兔单个植入前克隆胚胎cDNA文库的构建

人与小鼠和牛在正常胚胎植入前发育过程中基因活化的研究已经取得了长足的进展 ,但是还没有对同期克隆胚胎相关研究的报道 .利用单个家兔植入前移核重构胚胎成功地构建了MⅡ卵母细胞及发育至 4 、8 细胞期的胚胎和囊胚的特异性cDNA文库 .并用 β肌动蛋白和LAPTM4α证实这类文库是可靠的 .以 8 细胞期移核重构胚cDNA文库为例 ,随机挑取克隆进行测序分析 :其中 2 3的基因EST片段可以在GenBank或EST库中找到同源序列 ,约 1 3的EST片段属于未知的新片段 ,表明这是一类重要的新兴基因资源库 (期特异性EST库 ) .这种利用单胚胎构建cDNA文库的方法 ,解决了胚胎研究材料受限的问题 ,在时间上更加精确 ,更符合胚胎发育的规律 ,也能够更加准确地反映出一些克隆胚胎的异常表型 ,是研究早期胚胎发育基因表达以及克隆胚胎再程序化基因表达的一种有效手段 .     

兔植入前移核胚中发育相关基因的差异表达分析

与早期胚胎发育相关的一些重要基因异常表达致使克隆胚细胞核的再程序化过程受阻,是导致动物克隆失败的重要原因.为了分离鉴定再程序化相关基因,我们改进了mRNA差异显示技术,成功地建立了单胚差示技术体系.以不同发育时期的兔克隆胚（MⅡ卵、2细胞、4细胞、8～16细胞克隆胚胎）为材料进行单胚差示, 分离了80个差异片段.经反向RNA印迹验证、亚克隆、序列分析及NCBI GenBank数据库检索, 结果表明：A028片段与CstF3基因有93％的同源性, 在早期胚胎发育过程中的表达有阶段特异性, 该基因在兔克隆胚的早期发育过程中起重要作用.RNA印迹显示：该基因在所检测的组织中,只在卵巢中有表达.这项研究为再程序化相关基因全长的克隆及功能研究奠定了良好的基础.     

小鼠植入前胚胎紧密化相关基因Crg1的克隆

从已获得的运用抑制消减杂交技术(Suppression Subtractive Hybridization,SSH)分离、克隆和筛选代表8-细胞早期胚胎和紧密化8-细胞胚胎差别表达基因的ESTs片段(GenBank登录号：BQ740263、BQ740251)入手,经比较二者的同源性发现这两个EST末端反向互补,拼接成一个cDNA片段,经分析此序列包含一个完整的阅读框,提交给GenBank,登录号为AY134859。根据此序列设计引物从小鼠8-细胞紧密化胚胎cDNA中经PCR扩增出目的片段,克隆入pUCm—T载体后测序而获得全长cDNA,为小鼠植入前胚胎紧密化相关基因Crg1,分析比较证明Crg1基因与AY134859基本吻合。Crg1基因的cDNA全长为810bp,只有一个外显子,编码由150个氨基酸组成,分子量理论值为17．67kD的蛋白质。与最新的小鼠基因组工作草图进行电子杂交,该基因被定位在小鼠的14号染色体上。RT—PCR实验证明在小鼠植入前各个时期的胚胎、小鼠胚胎干细胞中均有表达,在小鼠胚胎成纤维细胞中没有表达。半定量RT—PCR实验证明Crg1基因在紧密化胚胎中表达较8—细胞胚胎高。采用Northern—blot手段分析Crg1基因在成年小鼠的8种组织中的表达情况,结果表明该基因只在小鼠卵巢中有微弱的表达,转录本大小为1．2kh,而在成年小鼠的脑、心脏、肾、睾丸、肝脏、肺、脾等中没有表达。研究表明,Crg1基因可能与小鼠胚胎紧密化及保持细胞的全能性相关。     

单个植入前胚胎mRNA差异显示方法的建立

在已有的研究基础上,优化和建立了单个植入前胚胎的mRNA差异显示（single preimplantation embryo differential display polymerase chain raction,SPEDDRT-PCR）.以小鼠单个成熟的卵母细胞和单个的2细胞期胚胎作为起始材料,比较二者之间基因的表达差异.选择在卵母细胞中不表达而在2细胞期表达的差异片段.进行GenBank检索和表达序列标签(EST)库电子克隆.与多胚研究方法一样,由线粒体编码的和2细胞特异表达的NADH脱氢酶亚单位2和ATPase 6证实了SPEDDRT-PCR方法是可行而且可靠的,是一种需要材料极少、应用广泛而有效的基因分离方法.     

