Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer

In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO-Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%,P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex-pressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neor genes were correctly expressed indicating that transgenic somatic cell lines and positive trans-genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and optimization, transgenic cash-mere goat embryos expressing red fluorescence and containing an IGF1 expression cassette were obtained, which was sufficient for production of transgenic cashmere goats.     

鸡胚胎干细胞的分离、培养和鉴定

SNL cells (permanent line of irradiated mouse fibroblast cells), primary mice embryonic fibroblasts (PMEF) cells and primary chicken embryonic fibroblasts (PCEF) cells were respectively used as the feeder cells for chicken embryonic stem cell culture. The isolated blastoderm cells front the stage X embryos of chicken were cultured in Dulercco‘‘ s Modified Eagle Medium (DMEM) supplemented with leukemia inhibitory factor (LIF, 1 000 IU/ml), basic fibroblast growth factor (bFGF 10 ng/ml) and stem cell factor (SCF, 5 ng/ml). The alkaline phosphatase (AKP) test, differentiation experiment in vitro and chimeric chicken production were carried out. The resuts showed that culture on feeder layer of PMEF yielded high quality CES cell colonies. The shape of typical CES clone showed as follows: nested aggregation (clone) with clear edge and round surface as well as close arrangement within the clone. Strong positive AKP reactive cellswere observed. On the other hand, the fourth passage CES cells could differentiate into various cells in the absence of feeder layer cells and LIF in vitro. The third and fourth passage cells were injected into the subgerminal cavity of recipient embryos at stage X. The manipulated embryos were incubated until hatching. Of 269 Hailan embryos injected with CES cells of Shouguang Chickens, 8.2 % (22/269) survived to hatching, 3 feather chimeras had been produced, which suggests that an effective culture systems were established and it could promote the growth of CES cells and maintain them in an undifferentiated state .     

Derivation and characterization of human embryonic stem cell lines from poor quality embryos

Poor quality embryos discarded from in vitro fertilization （IVF） laboratories are good sources for deriving human embryonic stem cell （hESC） lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses （ICMs） could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods （P〉0.05）. As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.     

Efficient production of omega-3 fatty acid desaturase (sFat-1)-transgenic pigs by somatic cell nuclear transfer

Omega-3(ω-3) fatty acid desaturase transgenic pigs may improve carcass fatty acid composition. The use of transgenic pigs is also an excellent large animal model for studying the role of ω-3 fatty acids in the prevention and treatment of coronary heart disease and cancer. Transgenic pigs carrying synthesized fatty acid desaturase-1 gene (sFat-1) from Caenorhabditis briggsae by somatic cell nuclear transfer (SCNT) were produced for the first time in China. Porcine fetal fibroblast cells were transfected with a sFat-1 expression cassette by the liposome-mediated method. Transgenic embryos were reconstructed by nuclear transfer of positive cells into enucleated in vitro matured oocytes. A total of 1889 reconstructed embryos were transferred into 10 naturally cycling gilts. Nine early pregnancies were established, 7 of which went to term. Twenty-one piglets were born. The cloning efficiency was 1.1% (born piglets/transferred embryos). The integration of the sFat-1 gene was confirmed in 15 live cloned piglets by PCR and Southern blot except for 2 piglets. Expression of the sFat-1 gene in 12 of 13 piglets was detected with RT-PCR. The data demonstrates that an efficient system for sFat-1 transgenic cloned pigs was developed, which led to the successful production of piglets expressing the sFat-1 gene.     

Cloned goats (Capra hircus) from adult ear cells

The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro for 20 h. 75.44% (906/1201) of matured oocytes with membrane evagination around the MII chromosomes were enucleated. Ear skin fibroblast cells were derived from an adult female dining Grey goat (C. hircus). The cells were cryopreserved in liquid nitrogen after passage 2. Thawed cells were further cultured for 3-6 passages and were subjected to serum starvation by 0.5% FBS for 2-10 d, then used as donor cells for nuclear transfer. 98.12% (889/906) of the enucleated oocytes were reconstructed by intracytoplasmic injection of karyoplast. The reconstructed embryos were activated by 5μ mol/L ionomycin for 4.5 min and further activated by culturing with 6-dimethylaminopurine (6-DMAP) for 3 h. After 36 h of culture in mCR1aaBF, 76.69% (645/841) of the cloned embryos cleaved. There were no signifi     

