巢蛋白mRNA在小鼠中枢神经系统发育过程中的表达

巢蛋白属于中等纤维基因家族,在增殖较快的神经前体细胞中表达。该基因被克隆后,作为神经前体的标记基因得到广泛应用。本文中,我们根据小鼠巢蛋白ｃＤＮＡ序列,设计了一对引物,在确定了反轩录ＰＣＲ反应的最佳反应条件后,详细地考察了小鼠巢蛋白ｍＲＮＡ在中枢神经系统发育过程中的表达规律。     

竞争性PCR检测MLC_2-糜酶融合基因在转基因小鼠中的组织特异性表达

 用竞争性 Ｐ Ｃ Ｒ 方法定量检测了大鼠肌球蛋白轻链 ２ 启动子（ｍ ｙｏｓｉｎ ｌｉｇｈｔ ｃｈａｉｎ ２ ｐｒｏｍ ｏｔ ｅｒ, Ｍ Ｌ Ｃ２） 糜酶（ｃｈｙｍ ａｓｅ）融合基因在转基因小鼠不同组织中的表达情况,发现 Ｍ Ｌ Ｃ２ ｃｈｙｍ ａｓｅ融合基因在转基因小鼠的心脏中有较高水平的表达,在骨骼肌中表达水平较低,在肾脏中有微弱表达;而在肝脏和肺中未检测到．表明 Ｍ Ｌ Ｃ２ ｃｈｙｍ ａｓｅ 融合基因改变了野生型 ｃｈｙｍ ａｓｅ 在生物体内的表达特性,不仅提高了表达效率,而且赋予了一定的心脏组织特异性,为进一步研究 ｃｈｙｍ ａｓｅ 基因在心脏中的功能奠定了基础．     

神经钙粘着蛋白在P19神经元分化中的作用

利用ＲＴ－ＰＣＲ技术,我们检测Ｐ１９细胞体外神经元分化过程中神经钙粘着蛋白（Ｎ－ｃａｄｈｅｒｉｎ）的表达模式。结果显示,该基因在上述过程中存在上调和下调过程,与体内中枢神经系统发育过程的表达模式十分相近。在此基础上,我们将神经钙粘着蛋白基因ｃＤＮＡ全长转入Ｐ１９细胞,通过药物筛选,得到稳定表达钙粘着蛋白的细胞株。     

小鼠巢蛋白基因结构及其转录调控元件的初步鉴定

通过筛选小鼠基因组ＢＡＣ文库,得到小鼠巢蛋白基因组２３．３ｋｂ ＤＮＡ序列,其中约１５．３ｋｂ已完成测序。与小鼠ｃＤＮＡ序列比较结果表明,小鼠巢蛋白基因包含３个内含子。与大鼠及人巢蛋白基因相比,小鼠巢蛋白基因中３个内含子的位置及大小均很保守。利用神经发育的体外实验模型,检测小鼠巢蛋白基因第２个内含子对荧光素酶报告基因的调控活性。系列缺失质粒转染细胞显示,小鼠巢蛋白基因第２个内含子对报告基因的转录具     

过量表达Wnt-1基因诱导P19细胞的神经分化

Ｗｎｔ－１基因在小鼠神经发育过程中起着重要的作用。该基因在胚胎性癌细胞Ｐ１９细胞经分化过程中存在瞬时性表达。利用克隆到的Ｗｎｔ－１基因转染Ｐ１９细胞,可使细胞不经视黄酸诱导,自发向神经细胞方向分化。     

Wnt信号通路关键基因β—catenin在RA诱导的P19胚胎性癌细胞神经分化 …

Ｗｎｔ信号参与了小鼠早期神经发育。我们以往的实验结果表明,Ｗｎｔ信号可引起Ｐ１９胚胎性癌细胞的神经分化。为进一步了解Ｗｎｔ信号在Ｐ１９神经分化过程中行使功能的时间,我们以Ｗｎｔ信号通路关键成员β－ｃａｔｅｎｉｎ是否定位在细胞核中作为考察Ｗｎｔ信号是否能传递到细胞核内调控下游基因活性的指标,分析了Ｗｎｔ信号在Ｐ     

