HSPC117 deficiency in cloned embryos causes placental abnormality and fetal death

Somatic cell nuclear transfer (SCNT) has been successfully used in many species to produce live cloned offspring, albeit with low efficiency. The low frequency of successful development has usually been ascribed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Elucidating the genetic differences between normal fertilized and cloned embryos is key to understand the low efficiency of SCNT. Here, we show that expression of HSPC117, which encodes a hypothetical protein of unknown function, was absent or very low in cloned mouse blastocysts. To investigate the role of HSPC117 in embryo development, we knocked-down this gene in normal fertilized embryos using RNA interference. We assessed the post-implantation survival of HSPC117 knock-down embryos at 3 stages: E9 (prior to placenta formation); E12 (after the placenta was fully functional) and E19 (post-natal). Our results show that, although siRNA-treated in vivo fertilized/produced (IVP) embryos could develop to the blastocyst stage and implanted without any difference from control embryos, the knock-down embryos showed substantial fetal death, accompanied by placental blood clotting, at E12. Furthermore, comparison of HSPC117 expression in placentas of nuclear transfer (NT), intracytoplasmic sperm injection (ICSI) and IVP embryos confirmed that HSPC117 deficiency correlates well with failures in embryo development: all NT embryos with a fetus, as well as IVP and ICSI embryos, had normal placental HSPC117 expression while those NT embryos showing reduced or no expression of HSPC117 failed to form a fetus. In conclusion, we show that HSPC117 is an important gene for post-implantation development of embryos, and that HSPC117 deficiency leads to fetal abnormalities after implantation, especially following placental formation. We suggest that defects in HSPC117 expression may be an important contributing factor to loss of cloned NT embryos in vivo. (C) 2010 Elsevier Inc. All rights reserved.

Identification of Differentially Expressed Genes Between Cloned and Zygote-Developing Zebrafish (Danio rerio) Embryos at the Dome Stage Using Suppression Subtractive Hybridization

Comparative analyses of differentially expressed genes between somatic cell nuclear transfer (SCNT) embryos and zygote-developing (ZD) embryos are important for understanding the molecular mechanism underlying the reprogramming processes. Herein, we used the suppression subtractive hybridization approach and from more than 2900 clones identified 96 differentially expressed genes between the SCNT and ZD embryos at the dome stage in zebrafish. We report the first database of differentially expressed genes in zebrafish SCNT embryos. Collectively, our findings demonstrate that zebrafish SCNT embryos undergo significant reprogramming processes during the dome stage. However, most differentially expressed genes are down-regulated in SCNT embryos, indicating failure of reprogramming. Based on Ensembl description and Gene Ontology Consortium annotation, the problems of reprogramming at the dome stage may occur during nuclear remodeling, translation initiation, and regulation of the cell cycle. The importance of regulation from recipient oocytes in cloning should not be underestimated in zebrafish.

Expression pattern and developmental behaviour of cellular nucleic acid-binding protein (CNBP) during folliculogenesis and oogenesis in fish

In vertebrates, folliculogeneis establishes an intricate system for somatic cell-oocyte interaction, and ultimately leads to the acquisition of their respective competences. Although the formation process and corresponding interactions are strikingly similar in diverse organisms, knowledge of genes and signaling pathways involved in follicle formation is very incomplete and the underlying molecular mechanisms remain enigmatic. CNBP has been identified for more than ten years, and the highest level of CNBP transcripts has been observed in adult zebrafish ovary, but little is known about its functional significance during folliculogeneis and oogenesis. In this study, we clone CNBP cDNA from gibel carp (Carassius auratus gibelio), and demonstrate its predominant expression in gibel carp ovary and testis not only by RTPCR but also by Western blot. Its full-length cDNA is 1402 bp, and has an ORF of 489 nt for encoding a peptide of 163 aa. And its complete amino acid sequence shared 68.5%-96.8% identity with CNBPs from other vertebrates. Based on the expression characterization, we further analyze its expression pattern and developmental behaviour during folliculogeneis and oogenesis. Following these studies, we reveal an unexpected discovery that the CagCNBP is associated with follicular cells and oocytes, and significant distribution changes have occurred in degenerating and regenerating follicles. More interestingly, the CagCNBP is more highly expressed in some clusters of interconnected cells within ovarian cysts, no matter whether the cell clusters are formed from the original primordial germ cells or from the newly formed cells from follicular cells that invaded into the atretic oocytes. It is the first time to reveal CNBP relevance to folliculogeneis and oogenesis. Moreover, a similar stage-specific and cell-specific expression pattern has also been observed in the gibel carp testis. Therefore, further studies on CNBP expression pattern and developmental behaviour will be of significance for understanding functional roles of CNBP during gametogenests. (c) 2005 Elsevier B.V. All rights reserved.