乌羊和小香羊的RAPD分析

用40条多态引物对乌羊、小香羊、南江黄羊、黑山羊、川东白山羊、波尔山羊和马头山羊7个品种(或群体)进行RAPD分析,其中28条引物扩增出多态性谱带,并用于进一步对12只乌羊个体和12只小香羊个体基因组进行扩增.扩增产物以1.5%琼脂糖凝胶(含0.5μg/ml溴化乙锭)电泳分离.Nei氏公式计算品种间的遗传距离指数和品种内的遗传相似指数,NJ法构建系统聚类图.结果表明:乌羊和川东白山羊间的遗传距离最小,亲缘关系较近,而小香羊与各品种间的遗传距离都较大,亲缘关系较远.乌羊群体及小香羊品种都具有一定的遗传稳定性。 Abstract:A total of 40 primers generated polymorphic loci were used in random amplified polymorphism DNA(RAPD)analysis in seven goat breeds(or populations)including Wu goat,Small-Xiang goat,Nanjiang Brown goat,Black goat,Chuandong white goat,Boer goat,Matou goat.28 of them were selected for further amplification in individuals of 12 Wu goats and 12 Small-Xiang goats because of their different amplified patterns among seven goat breeds.Amplification products were separated by 1.5% agarose gel(contain 0.5μg/ml ethidium bromide)electrophoresis.Genetic distance indexes among breeds and genetic similarity indexes within breed were calculated by Nei,and the phylogenetic tree was constructed by NJ method.The results indicate that the genetic distance index between Wu goat population and Chuandong white goat breed is smallest,therefore Wu goat population is closely related to Chuandong white goat breed.While the genetic distance indexes between Small-Xiang goat breed and any other goat breeds are all big,so Small-Xiang goat breed is far related to any other goat breeds.The results also indicate that the genetic stability either among the Wu goat population or the Small-Xiang goat breed reaches some extent.     

PCR方法用于奶牛早期胚胎的性别鉴定

根据牛Y染色体上的特异DNA序列合成一对引物, 通过PCR反应对奶牛早期胚胎进行性别鉴定。预测性别与移植胚胎产犊的实际性别相符率为80%。 Abstract:We have obtained the specific DNA segment from the bovine Y-chromosome and used it to design a pair of primer.The sex of embryos at the preimplantation stage have determined by using the polymerase chain reaction.10 months after uterine transfer showed that the rate of accuracy is 80%.     

Search for nave human pluripotent stem cells

Normal mouse pluripotent stem cells were originally derived from the inner cell mass(ICM) of blastocysts and shown to be the in vitro equivalent of those pre-implantation embryonic cells, and thus were called embryonic stem cells(ESCs). More than a decade later, pluripotent cells were isolated from the ICM of human blastocysts. Despite being called human ESCs, these cells differ significantly from mouse ESCs, including different morphology and mechanisms of control of pluripotency, suggesting distinct embryonic origins of ESCs from the two species. Subsequently, mouse pluripotent stem cells were established from the ICMderived epiblast of post-implantation embryos. These mouse epiblast stem cells(Epi SCs) are morphological and epigenetically more similar to human ESCs. This raised the question of whether cells from the human ICM are in a more advanced differentiation stage than their murine counterpart, or whether the available culture conditions were not adequate to maintain those human cells in their in vivo state, leading to a transition into Epi SC-like cells in vitro. More recently, novel culture conditions allowed the conversion of human ESCs into mouse ESC-like cells called nave(or ground state) human ESCs, and the derivation of nave human ESCs from blastocysts. Here we will review the characteristics of each type of pluripotent stem cells, how(and whether) these relate to different stages of embryonic development, and discuss the potential implications of nave human ESCs in research and therapy.     

3个山羊群体中4个微卫星DNA多态性及其与杂种优势的关系

利用4个微卫星标记（OarFCB11,OarAE101,McM218,McM38）对波尔山羊、太行山羊和河北奶山羊的等位基因频率、群体多态信息含量、有效等位基因数、杂合度和遗传距离进行了遗传检测,并测定了波尔山羊与河北奶山羊及太行山羊的杂交效果。结果表明：4个微卫星标记在波尔山羊、太行山羊和河北奶山羊3个品种中存在多态性,可以用于山羊遗传多样性的评估;从不同品种来看,太行山羊的遗传变异程度最大,而波尔山羊的遗传变异程度相对较小;波尔山羊与河北奶山羊的遗传距离大于与太行山羊,波尔山羊与河北奶山羊的杂种优势高于与太行山羊,与实际杂种优势测定结果相符。 Abstract: Gene frequency, polymorphism information contents, number of effective alleles, heterozygosity and genetic distances were studied in Boer goat, Taihang goat and Hebei dairy goat using four microsatellite markers（OarFCB11,OarAE101,McM218,McM38）. The crossing effects on Hebei dairy goat and Taihang goat with Boer goat were tested. The results indicated that there are genetic polymorphisms at four microsatellite markers in three goat breeds. Four microsatellite markers can be used for genetic diversity evaluation in goat breeds. The genetic variability of Taihang goat is the highest, and Boer goat is the lowest in three goat breeds. Genetic distances between Boer goat and Hebei dairy goat is bigger than that between Boer goat and Taihang goat. The heterosis between Boer goat and Hebei dairy goat is higher than that between Boer goat and Taihang goat. It accords with testing results on actual heterosis.     

