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。     

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.     

Thyrocyte-specific expression of Cre recombinase in transgenic mice

A transgenic mouse line that expresses Cre recombinase under control of the human thyroid peroxidase (TPO) gene promoter was established. The activity and specificity of the TPO-driven Cre recombinase were examined by using Northern blotting and by crossing with the ROSA26 reporter transgenic mouse line. In the latter mice, Cre-mediated recombination occurred only in the thyrocytes, and recombination commenced around embryonic day 14.5, at the time during thyroid organogenesis when TPO expression begins. This study demonstrates that the TPO-Cre transgenic mouse is a powerful tool to specifically delete loxP-inserted (floxed) genes in thyrocytes and will be of great value in the study of thyrocyte-specific genes during development and/or in adult thyroids.     

Dorsal telencephalon-specific expression of Cre recombinase in PAC transgenic mice

The ability to restrict gene expression or disruption to specific regions of the brain would enhance understanding of the molecular basis for brain development and function. For this purpose, brain region-restricted promoters are essential. Here we report the isolation of a DNA fragment containing the Emx1 gene promoter, which is responsible for dorsal telencephalon-specific expression. The Cre recombinase gene was inserted into a mouse PAC (P1-derived artificial chromosome) Emx1-locus clone (PAC-Emx1#1 clone) and utilized to generate three transgenic mouse lines. In all three lines, especially Tg3, Cre-mediated recombination was highly restricted to Emx1-expressing cell lineages, from embryonic stages to adulthood. Immunohistochemical analyses showed that Cre protein is expressed in the dorsal telencephalon in all three lines in adulthood. Thus, the PAC-Emx1#1 clone contains essentially all regulatory elements necessary for Emx1 gene expression. Our results suggest that Emx1-Cre Tg3 mice and the PAC-Emx1#1 clone constitute powerful tools for dorsal telencephalon-specific gene manipulation.     

Characterization of melanocyte-specific inducible Cre recombinase transgenic mice

Conditional Cre-mediated recombination has emerged as a robust method of introducing somatic genetic alterations in an organ-specific manner in the mouse. Here, we generated and characterized mice harboring a 4-hydroxytamoxifen (OHT)-inducible Cre recombinase-estrogen receptor fusion transgene under the control of the melanocyte-specific tyrosinase promoter, designated Tyr::CreER(T2). Cre-mediated recombination was induced in melanocytes in a spatially and temporally controlled manner upon administration of OHT and was documented in embryonic melanoblasts, follicular bulb melanocytes, dermal dendritic melanocytes, epidermal melanocytes of tail skin, and in putative melanocyte stem cells located within the follicular bulge. Functional evidence suggestive of recombination in follicular melanocyte stem cells included the presence of Cre-mediated recombination in follicular bulb melanocytes 1 year after topical OHT administration, by which time several hair cycles have elapsed and the melanocytes residing in this location have undergone multiple rounds of apoptosis and replenishment. These Tyr:: CreER(T2) transgenic mice represent a useful resource for the evaluation of melanocyte developmental genetics, the characterization of melanocyte stem cell function and dynamics, and the construction of refined mouse models of malignant melanoma.     

A mouse model with tamoxifen‐inducible thyrocyte‐specific cre recombinase activity

We have created a mouse model expressing tamoxifen‐inducible Cre recombinase (CreER^T2) under the control of the thyroglobulin (Tg) gene promoter to be able to study the role of defined genetic modifications in the regulation of thyroid function. We chose the thyroglobulin promoter, as it is expressed specifically in the thyroid. In order to obtain reliable expression under the control of the Tg promoter, we used a P1 artificial chromosome (PAC) containing a large piece of the Tg promoter. A tamoxifen inducible CreER^T2 construct was selected to avoid the possible consequences of the gene deletion for the development of the thyroid gland, and to study the role of gene deletion in the adult thyroid. Transgenic lines (TgCreER^T2) carrying this construct were generated and analyzed by crossing the TgCreER^T2 mice with the ROSA26LacZ reporter strain. The activity and specificity of the Cre recombinase was tested by staining for β‐galactosidase activity and by immunohistochemistry using an anti‐Cre‐antibody. In the TgCreER^T2xROSA26LacZ reporter line, Cre‐mediated recombination occurred specifically in the thyrocytes only after tamoxifen administration, and no significant staining was observed in controls. The recombination efficiency was nearly complete, since almost all thyrocytes showed X‐gal staining. We could also induce the recombination in utero by giving tamoxifen to the pregnant female. In addition, mice expressing TgCreER^T2 had no obvious histological changes, hormonal alterations, or different response to growth stimuli as compared to controls. These results demonstrate that the TgCreER^T2 mouse line is a powerful tool to study temporally controlled deletion of floxed genes in the thyroid. genesis 52:333–340, 2014. © 2014 Wiley Periodicals, Inc.     