Margulis' theory on division of labour in cells revisited

Division of labour is a marked feature of multicellular organisms. Margulis proposed that the ancestors of metazoans had only one microtubule organizing center (MTOC), so they could not move and divide simultaneously. Selection for simultaneous movement and cell division had driven the division of labour between cells. However, no evidence or explanation for this assumption was provided. Why could the unicellular ancetors not have multiple MTOCs? The gain and loss of three possible strategies are discussed. It was found that the advantage of one or two MTOC per cell is environment-dependent. Unicellular organisms with only one MTOC per cell are favored only in resource-limited environments without strong predatory pressure. If division of labour occurring in a bicellular organism just makes simultaneous movement and cell division possible, the possibility of its fixation by natural selection is very low because a somatic cell performing the function of an MTOC is obviously wasting resources. Evolutionary biologists should search for other selective forces for division of labour in cells.

猕猴体细胞核移植影响因素的研究：着床前胚胎表观遗传重编程和供体细胞周期同步化

体细胞核移植（somatic cell nuclear transfer）克隆技术的成功，特别 是运用终末分化的淋巴细胞和嗅觉神经元细胞成功克隆出小鼠，证实了分化的体 细胞核潜在的发育全能性。该技术已经在多个物种上成功地得到克隆后代，在转 基因动物、基因敲除动物和疾病模型动物生产中也得到成功应用，在结合干细胞 技术的治疗性克隆和再生医学方面也取得了初步成果，展现出了具有深远意义的 应用前景。但是，目前该领域仍然存在着很多急待解决的重要问题：克隆成功率 低，克隆胚和克隆动物经常呈现发育异常，妊娠和出生前后的高死亡率。对哺乳 动物早期胚胎发育过程中DNA 甲基化、组蛋白修饰等表观遗传重编程 （epigenetic reprogramming）机制的深入了解，有助于研究体细胞核在去核卵 母细胞中的表观遗传重编程事件，进而改善克隆胚重编程效率和发育能力。 猕猴是一种重要的实验动物，在人类疾病模型和生物医药研究中有重要的意 义。本研究主要围绕猕猴体细胞克隆胚胎早期发育过程中的表观遗传重编程事件 和核移植前体细胞同步化处理这两方面展开。1），首次详细地了描绘了猕猴着床 前胚胎发育过程中整体水平的DNA 甲基化表观遗传重编程事件，研究发现在受精 卵中父本基因组形成原核后迅速地发生了去甲基化，在2 细胞期后的卵裂过程 中，母本基因组才开始逐渐地去甲基化，到桑葚胚达到最低水平，然后开始重新 （de novo）甲基化,到囊胚期时形成不对称的甲基化模式，滋养外胚层（TE）呈 现高甲基化状态，而内细胞团（ICM）呈现低甲基化状态,这一不对称模式可能是 灵长类动物特有的，其他哺乳动物呈现正好相反的不对称模式。2），研究发现， 大多数猕猴克隆胚胎的DNA 甲基化重编程存在异常,效率低。很多2 细胞期克隆 胚（67%）和8 细胞期克隆胚（50%）的核DNA 甲基化水平显著高于对应的体 外受精胚，8 细胞克隆胚之间呈现多种不同的表观遗传特征。大多数克隆囊胚的 ICM 细胞核的甲基化水平显著高于IVF 囊胚，这些异常可能是导致克隆胚胎移 植到代孕母体后发育时间不长就失败的原因。3），在核移植前对猕猴成纤维细 胞同步化处理的研究中发现，血清饥饿，细胞周期阻断剂DMSO（二甲基亚砜）、 roscovitine、aphidicolin 和indirubin 的处理都有显著的同步化效果，提高了G0+G1 期细胞的比例。经过BrdU 标记法证实了这几种处理方法抑制细胞增殖的效果，并且证实了这种周期阻滞作用是可逆的。用原位末端标记法（TUNEL）分析证 实，血清饥饿1 到4 天后细胞凋亡比例显著上升，在贴壁的细胞中约有6%发生 凋亡，而正常对照只有1%左右，而周期阻断剂处理没有增加细胞凋亡率，这提 示这些周期阻断剂可能是一种相对安全且有效的猕猴成纤维细胞处理方法。核移 植前对猕猴成纤维细胞进行处理，有助于优化体细胞核移植技术，也是改善体细 胞核在克隆胚中重编程效率的重要途径。