波尔山羊杂交后代及其亲本随机扩增多态DNA研究

利用随机扩增多态DNA技术研究了波尔山羊、唐山奶山羊、青龙本地山羊以及波尔山羊与这两个山羊群体杂交后代共计128个山羊个体的随机扩增多态DNA。结果表明：（1）总群体平均遗传多样性指数（Hsp）为0.6974,群体遗传分化指数为0.9706,山羊群体间平均遗传距离指数（0.1314～0.2052）明显大于群体内的相应值（0.0582～0.1440）,上述结果说明,所研究山羊群体不仅具有较为丰富的遗传多样性,而且其核基因组遗传变异主要存在于群体间。（2）山羊群体间的分子聚类关系与各群体间的亲缘关系基本一致。 Abstract:The random amplified polymorphic DNA（RAPD）of 128 individuals was studied,which were from Boer goat, Tangshan dairy goat,Qinglong native goat and their hybrids crossbred with Boer goat.The average index of genetic polymorphism for whole population（Hsp）and the index of genetic differentiation were 0.6974 and 0.9706,respectively.The average index of genetic distance between populations（0.1314～0.2052）was significantly higher than that within populations（0.0582～0.1440）.All of these indicated that the genetic polymorphism was not only abundant,but also the genetic variation was mainly existed between goat populations.The molecular dendrogram among goat populations was in accord with their genetic relationship.     

应用雄性激素诱导异育银鲫性转化的研究

用丙酸睾丸素溶液浸泡行雌核发育的异育银鲫(Allogynogenetic crucian carp)胚胎10～14天,诱导出了11.0％～13.6％的雄性鱼和10.5％～27.3％的兼性鱼。对50日龄的异育银鲫幼鱼投喂甲基睾丸素4个月,再继续饲养182～335天,诱导出了20％以上的雄性鱼。此外,部分实验鱼两侧卵巢的大小出现明显差异。 Abstract：The embryos of allogynogenetic crucian carp were soaked in testosterone propionate for 10～14 days,11.0％～13.6％ fry were induced to be physiological males and 10.5％～27.3％ fry developed into bisexual sterile.The 50-day old fish were fed diet containing methyltestosterone for 4 months and then reared for 182～335 days.More than 20％ fry developed into males.In addition,the ovaries in both sides of some treated fish were significantly different in size.     

17α，20β-二羟黄体酮降低虹鳟鱼血浆免疫球蛋白M的水平(英文)

本文首次报道１７α,２０β－二羟黄体酮对虹鳟免疫功能的影响。在对生殖季节虹鳟血浆１７α,２０β－二羟黄体酮、免疫球蛋白Ｍ和总蛋白水平的调查时发现,雌雄虹鳟血浆１７α,２０β－二羟黄体酮的水平与免疫球蛋白Ｍ的水平负相关,即在雌性相关系数ｒ＞－０．７９７,Ｐ＞０．０１;而在雄性ｒ＞－０．４７７,Ｐ＞０．０５。这提示１７α,２０β－二羟黄体酮可能抑制免疫球蛋白Ｍ的合成或分泌。产卵或排精后的血浆总蛋白量低     

广州汉族人群DYS19、DYS389Ⅰ/Ⅱ、DYS390多态性及其单体型

用PCR结合PAGE技术观察111例广州汉族男性DYS19、DYS389Ⅰ/Ⅱ、DYS390等位基因及单体型分布状况。结果显示：广州地区汉族男性DYS19基因座观察到5种等位基因,DYS389Ⅰ观察到4种等位基因,DYS389Ⅱ观察到5种等位基因,DYS390观察到5种等位基因;χ2检验表明上述各等位基因频率分布与其他地区人群存在明显的差异。此外,还观察到72种由上述基因座共同构成的单体型,单体型多样性达0.953。 Abstract: In order to apply a set of useful and high polymorphic Y?STRs in forensic practice and genetic analysis,we performed a population genetic study from Chinese.The allele distributions of the systems DYS19、DYS389Ⅰ/Ⅱ、and DYS390 were investigated in sample of 111 unrelated males from the area of Guangzhou, China.PCR products were detected using polyacrylamide gel electrophoresis and silver staining.5、4、5、5 alleles were observed in locus DYS19、DYS389Ⅰ、DYS389Ⅱ、DYS390 respectively.Different allele frequency distributions were observed when compared to other population.Haplotype frequency date of 72 different types were obtained.     