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。     

小鼠单个植入前胚胎SSH方法的可行性

为建立一种能够一次性分离任意两个植入前胚胎之间全部的差别表达的基因的方法 ,在已有的单胚胎操作技术的基础之上 ,对单个植入前胚胎抑制性消减杂交 (singlepreimplantationembryosuppressionsubtractivehybridization ,SPE SSH)方法进行了初步的探索 ,分离到OM2和MⅡ d 2的基因片段 ,经GenBank和文献检索发现 ,这两个基因具有在MⅡ期和 2细胞期特异性表达的特点 .利用cDNA阵列所进行的鉴定也获得了同样的结果 ,而且所使用的材料极少 ,说明SPE SSH是一种强有力的分离和识别早期发育相关基因片段的实验技术 .若与单个卵裂球分离技术相结合 ,还可用于分离和识别人类早期分子诊断的标记性基因     

Ywhaz和ATPase6在小鼠植入前胚胎中阶段特异性表达的分析

Ywhaz基因与卵裂球之间的通讯以及细胞通讯系统的建立有关,而ATPase6基因对1-细胞向2-细胞转变是非常关键的并在氧化磷酸化系统的建立中起重要作用。本文报道小鼠2-细胞期胚胎特异表达的基因的表达分析。对mRNA差异显示技术（DDRT-PCR）进行改进,使之在小鼠植入前胚胎发育领域得以应用。通过对感兴趣的差异片段进行回收,亚克隆,序列分析和反向Northern杂交,结果表明：Ywhaz和ATPase6是2-细胞期胚胎特异表达的基因。     

小鼠植入前胚胎GCN5和HDAC1表达模式及体外培养对其表达的影响

为探讨小鼠植入前胚胎组蛋白乙酰化酶GCN5（general control of nucleotide synthesis,GCN5）和组蛋白去乙酰化酶1（histone deacetylasel,HDAC1）的表达模式及常规体外培养对它们表达的影响,应用荧光免疫细胞化学技术,检测了体内和体外培养的小鼠2、4、8细胞期卵裂胚胎、桑葚胚和囊胚GCN5和HDAC1的表达。结果显示,GCN5在体内组各细胞期卵裂胚胎和桑葚胚的细胞浆内均呈高表达,细胞核内未见明显表达,而囊胚细胞的细胞浆和细胞核内均无表达：HDACl在体内组小鼠2细胞期胚胎中以细胞浆内表达为主,在其他各期胚胎均以细胞核内表达为主。囊胚期内细胞团部分细胞的细胞核内未见HDAC1表达。GCN5在体外组小鼠植入前各期胚胎均不表达。而HDAC1的表达强度明显低于体内组的。提示体外培养抑制小鼠植入前胚胎GCN5和明显降低HDAC1的表达,影响胚胎基因的正确性表达。     

斜带石斑鱼囊胚期胚胎和尾芽期胚胎差异表达基因的筛选及克隆

以斜带石斑鱼囊胚期胚胎和尾芽期胚胎分别作为检验组和驱动组,构建了石斑鱼囊胚期胚胎和尾芽期胚胎的抑制性差减杂交cDNA文库。以α-tubulin作为检测指标,显示差减效率分别高达28和27。分别取囊胚期胚胎和尾芽期胚胎各192和960个PCR阳性克隆进行斑点杂交,得到15个囊胚期和131个尾芽期的斑点杂交阳性克隆。测序和数据库比对分析表明,囊胚期15个阳性克隆中有11个已知基因的cDNA片段和没有同源性的4个cDNA片段;而在尾芽期的131个阳性克隆中,有123个已知基因的cDNA片段和8个没有同源性的cD-NA片段。用半定量RT-PCR技术分析了部分基因片段在胚胎发育过程中的表达规律和和组织分布情况。这些差异表达片段的呈现为进一步揭示石斑鱼胚胎发育、早期性别决定和性腺分化的分子机制奠定了基础。     

