Reconstruction of human embryos derived from somatic cells

Reconstruction of human nuclear transfer embryos is a necessary step of therapeutic cloning. In this study we injected somatic cell nuclei into M Ⅱ oocytes and activated reconstructed oocytes with calcium ionophore A23187 (CaA) and 6-dimethylaminopurine (6-DMAP). After oocyteactivation and 2PN formation, we removed the female PN.By using this method, we avoided the application of DNA fluorescent stain and ultraviolet light for oocyte enucleation,and over elimination of ooplasm was also mitigated. Some reconstructed embryos developed into the blastocyst stage in vitro.     

花背蟾蜍早期发育中内胚层细胞核的分化

以细胞核移植的方法,探讨花背蟾蜍早期发育中内胚层细胞核的分化。实验按供体胚胎的不同发育时期分7组,并以囊胚期动物极区细胞核的移植作对照。结果表明:原肠晚期以前的内胚层细胞核与囊胚期动物极区细胞核一样,是尚未分化的,它们仍具有发育的全能性。从神经胚期开始,细胞核有了明显的分化,但这种分化是渐进的,至摄食期的蝌蚪仍有部分细胞核保持着促使卵子发育的功能。     

成纤维细胞和ES细胞在小鼠和兔去核卵母细胞内发育

以昆明白小鼠成纤维细胞和胚胎干（ES）细胞作为供核细胞,以昆明白小鼠和日本大耳白兔的MⅡ期去核卵母细胞作为受体,采用核移植方法,构楚了克隆胚胎．在同种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率明显低于以成纤维细胞为供核细胞的克隆胚胎卵裂率（24．4％相对于56．9％,P〈0．05）,1．8％的ES细胞克隆胚胎发育到囊胚阶段,而成纤维细胞克隆胚胎没能发育到囊胚阶段;在异种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率（89．6％）和囊胚发育率（18．8％）明显高于以成纤维细胞为供核细胞的克隆胚胎卵裂率（54．2％）和囊胚发育率（4．2％）．     

小鼠早期胚胎的全胚原位杂交技术

目的:探索一种适合于早期胚胎基因表达型检测的全胚原位杂交技术.方法:收集小鼠囊胚、固定;制备VASP、IL1R2基因特异性地高辛标记的cRNA探针;对囊胚进行杂交前处理、全胚原位杂交、抗体处理、显色以及显色后处理与镜检.结果:VASP、IL1R2特异性表达在囊胚的内细胞团细胞和滋养层细胞中.结论:小鼠早期胚胎全胚原位杂交技术适用于早期胚胎基因时空表达型的检测.     

Effects of different nuclear transfer and activation methods on the development of mouse somatic cell cloned embryos

A group of adult somatic cell cloned mice were obtained by using cumulus cells as nuclei donor cells. To study the effect of different nuclear transfer (NT) and activation methods on the development of mouse cloned embryos, embryos were reconstructed using two traditional NT methods (electrofusion and direct injection) and four activation treatments (electric pulse, ethanol, SrCl2 and electric pulse combined with SrCl2). The data showed that the efficiency of reconstruction using the direct injection method is significantly higher (90.7%) than that of the electrofusion method (49.7%). Parthenogenetic embryos can develop to blastocyst stage with three activation conditions, including ethanol, electric pulse and SrCl2; however, the rates of development to blastocyst after ethanol and electric pulse acti-vation (52.4%, 54.2%) are significantly lower than after SrCl2 activation (76.9%). Treatment of embryos for 6 h with 10 mmol/L SrCl2 was found to be the best condition for activation of parthenogenetic as well as reconstructed embryos. By contrast, reconstructed embryos failed to develop to blastocyst stage after being activated by ethanol. The use of either injection or electrofusion for embryo reconstruction affected the pre-implantation development. However, after transfer in pseudopregnant mice, cloned mice were obtained from both methods.     

