早期胚胎发育母源基因的表达调控

精卵细胞是自身细胞系谱发生的产物 ,具有与双亲生物环境相互作用发育的先天性遗传。受精是新个体发育的起始点 ,是基因表达在胚胎发育过程中的选择性和时间上的规律顺序[1,2 ] 。基因组内各个基因表达的选择性和程度 ,无论是由单基因的突变引起或是多基因的复杂影响引起 ,都随时间、位置和环境条件的不同而发生改变[3 ] 。基因表达的变化是控制个体发生的细调节中心 ,决定着所有的生命过程。1 .早期胚胎发育的物质基础伴随卵母细胞生长的是核糖体和信使ＲＮＡｓ转录活化 ,Ｐｏｌｙ(Ａ)ｍＲＮＡ的合成约占总ＲＮＡ的 2 0 % ,小鼠大约在排卵…     

基因转录调控与早期三维生命体模式的构建

从分子生物学的角度而言,胚胎发育就是基因的时空调控过程,从而构建动物的三维模式.顺式调控是发育基因的基本原理;转录调控因子的激活与抑制决定了细胞分化与结构形成.     

Time-Resolved Human Kinome RNAi Screen Identifies a Network Regulating Mitotic-Events as Early Regulators of Cell Proliferation

Analysis of biological processes is frequently performed with the help of phenotypic assays where data is mostly acquired in single end-point analysis. Alternative phenotypic profiling techniques are desired where time-series information is essential to the biological question, for instance to differentiate early and late regulators of cell proliferation in loss-of-function studies. So far there is no study addressing this question despite of high unmet interests, mostly due to the limitation of conventional end-point assaying technologies. We present the first human kinome screen with a real-time cell analysis system (RTCA) to capture dynamic RNAi phenotypes, employing time-resolved monitoring of cell proliferation via electrical impedance. RTCA allowed us to investigate the dynamics of phenotypes of cell proliferation instead of using conventional end-point analysis. By introducing data transformation with first-order derivative, i.e. the cell-index growth rate, we demonstrate this system suitable for high-throughput screenings (HTS). The screen validated previously identified inhibitor genes and, additionally, identified activators of cell proliferation. With the information of time kinetics available, we could establish a network of mitotic-event related genes to be among the first displaying inhibiting effects after RNAi knockdown. The time-resolved screen captured kinetics of cell proliferation caused by RNAi targeting human kinome, serving as a resource for researchers. Our work establishes RTCA technology as a novel robust tool with biological and pharmacological relevance amenable for high-throughput screening.     

High Throughput Gene Expression Analysis Identifies Reliable Expression Markers of Human Corneal Endothelial Cells

Considerable interest has been generated for the development of suitable corneal endothelial graft alternatives through cell-tissue engineering, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as Descemet’s Stripping Endothelial Keratoplasty (DSEK) and Descemet’s Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types was also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type.     

AI429618基因在小鼠胚期表达的研究

目的:通过对与小鼠胚胎发育相关的新基因AI429618表达模式的初步分析为揭示小鼠胚胎发育机理提供研究基础.方法:利用Northern-blot和原位杂交方法对该基因进行表达谱分析.结果:Northern结果表明该基因在E12.5,E.15.5,E18.5三个时期都有所表达,并且在E12.5的小鼠胚胎中处于一个相对较高的转录水平,E15.5表达骤降并且基本上与E18.5(略高)持平;原位杂交结果显示E9.5,E10.5的小鼠胚胎中这一基因的表达集中在端脑、中脑、后脑、腮弓、前肢芽以及尾芽,E15.5的切片原位杂交中这一基因的表达信号在胸腺,肺,肝,肾,小肠中极为显著.结论:AI429618基因在小鼠胚胎发育期有着持续广泛的表达,可能对胚胎的正常发育起着重要的调控作用.     

