bFGF培养体系中添加不同因子对绵羊类胚胎干细胞多能性的影响

为筛选出能够维持绵羊类胚胎干细胞(oESC-like)多能性的因子,以oESC-like为材料,在N2B27/CH/bFGF培养体系中,分别加入PD、PD/hLIF、PD/BPM4、PD/hLIF/BMP4,培养细胞8 d,分析绵羊类胚胎干细胞多能性,确定最适培养体系。结果显示:在N2B27/CH/bFGF培养体系添加PD或PD/hLIF后,oESC-like细胞生长较好;所有培养液中oESC-like细胞的AKP染色及多能性候选基因Sox2和Oct4免疫荧光蛋白染色结果均呈阳性;添加PD后,多能性候选基因Oct4与Klf4 mRNA的表达量显著高于N2B27/CH/bFGF培养体系中的(P<0.01),Nestin与Lin28的表达量显著降低(P<0.01),Sox2与c-Myc升高但差异不显著。添加PD/hLIF后,Oct4的表达量显著高于N2B27/CH/bFGF培养体系中的(P<0.01),c-Myc、Klf4、Lin28表达量升高但不显著,Nestin和Sox2表达量降低但也不显著。以上结果表明:在oESC-like培养体系中,添加PD或PD/hLIF可能有利于维持绵羊类胚胎干细胞的多能性。     

Role of histone H3 lysine 27 methylation in X inactivation

The Polycomb group (PcG) protein Eed is implicated in regulation of imprinted X-chromosome inactivation in extraembryonic cells but not of random X inactivation in embryonic cells. The Drosophila homolog of the Eed-Ezh2 PcG protein complex achieves gene silencing through methylation of histone H3 on lysine 27 (H3-K27), which suggests a role for H3-K27 methylation in imprinted X inactivation. Here we demonstrate that transient recruitment of the Eed-Ezh2 complex to the inactive X chromosome (Xi) occurs during initiation of X inactivation in both extraembryonic and embryonic cells and is accompanied by H3-K27 methylation. Recruitment of the complex and methylation on the Xi depend on Xist RNA but are independent of its silencing function. Together, our results suggest a role for Eed-Ezh2-mediated H3-K27 methylation during initiation of both imprinted and random X inactivation and demonstrate that H3-K27 methylation is not sufficient for silencing of the Xi.     

Autosomal dominant mutations affecting X inactivation choice in the mouse

X chromosome inactivation is the silencing mechanism eutherian mammals use to equalize the expression of X-linked genes between males and females early in embryonic development. In the mouse, genetic control of inactivation requires elements within the X inactivation center (Xic) on the X chromosome that influence the choice of which X chromosome is to be inactivated in individual cells. It has long been posited that unidentified autosomal factors are essential to the process. We have used chemical mutagenesis in the mouse to identify specific factors involved in X inactivation and report two genetically distinct autosomal mutations with dominant effects on X chromosome choice early in embryogenesis.     

Sall4表达调控及其启动子核心调控区的筛选

【目的】Sall4是一种锌指结构转录因子,对建立和维持动物多能干细胞具有重要意义。为探索猪 Sall4 的表达和转录调控机制,克隆了猪Sall4启动子,对其进行了功能验证和核心调控区的筛选。【方法】从猪基因组中克隆获得2.1 kb Sall4启动子片段,并构建相应的报告载体;将报告载体pE2.1转染不同种类的细胞,进行细胞特异性表达检测;采用实时荧光定量PCR方法,检测Sall4在猪不同组织和细胞中的表达变化;通过生物信息学方法,分析Sall4启动子上各种关键顺式作用元件和潜在的转录结合位点;将多能转录因子Oct4、Sox2、Klf4、Myc、Esrra/b和Smad与Sall4启动子共转染293T细胞,利用双萤光素酶报告系统检测Sall4启动子活性;采用DNA片段缺失的方法,构建一系列Sall4启动子片段缺失报告载体,将缺失片段载体分别转入293T细胞中,并利用双萤光素酶报告系统检测启动子的活性。【结果】Sall4在多能干细胞、生殖细胞和癌化肿瘤细胞中：包括P19、293T、CHO和Hela等细胞中特异性高表达。另外,通过实时荧光定量PCR检测结果显示,Sall4的表达具有明显的组织特异性,其在猪iPS细胞和生殖相关组织,如睾丸和卵巢组织中表达最高,而在脑、心、肝和肌肉等组织中的表达较低,表明该基因与细胞多能性维持具有互作关系。应用JASPAR和GPMiner软件分析发现,Sall4启动子上有TATA box、GC box、CAAT box等顺式作用元件,以及Oct4、Sox2、Klf4、Myc、Esrra/b、Stat3和Smad等多能转录因子的预测结合位点。其中,Oct4、Sox2和TGF-beta信号通路因子对Sall4启动子活性有较显著的激活作用,与对照组比较Sall4启动子活性提高了3倍;而Klf4因子对Sall4启动子活性有一定的抑制作用。采用分段PCR的方法,对2.1 kb启动子进行了片段缺失,分别构建了pL2.1、 pL1.0和pL0.5等3个报告载体,检测结果发现,在pL2.1与pL1.0之间相差1 kb左右,但是启动子的活性没有显著差异。载体pL1.0与pL0.5之间多了486 bp片段,启动子活性提高了1倍多。另外,pL0.5的启动子活性与对照组比较提高了8倍。这些结果表明,猪Sall4启动子在-367 至-852 bp和-1 至-366 bp两个区域内存在核心调控区。【结论】克隆了猪Sall4启动子,证明Sall4的表达具有明显的组织特异性;Sall4启动子序列上存在众多潜在的转录因子结合位点和启动子核心调控域,是参与调控 Sall4 表达的重要序列,这些转录因子与Sall4相互作用对Sall4在多能细胞中的表达调控发挥重要作用。     