人CD55基因的克隆及其转基因构件的组装

本实验采用人肝组织作为RNA的来源 ,经RT -PCR扩增得到CD55基因的cDNA片段。与人α -珠蛋白启动子及其polyA序列重组 ,插入质粒载体pGEM - 5zf,获得了可用于受精卵原核显微注射的基因构件 ,为建立人CD55转基因动物模型奠定了基础。     

云南普洱茶原料晒青毛茶的化学成分

从云南临沧地区生产普洱茶的原料晒青毛茶中分离到 2 1个化合物 ,通过波谱分析分别鉴定为 :(- )表儿茶素、 (- )表没食子儿茶素、 (- )表没食子儿茶素 - 3-O -没食子酸酯、 (- )表儿茶素 - 3-O -没食子酸酯、 (- )表阿福豆素 - 3-O -没食子酸酯、 ( )儿茶素、 (± )没食子儿茶素、槲皮素、槲皮素 - 3-O - β -D -葡萄吡喃糖甙、芦丁、山奈酚、山奈酚 - 3-O - β -D -葡萄吡喃糖甙、山奈酚 - 3-O -芦丁糖甙、小木麻黄素 (strictinin)、 1,6 -O -二没食子酰基 - β -D -葡萄吡喃糖、茶倍素、绿原酸、 3α ,5α -二羟基 - 4 -α -O -咖啡酰基金鸡纳酸、松柏醇甙、没食子酸和咖啡因     

西双版纳片断季节性雨林蚂蚁物种多样性研究

以西双版纳勐仑自然保护区为对照,采用陷井诱捕法和亲方调查法比较研究了３个不同面积、不同环境状况和片断季节性雨林蚂蚁的群落组成及物种多样性变化。结果表明：所获蚂蚁隶属４亚科２４属５０种,以城子龙山物种数量多。各样地优势种均不相同。４类样地间的共有物种较少,只有４种;相似性较低,相似性系 ０．１６２２～０．３５４８。聚类结果显示,植物园保护区与城子龙山最先聚为一类,然后与自然保护区聚为一类,最后才与曼     

妊娠小鼠子宫内膜LIF基因表达的研究

本文对妊娠第4天(Ⅰ组)、第7天(Ⅱ组)、第10天(Ⅲ组)的小鼠(各20只)子宫内膜LIF基因表达进行了研究。Ⅰ组20只小鼠子宫内膜全部存在LIF基因的表达、Ⅱ组有5只小鼠表达、Ⅲ组仅有1只小鼠表达。文中对不同孕期LIF基因的表达程度与胚胎着床的关系进行了讨论。 Abstract:Leukemia inhibitory factor(LIF)is a glycoprotein with multiple activities and is essential for blastocyst implantation in mouse.We have examined LIF gene expression in mice endometrium on day 4(group Ⅰ),day 7(group Ⅱ),day 10(group Ⅲ)of pregnancy.In group Ⅰ all had LIF gene expression,5 mice had LIF gene expression in group Ⅱ,only one mouse had LIF gene expression in group Ⅲ.We discussed the relation between level of LIF gene expression and embryonic implantation.     

小鼠肝炎病毒RT-PCR检测方法的建立和比较研究

用小鼠肝炎病毒的MHV1、MHV3、A59、JHM四株分别感染DBT细胞 ,收获病毒 ,提取病毒RNA。依据小鼠肝炎病毒的病毒基因、M结构蛋白、mRNA的基因保守区分别设计出多对引物 ,按各对引物的条件建立了RT -PCR方法 ,比较了不同引物敏感性和特异性 ,并比较选择与MHV同为冠状病毒的传染性支气管炎 (IBV)标准株和野毒株没有交叉的最佳特异引物。敏感性实验检测到 1 0pg的小鼠肝炎病毒RNA。     

藓类植物传孢类型及其系统演化关系

藓纲包括３个亚纲（Ｂｒｏｔｈｅｒｕｓ,１９２４－１９２５）。泥炭藓亚纲、黑藓亚纲,真藓亚纲,全世界约１００个科,近８００属,约１５０００种。藓类的传孢类型和藓类植物系统演化有密切关系。我们认为传孢不同类型,直接反映了他们的演化程度。我们根据藓类孢子体的分化、蒴齿分化与其机能,分为５种传孢类型。即腐媒传孢型,风媒孢型,汽－风媒传孢型,水媒传孢型,和虫媒传孢型等５种。根据它们的演化程度比较,我们提出了