High-throughput Functional Genomics Identifies Regulators of Primary Human Beta Cell Proliferation

The expansion of cells for regenerative therapy will require the genetic dissection of complex regulatory mechanisms governing the proliferation of non-transformed human cells. Here, we report the development of a high-throughput RNAi screening strategy specifically for use in primary cells and demonstrate that silencing the cell cycle-dependent kinase inhibitors CDKN2C/p18 or CDKN1A/p21 facilitates cell cycle entry of quiescent adult human pancreatic beta cells. This work identifies p18 and p21 as novel targets for promoting proliferation of human beta cells and demonstrates the promise of functional genetic screens for dissecting therapeutically relevant state changes in primary human cells.     

斑马鱼胚胎发育过程中FGF3基因的表达

为了解斑马鱼胚胎发育过程中FGF3基因的时空性表达情况,并探讨其对胚胎发育的调控作用,该研究分别提取2,4,8,12,24,36,48,72hpf斑马鱼胚胎的总RNA,经逆转录成cDNA,实时荧光定量PcR检测FGF3基因mRNA表达量;扩增FGF3基因特异片段,构建pGEM-T／FGF3基因片段重组质粒,经克隆及测序验证后,合成地高辛标记的反义RNA探针,以整体原位杂交法检测斑马鱼胚胎FGF3基因的空间性表达。结果显示：FGF3P基因在2hp胚胎就有表达,并持续至胚胎孵化,12hpf胚胎FGF3表达量达到高峰（P〈0．01）;胚胎发育过程中心表达部位以头、尾、咽弓为主。由此得出结论,FGF3主要在胚胎发育早期表达,其表达可能与胚胎脑、眼、耳、咽弓及尾部器官的发育调控有关。     

人EYA基因家族的克隆、表达及转录活性分析

采用PCR方法从人组织或细胞cDNA文库中扩增Eya基因家族的完整编码序列,将所扩增的基因克隆到带FLAG标签的真核表达载体上,转染人胚肾293T细胞,Western blot鉴定Eya基因家族的表达。检测Eya基因及其与Six基因共转染对MEF3-Luc转录活性的影响。经限制性内切酶分析和DNA序列测定鉴定构建的重组表达载体正确,通过Western blot实验证明Eya基因家族真核表达载体构建成功。荧光素酶活性测定显示Eya能协同Six激活肌细胞生成素的表达。本研究进一步证实Eya是Six的转录共激活因子,能提高肌细胞增强因子的活性。     

人早胚发育相关蛋白ZNF268的抗体制备

目的:获得纯化抗原用于制备ZNF268的多克隆抗体。方法:PCR扩增目的基因片Spacer区序列,亚克隆入融合蛋白表达载体pET28a+,构建了重组质粒pET28a+/SPA。然后将该重组质粒转化E.coliDE3(Rosseta),IPTG诱导表达,SDS-PAGE分离,获纯化融合蛋白6His-SPA。用6His-SPA免疫家兔,颈动脉取血,分离血清,从血清中获得ZNF268特异的多克隆抗体。结论:通过构建融合蛋白重组表达质粒pET28a+/SPA,用获得的初步纯化融合蛋白6His-SPA,制备了特异的ZNF268的多克隆抗体6His-SPA Ab。     

大鼠胰腺胚胎发育功能相关基因表达谱的分析

目的:研究大鼠胰腺胚胎发育不同阶段的基因表达谱,对比其功能相关基因随大鼠胰腺发育的变化.方法:采用显微分离及提取技术获得胚胎发育不同阶段胰腺组织并提取RNA,采用高密度寡核普酸芯片(Affemetrix芯片)对胚胎发育至第12.5天、15.5天、18.5天胚胎胰腺及成年胰腺进行基因转录水平分析,用生物信息学方法分析具体基因的表达情况.结果:胰腺的生物学功能尤其beta细胞功能相关基因insulin RNA,amylopsin RNA,GLUT-2 RNA等在胚胎15.5及18.5天显著高表达.结论:E15.5到E18.5直至出生是胰腺功能完善和成熟的阶段,这个时期以细胞功能成熟为主.