Detection of virus-specific sequences in Drosophila melanogaster mutants induced by injection of RSV DNA into early embryos

The injection of purified Rous sarcoma virus (RSV) (Prague strain) into Drosophila melanogaster (Oregon R line) eggs changes the fly phenotype in certain cases, and RSV-specific sequences can be identified in the Drosophila genome (ref. 1 and preceding paper). Here we have used Southern blotting to analyse in greater detail the proviral DNA present in several mutant lines of D. melanogaster produced by microinjection of intact RSV or plasmid DNA containing the viral insert. In certain populations of flies, RSV provirus was found to be incorporated into cellular DNA, and in one mutant family the unintegrated form of plasmid DNA was identified. Generally, the presence of injected genetic material in fly cells correlated with morphological changes in Drosophila.     

腺病毒载体介导GFP基因在鸭胚和雏鸭体内的表达

本试验旨在探讨腺病毒在鸭胚和雏鸭体内的感染情况,为腺病毒作为外源基因载体在活体上进行RNA干涉奠定基础．将GFP基因重组腺病毒（Adv-GFP）静脉注射15日龄鸭胚和10日龄雏鸭,在感染后不同时间采集组织器官,制作冰冻切片,荧光显微镜观察GFP基因在鸭胚和雏鸭组织器官中的表达．结果发现,GFP基因在Adv．GFP感染后24-72h能在鸭胚肝脏及心肌细胞中表达,在感染后1-5d,GFP基因可在雏鸭肝脏、脾脏、腔上囊中稳定表达．本试验首次证明腺病毒作为安全的外源载体,可介导外源基因在鸭胚和雏鸭体内的靶器官中持续稳定表达,为在鸭上进行病毒病的基因治疗提供了基础数据．     

向日葵未成熟胚体细胞胚胎发生的研究

以不同基因型向日葵未成熟胚为材料,从幼胚的大小、蔗糖浓度、碳水化合物(糖类)、激素(2,4-D和6-BA)等方面探讨了向日葵体细胞胚胎发生的影响因素.结果表明:幼胚≤2 mm时,体细胞胚胎发生频率较高(45.5%);体细胞胚胎发生的适宜培养基为MS 0.4 mg/ L 2,4-D 120 g/ L蔗糖.     

丹酚酸B诱导分化的小鼠胎脑神经干细胞膜结构特征

目的:观察丹酚酸B诱导小鼠胎脑神经干细胞分化后细胞膜结构.方法:取孕13.5 d小鼠胎脑,机械分离神经干细胞,用Nestin免疫细胞化学进行鉴定.丹酚酸B诱导其分化为神经元样细胞,用原子力显微镜观察分化后的细胞膜结构.结果:神经干细胞经诱导分化为典型的神经元形态,细胞呈聚集悬浮生长,发出突起和分支.用原子力显微镜观察到对照组细胞膜有一些形态不规则,边缘光滑的窗孔样结构及小凹,膜表面粗糙,丹酚酸组亦有相似结构,窗孔样结构大而深且小凹聚集,其密度较对照组明显增大(P＜0.05),膜粗糙度与对照组无显著差异(P＞0.05).结论:丹酚酸B诱导神经干细胞分化的神经元细胞膜的窗孔样结构与小凹明显增多,可能与增强细胞内外物质交换功能和促进细胞内信号转导有关.     

Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification

The failure of complex mammalian organs, such as the kidney, to function following freezing to low temperatures is thought to be due largely to mechanical disruption of the intercellular architecture by the formation of extracellular ice. Classical approaches to the avoidance of ice formation through the imposition of ultra-rapid cooling and warming rates or by gradual depression of the equilibrium freezing point during cooling to -80 degrees C have not been adequate. An alternative approach relies on the ability of highly concentrated aqueous solutions of cryoprotective agents to supercool to very low temperatures. At sufficiently low temperatures, these solutions become so viscous that they solidify without the formation of ice, a process termed vitrification. When embryo suspensions are cryopreserved using conventional procedures, this supercooling behaviour allows intracellular vitrification, even in the presence of extracellular ice. We have therefore used mouse embryos to examine the feasibility of obtaining high survival following vitrification of both the intra- and extracellular solutions and report here that in properly controlled conditions embryos seem to survive in high proportions after cryopreservation in the absence of ice.     