Generation of a conditional mouse model to target Acvr1b disruption in adult tissues

Alk4 is a type I receptor that belongs to the transforming growth factor‐beta (TGF‐β) family. It takes part in the signaling of TGF‐β ligands such as Activins, Gdfs, and Nodal that had been demonstrated to participate in numerous mechanisms ranging from early embryonic development to adult‐tissue homeostasis. Evidences indicate that Alk4 is a key regulator of many embryonic processes, but little is known about its signaling in adult tissues and in pathological conditions where Alk4 mutations had been reported. Conventional deletion of Alk4 gene (Acvr1b) results in early embryonic lethality prior gastrulation, which has precluded study of Alk4 functions in postnatal and adult mice. To circumvent this problem, we have generated a conditional Acvr1b floxed‐allele by flanking the fifth and sixth exons of the Acvr1b gene with loxP sites. Cre‐mediated deletion of the floxed allele generates a deleted allele, which behaves as an Acvr1b null allele leading to embryonic lethality in homozygous mutant animals. A tamoxifen‐inducible approach to target disruption of Acvr1b specifically in adult tissues was used and proved to be efficient for studying Alk4 functions in various organs. We report, therefore, a novel conditional model allowing investigation of biological role played by Alk4 in a variety of tissue‐specific contexts. genesis 51:120–127, 2013. © 2012 Wiley Periodicals, Inc.     

Inducible Cre recombinase activity in mouse mature astrocytes and adult neural precursor cells

Two transgenic mouse lines expressing an inducible form of the Cre recombinase (CreER(TM)) under the control of the human GFAP promoter have been generated and characterized. In adult mice, expression of the fusion protein is largely confined to astrocytes in all regions of the central nervous system. Minimal spontaneous Cre activity was detected and recombination was efficiently induced by intraperitoneal administration of tamoxifen in adult mice. The pattern of recombination closely mirrored that of transgene expression. The percentage of astrocytes undergoing recombination varied from region to region ranging from 35% to 70% while a much smaller portion (<1%) of oligodendrocytes and neural precursor cells showed evidence of Cre activity. These mouse lines will provide important tools to dissect gene function in glial cells and in gliomagenesis.     

他莫昔芬诱导的Cre重组酶在hGfapCreERT2转基因鼠小脑中的表达研究

目的探讨他莫昔芬诱导的hGfapCreERT2转基因鼠小脑中表达Cre重组酶的细胞类型。方法 hGfapCre-ERT2/Rosa26R转基因小鼠在胚胎晚期和出生早期用他莫昔芬诱导Cre重组酶表达,对小脑组织切片行X-gal染色,然后用细胞种类特异性抗体进行免疫组织化学染色,并和X-gal染色双重标记。结果在出生后第7天(P7)、第14天(P14)和第60天(P60),X-gal阳性染色和胶质细胞抗体Blbp阳性染色共标记,和神经元抗体Neun、浦肯野细胞抗体Calbindin及少突胶质细胞前体细胞抗体NG2不共标。结论自胚胎晚期第17.5天(E17.5)后用他莫昔芬诱导hGfapCreERT2转基因鼠,发现Cre重组酶特异性在小脑星形胶质细胞中表达,不在神经元、浦肯野细胞、少突胶质细胞前体细胞中表达。     

Generation of a mouse line expressing Cre recombinase in glycinergic interneurons

In caudal regions of the CNS, glycine constitutes the major inhibitory neurotransmitter. Here, we describe a mouse line that expresses Cre recombinase under the control of a BAC transgenic glycine transporter 2 (GlyT2) promoter fragment. Mating of GlyT2‐Cre mice with the Cre reporter mouse lines Rosa26/LacZ and Rosa26/YFP and analysis of double transgenic offsprings revealed strong transgene activity in caudal regions of the central nervous system, i.e., brain stem and spinal cord. Some additional Cre expression was observed in cortical and cerebellar regions. In brain stem and spinal cord, Cre expressing cells were identified as glycinergic interneurons by staining with GlyT2‐ and glycine‐immunoreactive antibodies; here, >80% of the glycine‐immunoreactive cells expressed the Cre reporter protein. These data indicate that GlyT2‐Cre mice are a useful tool for the genetic manipulation of glycinergic interneurons. genesis 48:437–445, 2010. © 2010 Wiley‐Liss, Inc.     

A tetracycline‐inducible and skeletal muscle‐specific Cre recombinase transgenic mouse

We have generated a transgenic mouse that expresses Cre recombinase only in skeletal muscle and only following tetracycline treatment. This spatiotemporal specificity is achieved using two transgenes. The first transgene uses the human skeletal actin (HSA) promoter to drive expression of the reverse tetracycline‐controlled transactivator (rtTA). The second transgene uses a tetracycline responsive promoter to drive the expression of Cre recombinase. We monitored transgene expression in these mice by crossing them with ROSA26 loxP‐LacZ reporter mice, which express β‐galactosidase when activated by Cre. We find that the expression of this transgene is only detectable within skeletal muscle and that Cre expression in the absence of tetracycline is negligible. Cre is readily induced in this model with tetracycline analogs at a range of embryonic and postnatal ages and in a pattern consistent with other HSA transgenic mice. This mouse improves upon existing transgenic mice in which skeletal muscle Cre is expressed throughout development by allowing Cre expression to begin at later developmental stages. This temporal control of transgene expression has several applications, including overcoming embryonic or perinatal lethality due to transgene expression. This mouse is especially suited for studies of steroid hormone action, as it uses tetracycline, rather than tamoxifen, to activate Cre expression. In summary, we find that this transgenic induction system is suitable for studies of gene function in the context of hormonal regulation of skeletal muscle or interactions between muscle and motoneurons in mice. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Targeted expression of Cre recombinase in macrophages and osteoclasts in transgenic mice