Intersection of the RNA interference and X-inactivation pathways

In mammals, dosage compensation is achieved by X-chromosome inactivation (XCI) in the female. The noncoding Xist gene initiates silencing of the X chromosome, whereas its antisense partner Tsix blocks silencing. The complementarity of Xist and Tsix RNAs has long suggested a role for RNA interference (RNAi). Here, we report that murine Xist and Tsix form duplexes in vivo. During XCI, the duplexes are processed to small RNAs (sRNAs), most likely on the active X (Xa) in a Dicer-dependent manner. Deleting Dicer compromises sRNA production and derepresses Xist. Furthermore, without Dicer, Xist RNA cannot accumulate and histone 3 lysine 27 trimethylation is blocked on the inactive X (Xi). The defects are partially rescued by truncating Tsix. Thus, XCI and RNAi intersect, down-regulating Xist on Xa and spreading silencing on Xi.     

Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome

To equalize X-chromosome dosages between the sexes, the female mammal inactivates one of her two X chromosomes. X-chromosome inactivation (XCI) is initiated by expression of Xist, a 17-kb noncoding RNA (ncRNA) that accumulates on the X in cis. Because interacting factors have not been isolated, the mechanism by which Xist induces silencing remains unknown. We discovered a 1.6-kilobase ncRNA (RepA) within Xist and identified the Polycomb complex, PRC2, as its direct target. PRC2 is initially recruited to the X by RepA RNA, with Ezh2 serving as the RNA binding subunit. The antisense Tsix RNA inhibits this interaction. RepA depletion abolishes full-length Xist induction and trimethylation on lysine 27 of histone H3 of the X. Likewise, PRC2 deficiency compromises Xist up-regulation. Therefore, RepA, together with PRC2, is required for the initiation and spread of XCI. We conclude that a ncRNA cofactor recruits Polycomb complexes to their target locus.     

徐淮山羊多能性转录因子mRNA制备及在成纤维细胞中的表达

【目的】克隆徐淮山羊多能性转录因子Oct4、Sox2、Klf4和c-Myc的CDS片段,构建pMD19-T-oct4、pMD19-T-sox2、pMD19-T-klf4和pMD19-T-c-myc重组质粒,随后构建含T7启动子的pcDNA3-oct4、pcDNA3-sox2、pcDNA3-klf4和pcDNA3-c-myc重组质粒,通过体外转录,获得该4种多能性转录因子的mRNA,并使其在徐淮山羊成纤维细胞中稳定表达。【方法】应用RT-PCR方法分别从徐淮山羊睾丸组织、皮肤组织、小肠组织中扩增多能性转录因子Oct4、Sox2、Klf4和c-Myc基因编码全序列,将该4种基因分别克隆到pMD19-T载体,构建pMD19-T-oct4、pMD19-T-sox2、pMD19-T-klf4和pMD19-T-c-myc重组质粒。然后将pcDNA3.0载体和pMD19-T重组载体双酶切后用T4连接酶连接,构建含有T7启动子的真核表达载体pcDNA3-oct4、pcDNA3-sox2、pcDNA3-klf4和pcDNA3-c-myc重组质粒。各重组质粒分别用限制性内切酶XhoI、XbaI单酶切,酶切后质粒模版按照体外转录试剂盒说明体外转录获得各多能性转录因子的mRNA,并对获得的mRNA进行检测,确定其稳定性和浓度。按照脂质体（体积）﹕mRNA（质量）为1﹕1的比例用脂质体2000转染。转染24 h后,利用Western blot技术、间接免疫荧光实验检测徐淮山羊多能性转录因子Oct4、Sox2、Klf4和c-Myc基因的mRNA在成纤维细胞中的表达。【结果】①克隆得到的徐淮山羊Oct4、Sox2、Klf4和c-Myc基因编码序列全长分别为1 083、962、1 434和1 320 bp,并经TA克隆测序验证,其CDS 序列与绵羊、人、牛和猪等的序列相似性在89%以上;②体外转录获得的4种多能性转录因子的mRNA经脂质体转染徐淮山羊成纤维细胞,在成纤维细胞中均定位于细胞核;③4种多能性转录因子的mRNA在徐淮山羊成纤维细胞中表达的蛋白与预期大小一致,分别为38、34、50和48 kd。【结论】成功克隆了徐淮山羊Oct4、Sox2、Klf4和c-Myc基因,该4种多能性转录因子的mRNA能够在徐淮山羊成纤维细胞中稳定表达,为进一步研究Oct4、Sox2、Klf4和c-Myc基因的功能和山羊体细胞的重编程奠定基础。     