小鼠植入前胚胎致密化与囊胚形成的分子调控

哺乳动物胚胎植入前的发育中致密化和囊胚形成分别标志着第一次、第二次细胞分化（即细胞命运决定）的起始,是胚胎正常发育的必要条件。因此对影响致密化和囊胚形成的蛋白及调节因子的研究尤为重要。本文探讨了与致密化相关的细胞黏附蛋白、连接蛋白、细胞骨架等分子和囊胚形成相关的紧密连接蛋白、钠钾三磷酸腺苷激酶等分子的一系列调控,以及致密化和囊胚形成在细胞命运决定中的重要作用。     

Ratio of the zygote cytoplasm to the paternal genome affects the reprogramming and developmental efficiency of androgenetic embryos

Uniparental embryos have uniparental genomes and are very useful models for studying the specific gene expression of parents or for exploring the biological significance of genomic imprinting in mammals. However, the early developmental efficiency of androgenetic embryos is significantly lower than that of parthenogenetic embryos. In addition, oocytes are able to reprogram sperm nuclei after fertilization to guarantee embryonic development by maternally derived reprogramming factors, which accumulate during oogenesis. However, the importance of maternal material in the efficiency of reprogramming the pronucleus of androgenetic embryos is not known. In this study, androgenetic embryos were constructed artificially by pronucleus transfer (PT) or double sperm injection (DS). Compared with DS embryos, PT embryos that were derived from two zygotes contained more maternal material, like 10–11 translocation methylcytosine deoxygenase 3 (Tet3) and histone variant 3.3 (H3.3). Our experiments confirmed the better developmental potential of PT embryos, which had higher blastocyst rates, a stronger expression of pluripotent genes, a lower expression of apoptotic genes, and superior blastocyst quality. Our findings indicate that the aggregation of more maternal materials in the paternal pronucleus facilitate the reprogramming of the paternal genome, improving embryonic development in PT androgenesis.     

Age-Related Effect of Cells as Donors of Nuclei: On the Efficiency of the Development of Cloned Rabbit Embryos

We studied the capacity of the nuclei of rabbit fibroblasts taken from various developmental stages for reprogramming in the cytoplasm of mature aging enucleated oocytes and the development of the cloned embryos to the preimplantation stages. A negative correlation was found between the age of an animal donor of fibroblasts and the efficiency of the development of cloned embryos (r _{morula-blastocyst}= –0.826, r _blastocyst= –0.7139). A reliably decreased capacity for reprogramming of the nuclei of donor fibroblasts was shown upon the transition from prenatal development to postnatal development, as well as a trend to a decreased capacity of nuclei for reprogramming during aging. The aging of cells in the culture, at least until the tenth passage, did not affect the capacity of the nuclei of fetal fibroblasts for reprogramming and the development of cloned embryos.     

正常胚胎与核移植重构胚发育中的生物学变化比较

本文介绍了正常胚胎和核移植重构胚发育过程中的生物学变化,从细胞形态学和分子机理两方面阐述了二者之间的差异,总结了影响核移植重构胚胎发育的主要因素。在细胞形态学上着重探讨了卵子染色体结构变化对于卵重编程作用的影响,在分子水平上对卵子组蛋白与供核细胞组蛋白的置换进行了讨论,理论上为核移植效率的提高提供了借鉴。     

Analysis of nuclear reprogramming in cloned miniature pig embryos by expression of Oct-4 and Oct-4 related genes

Xenotransplantation is a rapidly expanding field of research and cloned miniature pigs have been considered as a model animal for it. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several kinds of aberrant development. A possible explanation for the developmental failure of SCNT embryos is insufficient reprogramming of the somatic cell nucleus by the oocyte. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and embryonic germ cell nuclei with Oct-4 and Oct-4 related genes (Ndp5211, Dppa2, Dppa3, and Dppa5), which are important for embryonic development, Hand1 and GATA-4, which are important for placental development, as molecular markers using RT-PCR. The Oct-4 expression level was significantly lower (P<0.05) in cloned hatched blastocysts derived from fibroblasts and many of fibroblast-derived clones failed to reactivate at least one of the tested genes, while most of the germ cell clones and control embryos correctly expressed these genes. In conclusion, our results suggest that the reprogramming of fibroblast-derived cloned embryos is highly aberrant and this improper reprogramming could be one reason of the early lethality and post-implantation anomalies of somatic cell-derived clones.     