哺乳动物体细胞核移植影响因素的研究

题。 在已有的基础上，建立了基于单个植入前胚胎的逆转录-聚合酶链反应（RT-PCR）技术，成功实现了针对单个猕猴植入前胚胎进行可重复的，多个特定基因的表达检测。在此基础上，对一些重要的早期胚胎发育相关基因在猕猴体细胞核移植胚胎和体外受精胚胎中的表达进行了比较研究。这其中包括核仁相关蛋白基因nucleolin，nucleophosmin，fibrillarin，PAF53，UBF和mRNA早期装配相关蛋白snrpn，这些基因被认为与植入前胚胎的再程序化有着非常密切的关系。我们的研究结果发现，相对于体外受精胚胎，被检测基因在核移植胚胎中的转录出现了不同程度的异常，表现为表达丰度相对较低，这意味着体细胞核移植植入前胚胎的再程序化，或者说供体核的再程序化可能出现了异常。通过进一步对fibrillarin（胚胎合子基因组启动的标志基因）的表达研究，比较了合子基因组启动这一再程序化分子事件在核移植胚胎和体外受精胚胎中的发生时序。研究结果证实，体细胞核移植胚胎的合子基因组启动出现了明显的滞后。我们还发现这与核移植胚胎的异常卵裂速度有着一定的关联，同时说明某些源自核移植操作技术和方法的未知因素可能是造成核移植胚胎再程序化异常的主要原因。为了验证合子基因组滞后对核移植胚胎后续发育的影响，本论文研究分析了着床相关基因Mamu-AG在猕猴核移植胚胎中的表达。研究首次发现Mamu-AG mRNA只在囊胚期开始出现，这与在人类中的情况有所不同（始于8细胞时期）。在正常体外受精囊胚中，不论发育时间如何，第6和第7天的囊胚均正常表达Mamu-AG mRNA。而在核移植囊胚中，仅有第7天的囊胚检测到该mRNA，第6天的囊胚中则没有，这进一步说明了胚胎合子基因组启动的延迟是影响核移植胚胎发育的重要环节。 本研究从分子水平上对猕猴植入前胚胎基因转录和表达进行了分析研究，首次发现了在猕猴体细胞核移植胚胎中发育相关基因表达异常，以及合子基因组启动滞后的分子证据，这为我们改进现有的猕猴核移植操作程序提供了新的思路，同时也为进一步研究猕猴植入前胚胎发育的再程序化奠定了基础。