The Xist RNA gene evolved in eutherians by pseudogenization of a protein-coding gene

The Xist noncoding RNA is the key initiator of the process of X chromosome inactivation in eutherian mammals, but its precise function and origin remain unknown. Although Xist is well conserved among eutherians, until now, no homolog has been identified in other mammals. We show here that Xist evolved, at least partly, from a protein-coding gene and that the loss of protein-coding function of the proto-Xist coincides with the four flanking protein genes becoming pseudogenes. This event occurred after the divergence between eutherians and marsupials, which suggests that mechanisms of dosage compensation have evolved independently in both lineages.     

Generation of mouse induced pluripotent stem cells without viral vectors

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by introducing Oct3/4 and Sox2 with either Klf4 and c-Myc or Nanog and Lin28 using retroviruses or lentiviruses. Patient-specific iPS cells could be useful in drug discovery and regenerative medicine. However, viral integration into the host genome increases the risk of tumorigenicity. Here, we report the generation of mouse iPS cells without viral vectors. Repeated transfection of two expression plasmids, one containing the complementary DNAs (cDNAs) of Oct3/4, Sox2, and Klf4 and the other containing the c-Myc cDNA, into mouse embryonic fibroblasts resulted in iPS cells without evidence of plasmid integration, which produced teratomas when transplanted into mice and contributed to adult chimeras. The production of virus-free iPS cells, albeit from embryonic fibroblasts, addresses a critical safety concern for potential use of iPS cells in regenerative medicine.     

Transient homologous chromosome pairing marks the onset of X inactivation

Mammalian X inactivation turns off one female X chromosome to enact dosage compensation between XX and XY individuals. X inactivation is known to be regulated in cis by Xite, Tsix, and Xist, but in principle the two Xs must also be regulated in trans to ensure mutually exclusive silencing. Here, we demonstrate that interchromosomal pairing mediates this communication. Pairing occurs transiently at the onset of X inactivation and is specific to the X-inactivation center. Deleting Xite and Tsix perturbs pairing and counting/choice, whereas their autosomal insertion induces de novo X-autosome pairing. Ectopic X-autosome interactions inhibit endogenous X-X pairing and block the initiation of X-chromosome inactivation. Thus, Tsix and Xite function both in cis and in trans. We propose that Tsix and Xite regulate counting and mutually exclusive choice through X-X pairing.     

猪L-Myc基因的分子克隆及在细胞重编程中的应用

【目的】克隆猪L-Myc基因,并在蛋白水平表达的情况下探索其在细胞重编程中的作用,为深入研究猪L-Myc基因替代c-Myc诱导多能干细胞（induced pluripotent stem cell,iPSC）奠定基础。【方法】先通过NCBI序列比对,采用RT-PCR克隆猪L-Myc基因cDNA,生物信息学分析猪L-Myc基因与人和小鼠的同源性,构建融合表达载体pEGFP/L-Myc-C1,通过载体转染和免疫印记检测猪L-Myc基因cDNA的蛋白水平表达;再将L-Myc基因装入逆转录病毒载体中,分别使用不同的转录因子诱导猪胎儿成纤维细胞（porcine embryo fibroblast,PEF）,通过形态变化和碱性磷酸酶（AP）染色验证猪L-Myc基因在细胞重编程中的作用。【结果】①获得了1 113 bp的猪L-Myc基因cDNA,编码364个氨基酸,理论分子质量为40 kD;②生物信息学分析显示猪L-Myc基因与人和小鼠高度同源;③免疫印记检测结果说明猪L-Myc基因cDNA能够在蛋白水平表达;④细胞诱导试验和AP染色结果显示转录因子Oct4、Sox2、Klf4和L-Myc（OSKL）组合诱导的细胞阳性克隆率明显高于Oct4、Sox2和Klf4（OSK）组合的阳性克隆率。【结论】获得了猪L-Myc基因,并且该基因在蛋白水平表达且在细胞重编程过程中起到了重要的作用。     