To develop specific conditional gene ablation in the hematopoietic myeloid-osteoclast lineage, transgenic mice expressing Cre recombinase under the control of the CD11b promotor were generated on the C57BL/6 background. The cellular specificity of Cre activity following recombination was quantified in the Z/EG reporter transgenic mice by FACS analysis with lineage-specific markers and EGFP coexpression. A high degree of recombination, as evidenced by EGFP-positive cells, was demonstrated in macrophages and granulocytes of bone marrow and spleen by the presence of double-positive cells CD11b/EGFP and Gr1/EGFP, respectively. Interestingly, the peritoneal macrophage population showed almost complete DNA recombination at large. Most important, mature osteoclast cells derived from the double transgenic bone marrow and spleen progenitors were EGFP-positive. Hence, these CD11b-Cre mice will provide a unique tool to unravel novel gene function and activities involved during osteoclast and macrophage differentiation and maturation processes.     

Generation of a germ cell-specific mouse transgenic Cre line, Vasa-Cre

Cell type-specific genetic modification using the Cre/loxP system is a powerful tool for genetic analysis of distinct cell lineages. Because of the exquisite specificity of Vasa expression (confined to the germ cell lineage in invertebrate and vertebrate species), we hypothesized that a Vasa promoter-driven transgenic Cre line would prove useful for the germ cell lineage-specific inactivation of genes. Here we describe a transgenic mouse line, Vasa-Cre, where Cre is efficiently and specifically expressed in germ cells. Northern analysis showed that transgene expression was confined to the gonads. Cre-mediated recombination with the Rosa26-lacZ reporter was observed beginning at approximately e15, and was >95% efficient in male and female germ cells by birth. Although there was a potent maternal effect with some animals showing more widespread recombination, there was no ectopic activity in most adults. This Vasa-Cre transgenic line should thus prove useful for genetic analysis of diverse aspects of gametogenesis and as a general deletor line.     

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.     

Transgenic mouse lines expressing Cre recombinase specifically in posterior notochord and notochord

During development, the organizer provides instructive signals to surrounding cells as well as contributing cells to axial structures. To dissect organizer function at different developmental stages, conditional approaches such as the Cre/loxP system for conditional mutagenesis are particularly useful. Here we describe two new Cre transgenic mouse lines, Foxa2 NFP-Cre and Nodal PNC-Cre, with activity in two organizer domains, the posterior notochord (PNC) and notochord. These lines were made using defined regulatory elements from the Foxa2 and Nodal genes that direct Cre expression in overlapping domains of the PNC and notochord. Our detailed analysis of the timing and location of Foxa2 NFP-Cre and Nodal PNC-Cre activity indicates that these lines are appropriate for conditional mutagenesis of genes expressed from early somite stages onward.     

Site-specific DNA excision in transgenic rice with a cell-permeable Cre recombinase

The removal of selected marker genes from transgenic plants is necessary to address biosafety concerns and to carry out further experiments with transgenic organisms. In the present study, the 12-amino-acid membrane translocation sequence (MTS) from the Kaposi fibroblast growth factor (FGF)-4 was used as a carrier to deliver enzymatically active Cre proteins into living plant cells, and to produce a site-specific DNA excision in transgenic rice plants. The process, which made cells permeable to Cre recombinase-mediated DNA recombination, circumvented the need to express Cre under spatiotemporal control and was proved to be a simple and efficient system to achieve marker-free transgenic plants. The ultimate aim of the present study is to develop commercial rice cultivars free from selected marker genes to hasten public acceptance of transgenic crops.     

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。     

软骨组织特异性表达Cre重组酶转基因小鼠的研制和鉴定

构建了含有软骨组织特异性Ⅱ型胶原A1启动子和Cre重组酶基因的转基因载体pcol2Al-Cre。323枚小鼠受精卵经显微注射引入转基因片段后,分别移植至14只假孕母鼠的输卵管使其发育。共得到仔鼠52只,PCR结果显示其中10只小鼠基因组上有Cre基因的整合,整合率为19．2％。用整合有Cre基因的转基因小鼠与基因组上携带LoxP位点的条件基因打靶小鼠交配,以检测Cre酶介导的重组及其组织特异性。PCR结果表明：col2Al-Cre转基因小鼠软骨组织中表达的Cre重组酶成功地介导了LoxP之间的重组。此结果通过Southern杂交得到了进一步的证实。     

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.