial mesoderm. The Cre domain apparently includes cells dorsal to the heart that have been shown to constitute a secondary heart field, contributing myocardium to the outflow tract. Other sites of Nkx2-5 expression, including pharyngeal endoderm and its derivatives, branchial arch epithelium, stomach, spleen, pancreas and liver, also showed efficient deletion. Our data suggest that the Nkx2-5IRESCre strain will be useful for genetic dissection of the multiple tiers of lineage allocation to the forming heart as well as of molecular interactions within the heart fields and heart tube.     

Myocardial deletion of Smad4 using a novel α skeletal muscle actin Cre recombinase transgenic mouse causes misalignment of the cardiac outflow tract

SMAD4 acts as the converging point for TGFβ and BMP signaling in heart development. Here, we investigated the role of SMAD4 in heart development using a novel α skeletal muscle actin Cre recombinase (MuCre) transgenic mouse strain. Lineage tracing using MuCre/ROSA26^LacZ reporter mice indicated strong Cre-recombinase expression in developing and adult heart and skeletal muscles. In heart development, significant MuCre expression was noted at E11.5 in the atrial, ventricular, outflow tract and atrioventricular canal myocardium, but not in the endocardial cushions. MuCre-driven conditional deletion of Smad4 in mice caused double outlet right ventricle (DORV), ventricular septal defect (VSD), impaired trabeculation and thinning of ventricular myocardium, and mid-gestational embryonic lethality. In conclusion, MuCre mice effectively delete genes in both heart and skeletal muscles, thus enabling the discovery that myocardial Smad4 deletion causes misalignment of the outflow tract and DORV.     

Pan-myocardial expression of Cre recombinase throughout mouse development

Mouse-lines expressing Cre recombinase in a tissue-specific manner are a powerful tool in developmental biology. Here, we report that a 3 kb fragment of the Xenopus laevis myosin light-chain 2 (XMLC2) promoter drives Cre recombinase expression in a cardiac-restricted fashion in the mouse embryo. We have isolated two XMLC2-Cre lines that express recombinase exclusively within cardiomyocytes, from the onset of their differentiation in the cardiac crescent of the early embryo. Expression is maintained throughout the myocardium of the embryonic heart tube and subsequently the mature myocardium of the chambered heart. Recombinase activity is detected in all myocardial tissue, including the pulmonary veins. One XMLC2-Cre line shows uniform expression while the other only expresses recombinase in a mosaic fashion encompassing less than 50% of the myocardial cells. Both lines cause severe cardiac malformations when crossed to a conditional Tbx5 line, resulting in embryonic death at midgestation. Optical projection tomography reveals that the spectrum of developmental abnormalities includes a shortening of the outflow tract and its abnormal alignment, along with a dramatic reduction in trabeculation of the ventricular segment of the looping heart tube.