胰岛素样生长因子及其受体mRNAs在绵羊生殖系统中的表达和分布

为检测胰岛素样生长因子（IGFs）及其受体（IGFR）mRNAs在绵羊发情周期早期卵巢、子宫和输卵管中的表达,探讨绵羊胚胎早期发育过程中其发育环境——生殖道中生长因子的表达、分泌及其作用,取绵羊发情周期早期卵巢、子宫和输卵管,经固定、切片、免疫染色,观察IGFs mRNAs的表达和分布情况。同时用RT-PCR技术研究了各组织中IGF-Ⅰ、IGF-Ⅱ、IGF-ⅠR、IGF-ⅡR mRNAs的表达情况。结果表明,IGFs mRNAs在绵羊发情周期早期的卵巢、子宫和输卵管中都有表达,4种因子表达模式相似：在卵巢中,IGFs主要定位于卵泡颗粒细胞,间质细胞亦有少量表达。在输卵管中,上皮细胞免疫染色呈阳性;在子宫中,腺细胞及上皮细胞的阳性信号强于固有层。RT-PCR检测表明IGFs mRNAs在3种组织中均有表达。     

磷酸二酯酶亚型抑制剂与细胞周期蛋白抑制剂对绵羊卵母细胞体外成熟的影响

选用磷酸二酯酶3（PDE3）特异性抑制剂Milrinone和PDE4特异性抑制剂Rolipram以及细胞周期蛋白依赖性激酶（cdk）抑制剂Roscovitine（ROSC）对绵羊卵丘卵母细胞复合物（COCs）的体外自发成熟进行研究，拟建立一种模拟体内卵泡环境的绵羊卵母细胞体外培养模型。以M-199为基础培养液，绵羊COGs在含有50μmol／L的Milrinone、Rolipram、ROSC或不含上述任何抑制剂的培养液中分别培养24h。COCs在含有50μmol／LROSC的培养液中培养24h，转移到含有20IU／L FSH的培养液继续培养24h检查核相。结果表明：COGs在M-199基础培养液和含有Rolipram的培养液中都可以自发恢复减数分裂，但是在含有Milrinone或ROSC的培养液中减数分裂受到阻滞；并且ROSC对绵羊COCs体外自发成熟的抑制作用要远高于Milrinone（P〈0．05）。当COGs从ROSC转移到含有20Iu／LFSH的培养液继续培养24h后．90．4％卵母细胞恢复到第2次减数分裂中期（MⅡ）。上述结果表明PDE3和cdk在绵羊减数分裂恢复中具有重要的作用。     

Matrix attachment regions included in a bicistronic vector enhances and stabilizes follistatin gene expressions in both transgenic cells and transgenic mice

In the present study, follistatin (FST) gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region (MAR) were constructed and transfected to bovine fetal fibroblasts. Evaluations of both the integration and expression of exogenous FST indicated that the pMAR-CAG-FST-IRES-AcGFP1-polyA-MAR (pMAR-FST) vector had higher capacity to form monoclonal transgenic cells than the vector without MAR, though transient transfection and integration efficiency were similar with either construct. Remarkably, protein expression in transgenic cells with the pMAR-FST vector was significantly higher than that from the bicistronic vector. Exogenous FST was expressed in all of the pMAR-FST transgenic mice at F₀, F₁ and F₂. Total muscle growth in F₀ mice was significantly greater than in wild-type mice, with larger muscles in fore and hind limbs of transgenic mice. pMAR-FST transgenic mice were also found with more evenly distributed muscle bundles and thinner spaces between sarcolemma, which suggests a correlation between transgene expression-associated muscle development and the trend of muscle growth. In conclusion, a pMAR-FST vector, which excluded the resistant genes and frame structure, enhances and stabilizes FST gene expressions in both transfected cells and transgenic mice.     

牛体细胞中端粒酶组分的表达及端粒酶活性的研究

端粒酶是一种能够以自身的RNA组分为模板在染色体末端添加端粒重复序列的核酸蛋白复合体酶，主要由端粒酶RNA组分（TERC）与端粒酶逆转录酶组分（TERT）构成。通过对成年牛组织中端粒酶的这两种组分的表达情况及端粒酶活性状态进行检测，探讨牛端粒酶组分的组织特异性表达以及与端粒酶活性之间的关系。应用RT-PCR检测了成年牛的心脏、肾脏、肝脏及睾丸中TERC和TERT基因的mRNA表达情况，利用TRAP银染法检测各组织中的端粒酶活性状态。结果表明砸RC基因在这4种组织中均有表达，但转录水平在各组织之间有差异；TERT基因仅在睾丸中表达，在其他3种组织中均没有表达，端粒酶活性同样只在睾丸中检测到。由此可见牛端粒酶活性在体细胞中普遍受到抑制，仅在生殖腺中具有活性。端粒酶两种组分的转录是相互独立的，逆转录酶TERT组分是端粒酶活性的关键成分。     