Cleavage rate of haploid and diploid parthenogenetic mouse embryos during the preimplantation period.

The lack of a paternal genome in parthenogenetic embryos clearly limits their postimplantation development, but apparently not their preimplantation development, since morphologically normal blastocysts can be formed. The cleavage rate of these embryos during the preimplantation period gives a better indication of the influence of their genetic constitution than blastocyst formation. Conflicting results from previous studies prompted us to use a more suitable method of following the development of haploid and diploid parthenogenetic embryos during this period. Two classes of parthenogenetic embryos were analysed following the activation of oocytes in vitro with 7% ethanol: 1) single pronuclear (haploid) embryos and 2) two pronuclear (diploid) embryos. Each group was then transferred separately during the afternoon to the oviducts of recipients on the 1st day of pseudopregnancy. Control (diploid) 1-cell fertilised embryos were isolated in the morning of finding a vaginal plug, and transferred to pseudopregnant recipients at approximately the same time of the day as the parthenogenones. Embryos were isolated at various times after the HCG injection to induce ovulation, from each of the three groups studied. Total cell counts were made of each embryo, and the log mean values were plotted against time. The gradient of the lines indicated that 1) the cell doubling time of the diploid parthenogenones was 12.25 +/- 0.34 h, and was not significantly different from the value obtained for the control group (12.74 +/- 1.17 h), and that 2) the cell doubling time of the haploid parthenogenones (15.25 +/- 0.99 h) was slower than that of the diploid parthenogenones and the control diploid group.(ABSTRACT TRUNCATED AT 250 WORDS)     

DCAF13 promotes pluripotency by negatively regulating SUV39H1 stability during early embryonic development

Mammalian oocytes and zygotes have the unique ability to reprogram a somatic cell nucleus into a totipotent state. SUV39H1/2‐mediated histone H3 lysine‐9 trimethylation (H3K9me3) is a major barrier to efficient reprogramming. How SUV39H1/2 activities are regulated in early embryos and during generation of induced pluripotent stem cells (iPSCs) remains unclear. Since expression of the CRL4 E3 ubiquitin ligase in oocytes is crucial for female fertility, we analyzed putative CRL4 adaptors (DCAFs) and identified DCAF13 as a novel CRL4 adaptor that is essential for preimplantation embryonic development. Dcaf13 is expressed from eight‐cell to morula stages in both murine and human embryos, and Dcaf13 knockout in mice causes preimplantation‐stage mortality. Dcaf13 knockout embryos are arrested at the eight‐ to sixteen‐cell stage before compaction, and this arrest is accompanied by high levels of H3K9me3. Mechanistically, CRL4‐DCAF13 targets SUV39H1 for polyubiquitination and proteasomal degradation and therefore facilitates H3K9me3 removal and zygotic gene expression. Taken together, CRL4‐DCAF13‐mediated SUV39H1 degradation is an essential step for progressive genome reprogramming during preimplantation embryonic development.     

In vitro development of one-cell embryos from outbred mice: Influence of culture medium composition

Summary One-cell embryos from outbred mice (CF1, CD-1, and Dub:ICR) were cultured in various modifications of egg culture medium (ECM). The best development was observed in medium in which inorganic salts of modified T6 medium (mT6) replaced those of ECM. In this modification (TE), 66% of one-cell CF1 embryos developed into blastocysts, comared to 46 and 43% for ECM and mT6, respectively. Moreover, the cell numbers of blastocysts developing in TE (74.9±3.3) were higher than the cell numbers of those developing in ECM (55.1±2.4). The culture requirements of embryos varied between different stocks of mice: Fewer CF1 embryos developed to the blastocyst stage than either Dub:ICR embryos (90%) or CD-1 embryos (84%). Lowering the osmolarity of the medium from 300 to 280 mOsm, increasing the concentration of KC1 from 1.42 to 25 mM, or omitting lactate from the medium during Day 1 of culture did not further improve development of embryos, in contrast to previous reports. However, the time at which embryos were transferred to outgrowth medium influenced their postblastocyst development. The best development was observed when embryos were transferred on Day 4 of culture at the late morula-early blastocyst stage. This work was supported by the Office of Health and Environmental Research, U.S. Department of Energy, Washington, DC, contract DE-AC03-76-SF01012.