小鼠植入前胚胎及几种组织中IFRG15的差异表达研究

为进一步研究干扰素α应答基因IFRG15（Interferon responsive gene 15）在小鼠整个发育过程中的表达规律,从植入前胚胎及2、5、16周龄的雌、雄昆明小白鼠心、肝、脾、肺、肾、肌肉、卵巢或睾丸等组织中提取总RNA,以HPRT1（Hypoxanthine phosphoribosyltransferase 1）为内参基因,利用RT-PCR的方法进行目的片段的扩增及差异性分析。结果表明,IFRG15在植入前胚胎8-细胞期,桑葚胚期开始显著高表达于受精卵、2-细胞期、4-细胞期（p〈0.05）,在囊胚期表达量达到最高,且显著高于其他各期（p〈0.05）;在雌雄小鼠几个组织体外发育过程中均检测到表达,但表达量有所不同,在雄性小鼠各组织中的表达无显著规律性差异;在5周龄雌性小鼠组织中达到最高（p〈0.05）,卵巢组织尤为明显,推测该基因对卵巢的成熟有重要的促进作用;本实验成功获得IFRG15在小鼠植入前各期胚胎及体外发育过程中的表达模式,为进一步探究该基因在小鼠克隆胚发育过程中的作用奠定基础。     

pINC－lacZ逆转录病毒载体的构建及其在小鼠胚胎干细胞中的表达

小鼠胚胎干细胞系（ＥＳ）是从囊胚的内细胞团中建立起来的多潜能胚胎干细胞系。ＥＳ细胞系目前正广泛用于将基因打靶后的突变和其它遗传变化导入小鼠的种系中。其中,嵌合鼠的获得是非常必要的一步。这就需要用一个可以广泛表达的基因对ＥＳ细胞进行标记。大肠杆菌β－半乳糖苷酶（β－ｇａｌ）基因的表达在细胞水平就能很容易地观察到,而且对哺乳动物细胞没有任何毒害作用,因而是一个被广泛地用于各种细胞基因表达研究中的报导基因。猿类巨细胞病毒早期（ＳｉＣＭＶＩＥ）启动子是一个广泛用于转基因小鼠研究中的强启动子。然而,该启动子在ＥＳ细胞方面应用的报道尚未见到。本研究用ＢａｍＨＩ和ＨｉｎｄⅢ双酶切,从ｐｓｖ－β－ｇａｌａｃｔｏｓｉｄａｓｅ中得到３．７Ｋｂ的ｌａｃＺ基因片断,将其插入到ｐＩＮＣ载体上（Ｆｉｇ．１）,得到ｐＩＮＣ－ｌａｃＺ（Ｆｉｇ．２）。用ＮｏｔⅠ线性化ｐＩＮＣ－ｌａｃＺ后,电击导入ＭＥＳＰＵ－１３细胞中。ＭＥＳＰＵ－１３为本实验室从１２９／Ｔｅｒ品系小鼠的囊胚中建立的一个ＥＳ细胞系。转化细胞在含有２５０μｇ／ｍｌＧ４１８的培养基上培养两周,进行ＭＥＳＰＵ－１３细胞稳定转化子的筛选。在一次转化实验中,从１ｘ１０７个转化的ＭＥＳ     

cAMP依赖性蛋白激酶调节亚单位类型1A(RIα)基因的分子生物学研究进展

ｃＡＭＰ依赖性蛋白激酶调节亚单位类型１Ａ（ＲＩα）基因是ｃＡＭＰ依赖性蛋白激酶（ＰＫＡ）基因家族的一个成员,因其在人生殖细胞的生长、发育、成熟等过程中独有其功效而受到研究者的关注。本文就近年来该基因的定位、结构、功能及表达调控等方面研究进展作一综述。     

参与小鼠神经发育的mCcd1基因的组织表达

介绍了一种可能在神经发育过程中起重要调节作用的基因mCcd1在小鼠发育过程中的mRNA和蛋白质水平的表达变化。通过对新生和成年小鼠多组织免疫杂交(Western Blot)和反转录PCR(RT-PCR)检测发现:该基因在多种组织中广泛表达,但脑部显示高表达,并且新生鼠各组织表达均高于成体。小鼠11.5 d胚胎切片免疫组化实验也支持这一结果。mCcd1在神经系统发育早期的表达暗示它可能参与了神经系统的发育过程。     

Structural characterization of Escherichia coli sialic acid synthase

Wnt-1, the vertebrate counterpart of the Drosophila wingless gene, plays an important role in the early morphogenesis of neural tissues. In this report, we have shown that overexpression of Wnt-1 can direct embryonic carcinoma P19 cells to differentiate into neuron-like cells in the absence of retinoic acid. Immunocytochemistry showed that these cells expressed neuronal markers, such as the neurofilament (NF) and microtubule-associated protein 2 (MAP2), but failed to express the glial cell marker, glial fibrillary acidic protein (GFAP). RT-PCR revealed that two basic helix-loop-helix (bHLH) genes, Mash-1 and Ngn-1, were up-regulated during the differentiation stage of Wnt-1-overexpressing P19 cells. These results suggest that the Wnt-1 gene promotes neuronal differentiation and inhibits gliogenesis during the neural differentiation of P19 cells, and that neural bHLH genes might be involved in this process.     

