Dlx5/6-enhancer directed expression of Cre recombinase in the pharyngeal arches and brain

Dlx5 and Dlx6, two members of the Distalless gene family, are required for development of numerous tissues during embryogenesis, including facial and limb development. This gene pair is expressed in tandem, transcribed toward each other and separated by a short intergenic region containing multiple putative enhancers. Targeted inactivation of Dlx5 and Dlx6 in mice results in multiple developmental defects in craniofacial and limb structures, suggesting that these genes are crucial for aspects of both neural crest and nonneural crest development. To further investigate potential developmental roles of Dlx5 and Dlx6, we used one of the Dlx5/6 intergenic enhancers to drive Cre recombinase expression in transgenic mice. Crossing Dlx5/6-Cre transgenic mice with mice from the R26R strain results in beta-galactosidase staining in the apical ectodermal ridge, brain, and neural crest-derived mesenchyme of the pharyngeal arches, with staining in term embryos observed in the facial skeleton and specific brain structures. However, in contrast to endogenous expression patterns of Dlx5 and Dlx6, Cre expression within the pharyngeal arches occurs during a very narrow window in early development. Our studies suggest that Dlx5/6-Cre mice may prove useful both in further understanding the function and regulation of Distalless genes during development and in studies of gene function in conditional knockout mice.     

Cre重组酶表达载体的构建及其在转基因小鼠胰腺中的表达

组织特异性表达Cre重组酶的转基因小鼠是进行组织特异性条件敲除研究的关键。采用PCR扩增大鼠胰岛素基因705bp启动子指导发胰岛细胞中特异表达;同时采用改构的Cre重组酶基因,在其5'端添加有真核核糖体结合序列和核定位序列使Cre重组酶能穿越核膜在细胞核能发挥功能;同时,为了保证原核基因Cre能在真核系统顺利表达,在其3'端添加含内含子的人生长激素基因。构建的表达载体在去除原核序列后用显微注射方法转基因小鼠,在出生的27只仔鼠中,PCR检测共获得7只Cre整合阳性的转基因小鼠,整合率26％。这种Cre转基因小鼠与基因组小携带LoxP位点的条件基因打靶小鼠交配,在胰腺组织中可以检测到Cre介导的重组,表明Cre在转基因小鼠胰腺中有表达。     

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.     

Characterization of a novel transgenic mouse line expressing Cre recombinase under the control of the Cdx2 neural specific enhancer

Several genetically modified mouse models have been generated in order to drive expression of the Cre recombinase in the neuroectoderm. However, none of them specifically targets the posterior neural plate during neurulation. To fill this gap, we have generated a new transgenic mouse line in which Cre expression is controlled by a neural specific enhancer (NSE) from the Caudal‐related homeobox 2 (Cdx2) locus. Analyses of Cre activity via breeding with R26R‐YFP reporter mice have indicated that the Cdx2NSE‐Cre mouse line allows for recombination of LoxP sites in most cells of the posterior neural plate as soon as from the head fold stage. Detailed examination of double‐transgenic embryos has revealed that this novel Cre‐driver line allows targeting the entire posterior neural tube with an anterior limit in the caudal hindbrain. Of note, the Cdx2NSE regulatory sequences direct Cre expression along the whole dorso‐ventral axis (including pre‐migratory neural crest cells) and, accordingly, YFP fluorescence has been also observed in multiple non‐cranial neural crest derivatives of double‐transgenic embryos. Therefore, we believe that the Cdx2NSE‐Cre mouse line represents an important novel genetic tool for the study of early events occurring in the caudal neuroectoderm during the formation of both the central and the peripheral nervous systems. genesis 51:777–784. © 2013 Wiley Periodicals, Inc.     

Specific and spatial labeling of P0‐Cre versus Wnt1‐Cre in cranial neural crest in early mouse embryos

P0‐Cre and Wnt1‐Cre mouse lines have been widely used in combination with loxP‐flanked mice to label and genetically modify neural crest (NC) cells and their derivatives. Wnt1‐Cre has been regarded as the gold standard and there have been concerns about the specificity of P0‐Cre because it is not clear about the timing and spatial distribution of the P0‐Cre transgene in labeling NC cells at early embryonic stages. We re‐visited P0‐Cre and Wnt1‐Cre models in the labeling of NC cells in early mouse embryos with a focus on cranial NC. We found that R26‐lacZ Cre reporter responded to Cre activity more reliably than CAAG‐lacZ Cre reporter during early embryogenesis. Cre immunosignals in P0‐Cre and reporter (lacZ and RFP ) activity in P0‐Cre/R26‐lacZ and P0‐Cre/R26‐RFP embryos was detected in the cranial NC and notochord regions in E8.0–9.5 (4–19 somites) embryos. P0‐Cre transgene expression was observed in migrating NC cells and was more extensive in the forebrain and hindbrain but not apparent in the midbrain. Differences in the Cre distribution patterns of P0‐Cre and Wnt1‐Cre were profound in the midbrain and hindbrain regions, that is, extensive in the midbrain of Wnt1‐Cre and in the hindbrain of P0‐Cre embryos. The difference between P0‐Cre and Wnt1‐Cre in labeling cranial NC may provide a better explanation of the differential distributions of their NC derivatives and of the phenotypes caused by Cre‐driven genetic modifications.     

Dual origin of melanocytes defined by Sox1 expression and their region‐specific distribution in mammalian skin

Melanocytes are pigment‐producing cells generated from neural crest cells (NCCs) that delaminate from the dorsal neural tube. The widely accepted premise that NCCs migrating along the dorsolateral pathway are the main source of melanocytes in the skin was recently challenged by the finding that Schwann cell precursors are the major cellular source of melanocytes in the skin. Still, in a wide variety of vertebrate embryos, melanocytes are exclusively derived from NCCs. In this study, we show that a NCC population that is not derived from Sox1⁺ dorsal neuroepithelial cells but are derived from Sox1^? cells differentiate into a significant population of melanocytes in the skin of mice. Later, these Sox1^? cells clearly segregate from cells that originated from Sox1⁺ dorsal neuroepithelial cell‐derived NCCs. The possible derivation of Sox1^? cells from epidermal cells also strengthens their non‐neuroepithelial origin.     

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.     

Highly restricted expression of Cre recombinase in cerebellar Purkinje cells

The Purkinje neuron, one of the most fascinating components of the cerebellar cortex, is involved in motor learning, motor coordination, and cognitive function. Purkinje cell protein 2 (Pcp2/L7) expression is highly restricted to Purkinje and retinal bipolar cells, where it has been exploited to enable highly specific, Cre recombinase-mediated, site-specific recombination. Previous studies showed that mice carrying a Cre transgene produced by insertion of Cre cDNA into a small 2.88-kb Pcp2 DNA fragment expressed Cre in Purkinje cells; however, some Cre activity was also observed outside the target tissues. Here, we used Red-mediated recombineering to insert Cre cDNA into a 173-kb BAC carrying the entire intact Pcp2 gene, and characterize the resultant BAC/Cre transgenic mice for Cre expression. We show that BAC/Cre transgenic mice have exclusive Cre expression in Purkinje and bipolar cells and nowhere else. These mice will facilitate Purkinje cell and retinal bipolar cell-specific genetic manipulation.     

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.     

Temporal regulation of Cre recombinase activity in neural stem cells

Neural stem cells are known to give rise to distinct subtypes of neurons and glial cells over time by changing their competency. However, precise characterization of neural stem cells at various developmental stages remains to be performed. For such analysis, a tool to manipulate neural stem cells at different time points is necessary. Here, we generated transgenic mice that express Cre-ER(T2) in the ventricular zone of the developing nervous system under the control of the nestin promoter and enhancer (Nes-CreER(T2)). In mice expressing Cre-ER(T2) at appropriate levels, Cre recombinase activity was mostly inactive but efficiently activated by tamoxifen within 1 day. When such mice were crossed with the ROSA-26 or Z/EG reporter mice, neural stem cells were permanently labeled after administration of tamoxifen. Thus, Nes-CreER(T2) mice offer a powerful tool to manipulate neural stem cells genetically at desired time points.     