Clone of Chinese Jinan red-cross yellow cattle and evaluation of reproductive characteristics of cloned calf

Somatic cell clone technology is a viable approach to preserving endangered livestock and wildlife genetic resources. In the present research, somatic cell nuclear transfer （SCNT） was performed using granulose cells from the critical endangered Chinese red-cross yellow cattle as donor cells. A total of 211 oocytes were manipulated and 166 （79%） of them were successfully enucleated. 112 （67.4%） SCNT embryos were reconstructed, 94 （83%） of them cleaved, and 48 （43 %） of them developed to blastocyst stage. SCNT blastocysts were transferred to 6 Holstein recipients, and 2 （33%） of them were found to be pregnant. One of them maintained to term and delivered a calf, whereas another aborted. Effect of different fusion buffer （mannitol vs. Zimmerman fusion buffer） and different activation methods （calcium ionophore＋6-DMAP vs. cycloheximide＋CB） on fusion rate and development of SCNT embryos were investigated. The results indicated that： （i） on condition of two DC pulses of 2.5 kV/cm for 10 μs each, fusion rates were higher in mannitol solution than in Zimmerman fusion buffer （71% vs. 61%, respectively, p 〈 0.05）, but the blastocysts rates did not differ between two treatments （36 % vs. 39 %, p〉0.05 ）; （ii） There was no significant difference in development rates to the blastocyst stage for SCNT embryos activated by calcium ionophore＋6-DMAP or by cycloheximide＋CB （42% vs. 46%, respectively, p〉0.05）. Microsatellite DNA analysis examining 28 loci confirmed that the cloned calf was genetically identical to the donor Jinan red-cross yellow cattle and different from the recipient females. Growth and reproductive performance of cloned cow were evaluated, and there were no difference i cross-red n it between cloned and normal control Jinan yellow cattle. Furthermore, the cloned yellow cow has delivered a healthy yellow calf.     

动物克隆技术应用及新发展

本文综述了国内外动物克隆技术的进展,并结合猪克隆对动物克隆技术的要点、路线以及当前存在的问题等方面作了较详细的论述.另外,根据现有动物克隆技术理论和实践,着重对该技术的应用和新的发展趋势进行了综述和讨论.     

影响体细胞核移植重构胚发育的因素

目前已有多种动物被成功克隆,但存在着诸多未知因素,使体细胞核移植重构胚发育至囊胚阶段的比例过低,克隆动物存在早衰等异常现象.该文围绕这产生这些现象的根本原因、高度分化的体细胞核移入卵质后所发生的分子事件以及其影响因素如体细胞的来源和培养代数、细胞周期、核质相互作用、细胞核再程序化、线粒体等方面对体细胞核移植重构胚发育的影响等进行综述.     

一种大肠杆菌胆碱脱氢酶活性的测定方法

本文提出了一种简单易行的测定微生物中胆碱脱氢酶活力的方法。通过测定549nm波长下电子传递体系PMS(吩嗪二甲酯硫酸盐)-cty.c(细胞色素C)光吸收值的增加,就可以计算出胆碱脱氢酶活力。利用此方法测定了大肠杆菌胆碱脱氢酶热激诱导前后的活力,结果发现受到诱导的大肠杆菌中胆碱脱氢酶的活力明显高于对照组。     

复合核裂变与微观裂变理论进展

核裂变过程是一个复杂的大幅度的量子多体动力学过程,对其理论描述一直是一个挑战.为了描述实验上的裂变可观测量,理论上发展了一些半唯象与近似的模型,但这些模型依赖唯象的参数.从有效核力出发发展微观的裂变理论,原则上无需自由参数,但要达到定量计算的阶段还很困难.本文介绍了复合核裂变的理论进展,特别讨论了微观计算复合核存活概率在超重核合成研究中的关键影响.此外比较了绝热和非绝热的微观裂变动力学模型.其中绝热裂变模型对低激发裂变的实验可观测量能较好描述,而非绝热动力学模型对深入研究裂变机制非常有用.本文分析了核裂变理论的问题和挑战,意识到裂变过程极其复杂,并展望了裂变理论的发展方向.     

用核裂变径迹法和热释光法研究瘦狗岭断裂活动年代

用核裂变径迹法和热释光法测定广州市郊瘦狗岭的断裂活动年代．结果表明：广州瘦狗岭断裂在中新代是处在较大规模较强烈的活动时期，而在第四纪早更新世及中更新世仍发生过较强烈的活动．最新的断裂活动是在４３×１０４ａ前．两种方法的数据可以互相印证