体细胞核移植影响因素的研究

尽管体细胞核移植(somatic cell nuclear transfer, SCNT)技术仍然处于 起步发展阶段，但是随着体细胞核移植技术在近些年来的飞速发展，人们已经得 到了多种哺乳动物体细胞核移植的存活后代。体细胞核移植技术在生物医药、农 业及其它领域的应用显示了这项技术的巨大发展前景。另一方面，人们发现体细 胞核移植效率低下并且体细胞克隆动物存在许多缺陷，这些主要是由于核移植技 术，供体细胞选择，体外培养系统以及卵母细胞的状态等的差异导致的。本研究 主要围绕着这些因素在体细胞核移植过程中对于克隆胚胎植入前后发育的影响 而开展，目的在于提高体细胞核移植的效率。 实验一，研究了几个因素对克隆胚胎发育和克隆效率的影响，为提高体细胞 核移植效率提供一些依据。这部分研究主要包括四倍体半克隆小鼠研究和蛋白酶 体抑制剂在克隆胚胎和孤雌激活胚胎发育中的影响。四倍体体细胞半克隆 （tetraploid semi-cloned , TSC）胚胎的体外发育结果表明，虽然TSC 胚胎可 能避免二倍体半克隆胚胎发育过程中的非整倍体现象，但是仍然不能形成胎儿。 另外，利用蛋白酶体抑制剂MG132 处理克隆胚胎的结果表明，虽然MG132 通过抑 制成熟促进因子（maturation promoting factors，MPF）活性的降低可以提高 克隆胚胎的体外发育率，但是没有改变克隆胚胎的质量。 实验二，研究了克隆技术在克隆胚胎构建和发育上的影响。首先，我们研究 了完整颗粒细胞注射到卵母细胞中所引起的变化。我们发现完整的颗粒细胞注入 到卵母细胞中之后很快引起细胞膜裂解和细胞核碎裂，从而导致激活后重构胚胎 的碎裂。这一实验表明完整供体细胞核注入的方法不适用于小鼠体细胞克隆。接 下来我们研究了不同的核移植技术――利用piezo 的直接注射法（piezoelectric microinjection，PEM）和电融合法（Electrofusion，EF）――对植入前后的克隆胚 胎以及出生后的克隆动物的影响。研究的结果发现，在PEM 法中，提高piezo 脉 冲的强度会导致供体细胞核DNA 断裂增加，使植入前克隆胚胎的细胞数目减少， 凋亡增加，从而降低克隆胚胎的质量。相反，在用EF 法产生的胚胎中细胞核DNA 的断裂很少，克隆胚胎质量相对较好。实验的结果表明，由于两种克隆技术利用 的原理不同，会对克隆胚胎的发育产生一定的影响，从而改变克隆的效率。 实验三，研究了猕猴体细胞克隆胚胎早期发育过程中细胞核内有丝分裂器蛋 白[Nuclear Mitotic Apparatus Protein，NuMA]的表达和分布。尽管在有些猕猴体 细胞核移植胚胎中没有检测到NuMA 的正确表达和分布并且有些胚胎出现了微 管组装异常，但是大多数猕猴体细胞核移植胚胎具有正常的NuMA 蛋白表达和 正常的核型。通过改进操作技术我们得到了可以发育到正常囊胚的猕猴体细胞核 移植胚胎。这些结果提示猕猴SCNT 胚胎发育失败的原因不是NuMA 等重要蛋 白的缺失。

猕猴胚胎干细胞自我更新机理及WNT 信号通路在猕猴胚胎早期发育中作用的研究

尽管大部分动物实验是在啮齿类动物上开展的，我们仍然相信涉及人类的许多问题，如胚胎干细胞的体内功能、调亡和肿瘤形成等只有在非人灵长类模型上才能得到最好的回答。猕猴(标准的非人灵长类动物模型)在解剖、生理和代谢方面都和人类非常相似。人类很多神经疾病，如阿尔茨海默氏病、帕金森病，只能在非人灵长类模型上才能精确建模。所以研究猕猴胚胎干细胞自我更新的原理及猕猴胚胎早期发育，对研究免疫排斥，检测基于胚胎干细胞的治疗的可行性，安全性和有效性具有重要意义。本文一方面对胚胎干细胞维持自我更新和多潜能性的机理研究进行了综述，另一方面对以下两个方面的内容进行了研究： 1）运用寡核苷酸芯片和定量PCR 验证的方法来分析五株猕猴饲养层细胞的表达模式，期望发现在支持性和非支持性的饲养层细胞中差异性表达的基因。我们着重定位于饲养层胞外空间和细胞膜上的细胞因子，因为这些因子可以通过直接接触或通过膜结合受体激活下游信号通路，并最终促进猕猴胚胎干细胞的自我更新。我们发现在支持性的饲养层中有八个基因是高表达的，他们是GREM2， bFGF，KITLG，DKK3，GREM1，AREG，SERPINF1 和LTBP1； 经定量PCR 验证的SCF，bFGF 和GREM2 的表达情况都和芯片数据吻合。 2）为了描述在IVF (in vitro fertilized, 体外受精)，ICSI (intracytoplasmic sperm injection, 单精注射)，SCNT (somatic cell nuclear transfer, 体细胞核移植)和孤雌生殖猕猴囊胚中WNT 信号通路的表达情况，我们运用了信号通路特异性PCR Array 系统及免疫细胞化学来检测mRNA 和蛋白表达水平。其中，ICSI 作为IVF 胚胎的参照组，以排除显微操作对胚胎质量的影响。结果，我们发现非经典WNT/JNK 信号，而不是经典WNT 信号通路，在IVF 正常胚胎发育中起作用。而体细胞核移植和孤雌生殖的胚胎的WNT 信号通路基因表达明显高于正常胚胎。WNT 信号通路基因的表达模式可以作为胚胎质量的一个指示标准，有助于回答为什么猕猴 SCNT 和孤雌生殖胚胎发育异常。