Capn8 promoter directs the expression of Cre recombinase in gastric pit cells of transgenic mice

Gastric pit cells are high‐turnover epithelial cells of the gastric mucosa. They secrete mucus to protect the gastric epithelium from acid and pepsin. To investigate the genetic mechanisms underlying the physiological functions of gastric pit cells, we generated a transgenic mouse line, namely, Capn8‐Cre, in which the expression of Cre recombinase was controlled by the promoter of the intracellular Ca²⁺‐regulated cysteine protease calpain‐8. To test the tissue distribution and excision activity of Cre recombinase, the Capn8‐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 and LacZ staining demonstrated that Capn8‐Cre transgenic mouse expressed Cre recombinase in the gastric pit cells. Cre recombinase activity was also detected in the liver and skin tissues. These data suggest that the Capn8‐Cre mouse line described here could be used to dissect gene function in gastric pit cells. genesis 47:674–679, 2009. © 2009 Wiley‐Liss, Inc.     

Generation of transgenic mice expressing Cre recombinase under the control of the Dll1 mesoderm enhancer element

To study paraxial mesoderm formation in the mouse, transgenic lines that can be used to either selectively delete or express genes of interest in the paraxial mesoderm are required. We have generated a transgenic mouse line that expresses Cre recombinase in the paraxial mesoderm (PAM) beginning at e7.5. A lacZ Cre recombinase reporter line showed that in addition to PAM and its derivatives, lateral plate and intermediate mesoderm derivatives were also exposed to Cre activity, while the node, notochord, and cardiac mesoderm were not. We further demonstrate that 70–75% of the fibroblasts generated from Dll1‐msd Cre, ROSA26‐rtTA embryos possess Cre recombinase activity. These mice can therefore be used in combination with tet‐responsive transgenic lines to generate mesoderm‐derived embryonic fibroblasts that inducibly express a gene of interest. genesis 47:309–313, 2009. © 2009 Wiley‐Liss, Inc.     

A Tlx2‐Cre mouse line uncovers essential roles for hand1 in extraembryonic and lateral mesoderm

Hand1 regulates development of numerous tissues within the embryo, extraembryonic mesoderm, and trophectoderm. Systemic loss of Hand1 results in early embryonic lethality but the cause has remained unknown. To determine if Hand1 expression in extraembryonic mesoderm is essential for embryonic survival, Hand1 was conditionally deleted using the HoxB6‐Cre mouse line that expresses Cre in extraembryonic and lateral mesoderm. Deletion of Hand1 using HoxB6‐Cre resulted in embryonic lethality identical to systemic knockout. To determine if lethality is due to Hand1 function in extraembryonic mesoderm or lateral mesoderm, we generated a Tlx2‐Cre mouse line expressing Cre in lateral mesoderm but not extraembryonic tissues. Deletion of Hand1 using the Tlx2‐Cre line results in embryonic survival with embryos exhibiting herniated gut and thin enteric smooth muscle. Our results show that Hand1 regulates development of lateral mesoderm derivatives and its loss in extraembryonic mesoderm is the primary cause of lethality in Hand1‐null embryos. genesis 48:479–484, 2010. © 2010 Wiley‐Liss, Inc.     

肾上腺皮质束状带特异性表达Cre重组酶转基因小鼠的构建

肾上腺是人体重要的内分泌器官。由于缺乏肾上腺皮质束状带特异性表达Cre酶的工具鼠,目前对肾上腺皮质束状带细胞中特异表达基因的功能缺乏深入的解析。CYP11B1基因编码类固醇11β-羟化酶,该酶是糖皮质激素合成的关键酶,在肾上腺皮质束状带中特异性表达。本研究旨在利用CYP11B1基因在束状带特异性表达的特点,构建在肾上腺皮质束状带中特异性表达Cre重组酶的转基因动物。采用CRISPR/Cas9技术在CYP11B1基因终止密码子位点定点敲入2A-GfpCre表达框,获得CYP11B1-2A-GfpCre同源重组载体,进而构建CYP11B1Cre小鼠,并通过mTmG和LacZ染色确定Cre酶主要表达在小鼠肾上腺皮质束状带。在此基础上,本研究还用该工具鼠与胱硫醚-γ-裂解酶(cystathionineγ-lyase, CTH)条件性敲除鼠交配,获得了肾上腺皮质束状带CTH特异性敲除的小鼠,并证实了该动物肾上腺皮质束状带中CTH表达缺失。以上结果充分说明肾上腺皮质束状带特异性表达Cre重组酶小鼠构建成功。该工具鼠的成功构建,为深入研究肾上腺皮质束状带相关功能提供了有力工具。