Homeotic factor ATBF1 induces the cell cycle arrest associated with neuronal differentiation

The present study aimed to elucidate the function of AT motif-binding factor 1 (ATBF1) during neurogenesis in the developing brain and in primary cultures of neuroepithelial cells and cell lines (Neuro 2A and P19 cells). Here, we show that ATBF1 is expressed in the differentiating field in association with the neuronal differentiation markers beta-tubulin and MAP2 in the day E14.5 embryo rat brain, suggesting that it promotes neuronal differentiation. In support of this, we show that ATBF1 suppresses nestin expression, a neural stem cell marker, and activates the promoter of Neurod1 gene, a marker for neuronal differentiation. Furthermore, we show that in Neuro 2A cells, overexpressed ATBF1 localizes predominantly in the nucleus and causes cell cycle arrest. In P19 cells, which formed embryonic bodies in the floating condition, ATBF1 is mainly cytoplasmic and has no effect on the cell cycle. However, the cell cycle was arrested when ATBF1 became nuclear after transfer of P19 cells onto adhesive surfaces or in isolated single cells. The nuclear localization of ATBF1 was suppressed by treatment with caffeine, an inhibitor of PI(3)K-related kinase activity of ataxa-telangiectasia mutated (ATM) gene product. The cytoplasmic localization of ATBF1 in floating/nonadherent cells is due to CRM1-dependent nuclear export of ATBF1. Moreover, in the embryonic brain ATBF1 was expressed in the cytoplasm of proliferating stem cells on the ventricular zone, where cells are present at high density and interact through cell-to-cell contact. Conversely, in the differentiating field, where cell density is low and extracellular matrix is dense, the cell-to-matrix interaction triggered nuclear localization of ATBF1, resulting in the cell cycle arrest. We propose that ATBF1 plays an important role in the nucleus by organizing the neuronal differentiation associated with the cell cycle arrest.     

Transient expression of the proto-oncogene int-1 during differentiation of P19 embryonal carcinoma cells.

In mouse embryos, the int-1 proto-oncogene is transiently expressed in areas of the developing neural system. Retinoic acid-treated P19 embryonal carcinoma cells have often been used as an in vitro model for the molecular basis of neural development. We shown here that int-1 is transiently expressed in differentiated P19 cells. The time course and retinoic acid dose dependence of int-1 expression suggest that the gene is specifically expressed during early neural differentiation. P19 cells may be a useful model to assist in the study, at the cellular level, of the role of int-1 in neural development.     

DIXDC1 Promotes Retinoic Acid-Induced Neuronal Differentiation and Inhibits Gliogenesis in P19 Cells

Human DIXDC1 is a member of Dishevelled-Axin (DIX) domain containing gene family which plays important roles in Wnt signaling and neural development. In this report, we first confirmed that expression of Ccd1, a mouse homologous gene of DIXDC1, was up-regulated in embryonic developing nervous system. Further studies showed that Ccd1 was expressed specifically in neurons and colocalized with early neuronal marker Tuj1. During the aggregation induced by RA and neuronal differentiation of embryonic carcinoma P19 cells, expressions of Ccd1 as well as Wnt-1 and N-cadherin were dramatically increased. Stable overexpression of DIXDC1 in P19 cells promoted the neuronal differentiation. P19 cells overexpressing DIXDC1 but not the control P19 cells could differentiate into Tuj1 positive cells with RA induction for only 2 days. Meanwhile, we also found that overexpression of DIXDC1 facilitated the expression of Wnt1 and bHLHs during aggregation and differentiation, respectively, while inhibited gliogenesis by down-regulating the expression of GFAP in P19 cells. Thus, our finding suggested that DIXDC1 might play an important role during neurogenesis, overexpression of DIXDC1 in embryonic carcinoma P19 cells promoted neuronal differentiation, and inhibited gliogenesis induced by retinoic acid. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. XT Jing and HT Wu contributed equally to this work.     

A role of N-cadherin in neuronal differentiation of embryonic carcinoma P19 cells.