评价水产动物生长性能的生理生化指标和分子生物学指标

以奥利亚罗非鱼(Oreochromis aureus)为实验对象，设计了3种不同的摄食类型，分别是鲜活饵料组、饥饿3周后饱食投喂组和人工饲料组。鲜活饵料组投喂冰冻赤子爱胜蚓，利用蚯蚓体内丰富的营养成分和活性物质，以期获得奥利亚罗非鱼良好的生长状况；饥饿后饱食组是指饥饿3周后，以人工饲料饱食投喂2周，用于研究饥饿与补偿生长获得快速生长时血液理化指标的变化情况；人工饲料组作为对照组。纯淡水条件下养殖，水温25±2℃。测定了奥利亚罗非鱼在3种摄食类型饲喂下某些血液生理生化指标变化的情况，并将指标变化情况与增重率做相关性分析，试图找出能够反映奥利亚罗非鱼生长性能的血液生理生化指标。 研究结果表明，奥利亚罗非鱼在饥饿3周后获得了补偿生长，补偿生长时的增重率和特定生长率显著高于人工饲料组(P<0.05)，高于鲜活饵料组，但差别不显著；相关性分析研究表明血清总蛋白、胆固醇、四碘甲状腺原氨酸（T4）与增重率极显著相关(P<0.01)，血红蛋白显著相关(P<0.05)，红细胞、白细胞、碱性磷酸酶高度相关(相关系数为0.580、0.551和0.557)，因此，建议血清总蛋白、胆固醇和血红蛋白可作为能够反映罗非鱼生长性能的新指标。 根据序列设计引物，PCR反应条件：变性温度：95 ℃，3 min；退火温度：57℃，20 sec；延伸温度：72℃，5 min，共36个循环，从牙鲆、黑鲪和鲈鱼中克隆出胰岛素样生长因子（IGF-Ⅰ）部分序列，首次证实了IGF-Ⅰ在3种海水鱼中的存在。 利用蛋氨酸与ZnSO4•7H2O，在pH 5.5、80℃下，反应1小时，采用蛋氨酸与硫酸锌2：1的配料比，合成出了产物蛋氨酸螯合锌，蛋氨酸螯合锌外观白色，粉状，室温下微溶于水，不溶于乙醇，并用原子吸收光谱法测定其含锌量为15%，螯合率为88.2%。

A Particle-in-cell scheme of the RFQ in the SSC-Linac

A 52 MHz Radio Frequency Quadrupole (RFQ) linear accelerator (linac) is designed to serve as an initial structure for the SSC-Linac system (injector into Separated Sector Cyclotron). The designed injection and output energy are 3.5 keV/u and 143 keV/u, respectively. The beam dynamics in this RFQ have been studied using a three-dimensional Particle-In-Cell (PIC) code BEAMPATH. Simulation results show that this R,FQ structure is characterized by stable values of beam transmission efficiency (at least 95%) for both zero-current mode and the space charge dominated regime. The beam accelerated in the RFQ has good quality in both transverse and longitudinal directions, and could easily be accepted by Drift Tube Linac (DTL). The effect of the vane error and that of the space charge on the beam parameters have been studied as well to define the engineering tolerance for RFQ vane machining and alignment.