白绒山羊性控胚胎生产及移植应用研究初探

应用X性控冷冻精液对超排供体白绒山羊进行人工授精,生产性控胚胎并进行鲜胚移植。结果发现,①用性控冻精和鲜精输精的供体母羊,平均卵子回收数分别为13.3和12.5枚,其中性控冻精组受精卵比例为29.6%（55/186）,极显著低于鲜精组94.0%（281/299）的比例（P<0.01）;②性控胚胎和普通胚胎移植受体母羊产羔率分别为42.2% 和58.1% ,二者差异极显著（P<0.01）;③性控胚胎移植受体母羊所产羔羊雌性所占百分数为100%,极显著高于普通胚胎移植（P<0.01）。表明通过白绒山羊X、Y精子分离、人工授精生产性控胚胎,同时结合胚胎移植技术,可以达到按照生产实际中的意愿得到性别控制子代白绒山羊的目的。     

供体细胞的传代次数和不同处理方法对异种核移植效率的影响

实验旨在探讨供体细胞培养代数及其所处的细胞周期对异种核移植效率的影响.以经Hochest33342染色后去核的牛卵母细胞为受体,以处于不同细胞周期的不同代数的人成纤维细胞为供体,采用电融合方法构建重构胚,6-DAMP和乙醇激活重构胚,SOFaa培养液中培养.第20代细胞非饥饿组的融合率、发育率显著低于第20代饥饿组（P＜0.05）,极显著的低于第8代细胞的饥饿组和非饥饿组（P＜0.01）,而8-细胞胚胎率,囊胚率4组间无显著差异.结果表明,饥饿处理对于培养代数低的细胞来说可能是非必要因素,但对于高代次细胞则是提高核移植效率的必要步骤,饥饿处理可使核移植融合率和发育率显著的提高.     

The ecological adaptability of cloned sheep to free-grazing in the Tengger Desert of Inner Mongolia,China

Since the birth of the first cloned sheep, somatic cell nuclear transfer technology has been successfully used to clone a variety of mammals. Cloned livestock have no apparent health risks, and the quality and safety of the cloned animal products are similar to non-cloned animals. The social behavior and environmental adaptability of postnatal cloned animals, especially when used for grassland farm production purposes, is unknown. In the present study, the cloned Dorper sheep equipped with GPS location devices were free-grazed in a harsh natural environment similar to conditions commonly experienced by Mongolian sheep. The main findings of this research were as follows. (1) Under free-grazing conditions, the cloned sheep showed excellent climatic and ecological adaptability. In extreme temperature conditions ranging from -30 to 40°C, the cloned sheep maintained acceptable body condition and behaved as other sheep. (2) The cloned sheep quickly adapted from a herd feeding strategy to the harsh environment and quickly exhibited a grazing regimen as other free-grazing sheep. (3) The cloned sheep exhibited free-grazing patterns and social behavior as other sheep. (4) The cloned sheep in the harsh environment thrived and produced healthy lambs. Overall, the cloned Dorper sheep exhibited excellent ecological adaptation, which is an important consideration for breeding meat sheep by cloning. The Dorper sheep readily adapted to the free-grazing conditions on the Mongolian plateau grassland, which attests to their ability to withstand harsh environmental conditions.     

In vivo and in vitro development of Tibetan antelope (Pantholops hodgsonii) interspecific cloned embryos

The Tibetan antelope is endemic to the Tibetan Plateau, China, and is now considered an endangered species. As a possible rescue strategy, the development of embryos constructed by interspecies somatic cell nuclear transfer (iSCNT) was examined. Tibetan antelope fibroblast cells were transferred into enucleated bovine, ovine and caprine oocytes. These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals. Less than 0.5% of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage. However, when the cloned antelope-bovine embryos were transferred to caprine oviducts, about 1.6% of the embryos developed to the blastocyst stage. In contrast, only 0.7% of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts. The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine. When the antelope-bovine embryos, constructed from oocytes treated with roscovitine or trichostatin A, were cultured in rabbit oviducts 2.3% and 14.3% developed to blastocysts, respectively. It is concluded that although some success was achieved with the protocols used, interspecies cloning of Tibetan antelope presents difficulties still to be overcome. The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming.