6—DMAP和胚胎干细胞代数对异种重构卵体外发育的影响（简报）

The development of reconstructed oocytes and the survival rate of cloned animal were affected by many factors during nuclear transfer. The genetic constitution and the genetic state of donor nucleus were proposed to be primary factors, which affected the survival rate of cloned animal. In addition, the survival rate of cloned animal might be influenced by nuclear transfer technique itself and passages of donor cells as well as the activation methods of oocytes. We reconstructed oocytes with outbreeding Kunming albino mouse ES cells and enucleated rabbit oocytes, and analyzed the effects of the passages of ES cells and 6-DMAP on the development of interspecific reconstructed oocytes. The interspecific reconstructed ES-rabbit oocytes were activated either by combined two set electric pulses and 6-DMAP or by two set electric pulses alone. The rate of cleavage was significantly higher for the group (86.2%) treated with 6-DMAP than the group (64.2%, P < 0.05) treated with electric pulses only, and the rate of blastocysts was 17.0% and 13.4% respectively, which were not significantly different between two groups. When ES cells that had been passed for 24 and 14 generations were used as donors, the cleavage rates of the reconstructed oocytes were 88.5% and 82.1%, respectively (P > 0.05), and the rates of blastolation were 16.7% and 15.4%, respectively (P > 0.05). The results show that 6-DMAP increases the cleavage rate of reconstructed oocytes derived from ES cells, and affects slightly the developmental rate of blastocysts. There are no differences when high passage and low passage ES cells are used as nuclear donors.     

小鼠的胚胎移植方法

全面而详细地描述了小鼠胚胎移植中所采用的输卵管移植及子宫移植方法,包括作者的改进。介绍了寄母小鼠的选择、配种及孕鼠的编号方法等,具有较强的实用意义。     

M16添加牛磺酸和EDTA支持昆明白小鼠体外受精并发育至囊胚

在以往研究工作的基础上证明了通过添加2.5mmol/L的牛磺酸和0.1mmol/L的EDTA至M16培养液中,可支持昆明白小鼠的体外受精（IVF）,并支持受精卵突破2－细胞阻滞发育至囊胚期。本研究进一步证明了牛磺酸和EDTA在昆明小鼠早期胚胎发育和克服2－细胞阻滞中起关键作用。 Abstract:The results of our early experiments show that the 2-cell block can be overcome by culturing zygote in modified M16 ,modified CZB and TE medium.Our research shows that the taurine and EDTA play key role in overcoming 2-cell block in Kunming mouse.The results show that the addition of taurine and EDTA to M16 medium can support the IVF and develoment to blastocyst in vitro in Kunming strain mice.This is the first report that M16 medium added with taurine and EDTA can be used in both IVF and culture medium to overcome the 2-cell block of embryo development in Kunming strain mouse.     

家猫及其他猫科动物的生殖工程研究

在脊椎动物中,猫科动物是一类比较特殊的动物类群,共有37种动物,除家猫外,其余均为珍稀或临近灭绝的濒危动物,因而了解家猫的生殖习性和生殖规律,尤其是了解家猫胚胎工程领域,如猫卵母细胞的体外成熟（in vitro maturation IVM),体外受精（in vitro fertilization,IVF),显微受精等,对于利用现代生殖技术研究和探讨非家猫猫科动物的繁殖和保护具有极为重要的借鉴意义。     

未经休眠处理的体细胞用于异种核移植

自“多莉”诞生以来,在全世界掀起了一场体细胞克隆的浪潮,许多体细胞克隆动物,如小鼠、山羊、牛、猪等纷纷问世。围绕体细胞克隆的供体细胞周期问题,学术界存在两种不同的观点,一是Wilmut等认为体细胞必须经过休眠处理,使细胞停滞在G0/G1期,或者采用以G0/G1期为主的活体细胞作为供体,这是克隆成功的关键,这一方面的报道已有很多。第二是Cibelli等认为不必对细胞作     

几种克服昆明小鼠2一细胞胚胎发育阻滞的培养液研究

比较了几种培养液对克服昆明小鼠胚胎２－细胞发育阻滞的效果。实验１的结果表明，在Ｍ１６及ＣＺＢ培养液基础上，加减几种成分得到的改进Ｍ１６培养液（用ｍＭ１６表示）和改进的ＣＺＢ培养液（用ｍＣＺＢ表示）均能有效克服２－细胞阻滞现象。除去Ｍ１６和ＣＺＢ培养液中的葡萄糖和磷酸盐后添加５．５５ｍｍｏｌ／Ｌ牛磺酸、１００或１１０μｍｏｌ／Ｌ ＥＤＴＡ、２ｍｍｏｌ／Ｌ谷氨酰胺和２％必需氨基酸（ＥＡＡ）及１％非必需     

不同因素对大鼠卵母细胞孤雌激活作用影响的研究

本实验比较了SrCl_2,放线菌酮(CHX),电刺激和乙醇等理化因素对SD大鼠卵母细胞激活的作用。结果表明,SrCl_2,CHX和电刺激均能有效激活SD大鼠卵母细胞,其最高激活率分别达到93.24％,91.89％和85.90％。8％乙醇对注射hCG 21小时后的卵母细胞激活率也达70％。SrCl_2在1.6和3.2mmol/L浓度,作用10—30分钟均有较好激活效果。电刺激强度在160V,80μs作用较佳。当SrCl_2与CHX联合作用时,激活率可有明显提高。但电刺激或乙醇与CHX的联合作用不能有效提高激活 率。本研究还比较了卵丘细胞的存在与否对激活的影响,发现卵丘-卵母细胞复合体中的卵母细胞不能被有效激活。刚离体的超排卵母细胞也不能被有效激活,须在体外培养一定时间后才能被激活。     

大熊猫供核体细胞在兔卵胞质中可去分化而支持早期重构胚发育

体外培养的成年大熊猫骨骼肌细胞、子宫上皮细胞和乳腺细胞 ,分别作为供核细胞移植进入去核兔卵母细胞中以构建异种重构胚 .3种组织来源的体细胞在去核兔卵质中均可去分化、恢复全能性和替代合子核进行卵裂 ,并支持早期重构胚发育 .其中以乳腺细胞效果最好 ,子官上皮细胞次之 ,骨骼肌细胞最差 .对比实验表明 ,胞质直接注射法结合重构卵在体培养可比电融合法加体外培养方案获得更大比例的囊胚率 .染色体分析表明异种重构胚的核遗传物质来自大熊猫供体体细胞核 .线粒体DNA分析表明重构囊胚中存在有大熊猫线粒体 .这些结果初步说明 :( 1 )卵胞质使体细胞核去分化不具种特异性 ;( 2 )哺乳动物异种重构胚早期发育中 ,异种细胞核与细胞质之间是相容的 .     

The giant panda (Ailuropoda melanoleuca) somatic nucleus can dedifferentiate in rabbit ooplasm and support early development of the reconstructed egg

The giant panda skeletal muscle cells, uterus epithelial cells and mammary gland cells from an adult individual were cultured and used as nucleus donor for the construction of interspecies embryos by transferring them into enucleated rabbit eggs. All the three kinds of somatic cells were able to reprogram in rabbit ooplasm and support early embryo development, of which mammary gland cells were proven to be the best, followed by uterus epithelial cells and skeletal muscle cells. The experiments showed that direct injection of mammary gland cell into enucleated rabbit ooplasm, combined with in vivo development in ligated rabbit oviduct, achieved higher blastoeyst development than in vitro culture after the somatic cell was injected into the perivitelline space and fused with the enucleated egg by electrical stimulation. The chromosome analysis demonstrated that the genetic materials in reconstructed blastocyst cells were the same as that in panda somatic cells. In addition, giant panda mitochondrial DNA (     

胚胎操作和显微操作的几点改进

改进的胚胎吸管包括外径为2.5mm的单根玻璃细管和连接支持管两大部分.与传统的巴氏管比较,具有容易制作,可使有关操作更为简便有效和经济适用的优点.通过配置上的优化,并利用国产倒置显微镜所装配的显微操作仪,与全套进口的显微操作仪相比,可节约三分之二以上的费用,实践证明这样的配置的显微操作仪完全可以满足有关显微操作的需要.同时,引入了一种新的操作系统--空气系统.本文还介绍了改进的显微操作小室和电融合槽的制作方法.