N-cadherin is one of the important molecules for cell to cell interaction in the development of the central nervous system (CNS). In this report, we have shown that N-cadherin mRNA and protein were increased rapidly in retinoic acid (RA)-induced neuronal differentiation of embryonic carcinoma P19 cells. To explore possible roles for N-cadherin during this process, N-cadherin-overexpressing P19 cell lines were established. These transfected cells could differentiate into neurofilament-expressing neurons in the absence of RA. RT-PCR revealed that the expression patterns of development-related genes, such as Oct-3/4, nestin, Notch-1, and Mash-1 were similar between the transfected P19 cells and the RA-induced wild-type P19 cells during their neuronal differentiation. On the contrary, the Wnt-1 gene was up-regulated in the N-cadherin-overexpressing P19 cells, but could not be detected in the wild-type P19 cells. These results suggest N-cadherin may play a role in neuronal differentiation of P19 cells, possibly through the Wnt-1 signaling pathway.     

The expression level of the orphan nuclear receptor GCNF (germ cell nuclear factor) is critical for neuronal differentiation

The germ cell nuclear factor (GCNF) is essential for normal embryonic development and gametogenesis. To test the prediction that GCNF is additionally required for neuronal differentiation, we used the mouse embryonal carcinoma cell line PCC7-Mz1, which represents an advantageous model to study neuronal cells from the stage of fate choice until the acquirement of functional competence. We generated stable transfectants that express gcnf sense or antisense RNA under the control of a tetracycline-regulated promoter. After retinoic acid-induced withdrawal from the cell cycle, sense clones developed a neuron network with changed properties, and the time course of neuron maturation was shortened. Consistent with these data, differentiation of neuronal precursor cells was impaired in antisense cultures. This involved a delay in 1) the down-regulation of nestin, a marker for undifferentiated neuroepithelial cells and stem cells of the central nervous system, and 2) up-regulation of the somatodendritic protein microtubule-associated protein 2 and the synaptic vesicle protein synaptophysin. Neuronal cells in the antisense cultures acquired functional competence, although with a significant delay. Our data propose that the level of GCNF is critical for differentiation and maturation of neuronal precursor cells.     

SVCT2 Is Expressed by Cerebellar Precursor Cells,Which Differentiate into Neurons in Response to Ascorbic Acid

Ascorbic acid (AA) is a known antioxidant that participates in a wide range of processes, including stem cell differentiation. It enters the cell through the sodium-ascorbate co-transporter SVCT2, which is mainly expressed by neurons in the adult brain. Here, we have characterized SVCT2 expression in the postnatal cerebellum in situ, a model used for studying neurogenesis, and have identified its expression in granular precursor cells and mature neurons. We have also detected SVCT2 expression in the cerebellar cell line C17.2 and in postnatal cerebellum-derived neurospheres in vitro and have identified a tight relationship between SVCT2 expression and that of the stem cell-like marker nestin. AA supplementation potentiates the neuronal phenotype in cerebellar neural stem cells by increasing the expression of the neuronal marker β III tubulin. Stable over-expression of SVCT2 in C17.2 cells enhances β III tubulin expression, but it also increases cell death, suggesting that AA transporter levels must be finely tuned during neural stem cell differentiation.     

A two-step strategy for neuronal differentiation in vitro of human dental follicle cells

Human dental follicle cells (DFCs) derived from wisdom teeth are precursor cells for cementoblasts. In this study, we recognized that naïve DFCs express constitutively the early neural cell marker β-III-tubulin. Interestingly, DFCs formed β-III-tubulin-positive neurosphere-like cell clusters (NLCCs) on low-attachment cell culture dishes in serum-replacement medium (SRM). For a detailed examination of the neural differentiation potential, DFCs were cultivated in different compositions of SRM containing supplements such as N2, B27, G5 and the neural stem cell supplement. Moreover, these cell culture media were combined with different cell culture substrates such as gelatin, laminin, poly-l-ornithine or poly-l-lysine. After cultivation in SRM, DFCs differentiated into cells with small cell bodies and long cellular extrusions. The expression of nestin, β-III-tubulin, neuron-specific enolase (NSE) and neurofilament was up-regulated in SRM supplemented with G5, a cell culture supplement for glial cells, and the neural stem cell supplement. DFCs formed NLCCs and demonstrated an increased gene expression of neural cell markers β-III-tubulin, NSE, nestin and for small neuron markers such as neuropeptides galanin (GAL) and tachykinin (TAC1) after cultivation on poly-l-lysine. For a further neural differentiation NLCC-derived cells were sub-cultivated on laminin and poly-l-ornithine cell culture substrate. After 2 weeks of differentiation, DFCs exposed neural-like cell morphology with small neurite-like cell extrusions. These cells differentially express neurofilament and NSE, but only low levels of β-III-tubulin and nestin. In conclusion, we demonstrated the differentiation of human DFCs into neuron-like cells after a two-step strategy for neuronal differentiation.