miRNA-145、Oct4和Sox2基因在小鼠早期胚胎发育分化中的表达情况

【目的】揭示miRNA-145、Oct4和Sox2基因与早期胚胎细胞表型间可能存在的关联,为阐明miRNA-145调控早期胚胎发育分化的分子机制奠定基础。【方法】利用手术法收集SPF级昆明小白鼠体内生产的2-细胞胚胎和囊胚,采用SYBR Green I实时荧光定量PCR检测小鼠2-细胞胚胎和囊胚中miRNA-145、Oct4和Sox2基因的表达情况。【结果】在小鼠早期胚胎由2-细胞胚胎发育至囊胚的过程中,miRNA-145在囊胚中的表达量显著高于在2-细胞胚胎中的表达量（P〈0.05,下同）,而Oct4和Sox2在囊胚中的表达量显著低于在2-细胞胚胎中的表达量。【结论】miRNA-145基因表达量上调与Oct4、Sox2基因表达量下调是随着小鼠早期胚胎发育分化的进行同时发生。     

Research on Isolation and Clone of Embryonic Stem Cell-Like in Bovine

Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary marine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS ,0.1μmol/L Na2SeO3, 0. 1mmol/L β-mercaptoethanol, 1 000ng/ml LIF,10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine), then, we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage marine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0. 125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification, karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency.     

ROCK选择性抑制剂Y-27632提高小鼠胚胎干细胞传代效率研究

以小鼠胚胎干细胞(ES细胞)为研究对象,探讨ROCK选择性抑制剂Y-27632在细胞传代过程中的作用,并对ES细胞相关生物学特性进行检测。结果表明:Y-27632能够引起ES细胞形态发生改变,通过Y-27632的添加能够使质地紧密的ES细胞集落变得松散、扁平。将Y-27632应用于ES细胞传代,能够显著提高传代效率。并且,在Y-27632长期存在的条件下,ES细胞依旧维持稳定的染色体数目,较强的碱性磷酸酶活性,在体内能够分化形成畸胎瘤,在体外能够分化形成心肌细胞。说明Y-27632的使用能够显著提高小鼠ES细胞的传代效率;紧密的细胞连接不是维持ES细胞多能性的必要条件。     

龙眼胚性愈伤组织长期继代培养及其染色体数目变异

从龙眼幼胚培养诱导并筛选出的松散型胚性愈伤组织 ,在继代培养基上已培养 6 a以上 ,所保持的若干愈伤组织仍具有强烈的体细胞胚胎发生能力 .经细胞学检查 ,其中个别愈伤组织系的部分愈伤组织团发现有2 n=3x=4 5,或 2 n=4 x=6 0等染色体数目的变异细胞 ,这对于离体筛选龙眼细胞突变体很有价值     

播期·钾肥及钙硼比对油菜根肿病的控病效应研究

[目的]为了明确不同的播期、钾肥和钙硼比例对油菜根肿病的控制效果。[方法]通过3因素5水平二次通用旋转组合设计方案,建立回归模型及对其进行解析、寻优和频数分析。[结果]结果表明,所设各试验因素对油菜根肿病控病效应的大小顺序为播种期（X1）〉钾肥（X3）〉钙硼比（X2）。[结论]3因素对防治油菜根肿病的最优配比方案：播种期为10月10日或者11日,钙硼比[氢氧化钙Ca（OH）2/硼砂]比值为26.85,钾肥（KCl）8.37 g/m^2。     

昆嵛山萱草属植物细胞学研究及其应用分析

研究了两种来自昆嵛山萱草属(H.emerocallis)植物的染色体数目、核型,结果表明:黄花菜(H.citrina)的体细胞中期染色体核型:2n=2X=22=16m+4sm+2T,核型不对称系数为58.9%,染色体相对长度组成:2n=2X=22=6L+10M1+6S,染色体总体积为105.39μm3;北萱草(H.esculenta)体细胞中期染色体核型:2n=2X=22=20m+2T,核型不对称系数为53.8%,染色体相对长度组成:2n=2X=22=4L+2M2+14M1+2S,染色体总体积为99.43μm3。同时对染色体的性状进行了巢式方差分析,表明居群内个体间、细胞间染色体的形态和大小具有一定程度的变异,且居群内的变异主要来自于个体间;最后分析了该属植物的应用前景。