克隆

The three types of reconstructed embryos(reconstructed embryo of goat-rabbit, goat-bovine and goat-goat) were produced respectively by nuclear transfer using goat ear firbroblast cells as donors and rabbit, bovine and goat ooctyes as recipients, and the factors of cytoplast environments influencing cloned embryo development in vitro, cytoplast effect in nuclear transfer, the methods of transferring cloned embryo and the effects of pregnancy factor on implantation of cloned embryo were studied in this experiment, in order to supply basis for solving the difficulty of implantation of inter-species cloned embryo in recipient and improving the efficiency of cloning.

本试验以波尔山羊耳成纤维细胞为供体，分别以牛、羊、兔的卵母细胞为受体进行体细胞核移植，构建了山羊-兔、山羊-牛和山羊-山羊三种重构胚。研究了不同的胞质环境对重构胚体外发育的影响与核移植中的胞质效应，并对异种克隆胚胎采用了去透明带移植和配种后再移植的方法，探讨移植方法和妊娠因子对异种克隆胚胎在受体动物体内发育的影响，旨在为解决异种克隆胚移植不易着床发育的难题和为提高动物克隆效率提供理论依据。

Italian scientist plans first mass human clone trial 意大利科学家准备首次大规模人类克隆试验 A controversial Italian embryologist is preparing to impregnate up to 200 women with cloned embryos in the world's first attempt to produce a human clone, The Sunday Times reported.

周日泰晤士报报道，一位好争议的意大利胚胎学家正着手把克隆的胚胎注入200位妇女体内，成为世界上首次克隆人的试验。

The different fishes which are employed in the present studies are wild-type salmon, cultured salmon of freshwater and seawater, sea perch and fat greenling. The complete CT gene sequences of salmons are obtained by PCR amplification. The partial CT sequences of sea perch and fat greenling are obtained by in vitro cloning PCR method. Alignment of obtained CT sequences with other fish CT shows that CT appears to be well conserved among the same family. And the relative in taxonomy is far away, the similarity of different fish CTs is low. On the contrary, the closer the relative in taxonomy is, the higher the similarity of different fish CTs is. The sCT is expressed in pGEX-4T-X by recombinant form . We also succeed in the research of sCT expression alone by expression PCR. In addition, sCT antiserum is obtained using GST-sCT as antigen, and the high titer is tested by double immunodiffusion. In the rat bioassay, administration of 50 μg recombinant protein evoked significant hypocalcemia at 1 h after the data are analyzed by t-test.

本文用PCR方法克隆了野生鲑鱼、养殖鲑鱼的降钙素基因，并应用体外克隆PCR的方法首次克隆出鲈鱼、六线鱼降钙素的部分基因序列，通过对克隆的降钙素序列的比较研究，结果显示同一科的鱼降钙素序列保守性较高，同时，根据降钙素的部分氨基酸序列进行了降钙素相似性的比较研究，结果显示在分类学上，分类地位较远的鱼，其降钙素相似性较低，分类地位越接近的鱼，其降钙素相似性越高；我们利用谷胱甘肽S-转移酶(Glutathions S-transferase，GST)融合表达载体pGEX-4T-X对克隆的鲑鱼降钙素基因进行了融合表达研究，应用表达PCR的方法对降钙素基因的独立表达进行了初步的研究探索，并将纯化的融合蛋白作为抗原，获得了高效价的兔抗鲑鱼降钙素免疫血清；生物活性研究表明，大肠杆菌表达的融合蛋白具有显著的降血钙作用。

The result showed that twocDNA fragments which expressed at high level both in shoot and radicle representedthe gene encoding beta-D-glucosidase; one cDNA fragment expressed specifically inshoot represented the gene encoding mitochondria HSP60; most clones of MF12 andMF17 fragments respectively represented the chloroplast genes encoding prp22 andprp19 proteins which are two components of ribosomal small subunit; while thededuced amino acid sequence from each exceptional one clone was respectivelyhomologous to CDC5 proteins and vesicle-associated membrane proteins; otherthree cDNA fragments expressed preferentially in shoot had no homologue inGenBank.

结果发现2个在胚芽和胚根中表达量都很高的cDNA片段代表的是编码玉米β-D-葡糖苷酶的基因；一个在胚芽中表达而在胚根中不表达的片段代表的是编码线粒体分子伴侣HSP60的基因；片段MFl2的大部分克隆测序结果是与叶绿体基因组中编码核糖体小亚基蛋白prp22的基因同源，但其中有一个克隆测定的cDNA片段序列推测的氨基酸序列与CDC5家族成员有较高的同源性；片段MF17的大部分克隆测序结果与叶绿体基因组中编码核糖体蛋白prp19的基因同源，而有一克隆测定的cDNA片段序列推测的氨基酸序列与参与信号转导的膜结合蛋白VAP27和VAMP有较高的同源性；另有3个优先在胚芽中表达的cDNA片段未查询到同源序列。

It also can be used as alternative antigen of newgeneration vaccine.In this experiment,we screen the major protective antigen hsp65 gene of MAP in order to developnew vaccine especially the DNA vaccine for the prevention of paratuberculosis disease.The hsp65 genewas amplified from MAP C-2 chromosomal DNA by using the PCR technique.We gained a hsp65 gene of 1 626bp.Then PCR product was cloned into pGEM-T vector by T-A clone technique and therecombinant clone was identified by plasmid size,enzyme digestion and PCR identification.The cloneplasmid of pGEM-T- hsp65 was successfully constructed.The nucleotide sequence and deduced aminoacid sequence ofclone gene was analyzed by DNASTAR software.The result indicated that the size ofhsp65 gene consist with M.paratuberculosis K-10 strain in GenBank and the sequential homogeneityreached 99.1%,the amino acid homogeneity reached 99.3%.The preceding analysis indicated that thehsp65 gene was very conservative in M.paratuberculosis.

为了研发预防副结核病的新型疫苗尤其是DNA疫苗及相关蛋白功能，本研究选择了MAP的主要保护性抗原Hsp65蛋白，以副结核分枝杆菌C-2株染色体DNA为模板，以hsp65基因的特异性引物进行PCR扩增，获得了1 626bp的hsp65基因，通过T-A克隆技术，将PCR产物克隆至pGEM-T Vector中，以质粒大小、酶切分析、PCR扩增及序列分析鉴定重组克隆，成功地构建出克隆质粒pGEM-T-hsp65，以DNASTAR软件分析了所克隆基因的核苷酸序列和推导的氨基酸序列，结果表明，所获得的hsp65基因与GenBank中MAPK-10株该基因核苷酸大小完全一致，两者核苷酸序列的同源性为99.1％，氨基酸序列的同源性为99.3％，表明该基因在副结核分枝杆菌中高度保守。

Results:(1)NSCs form typical neurospheres under adequate concentration in vitro, which are immunoreactive to Vimentin. Typically and terminally differentiated mature neural cells could not be found without the stimulus of mitogen or only under NSCs self-regulation and self-induction;(2)NSCs derived from hippocampus maintain the character of stem cells much longer with better biological behavior; NSCs passed to the 2-3 passage are the best to graft since they have not differentiated;(3)NSCs cultured in vitro could self-regulate and differentiate into neurospheres and progenitors positively immunoreactive to specific antibodies representing neurons, astrocytes, oligodendrocytes and Schwann cells;(4)There are widespread synaptic contacts between various kinds of descendent clones and cells;(5)Neurospheres could be formed without the stimulus of mitogen when NSCs and OECs are cocultured. Many neurospheres and cells immunoreactive to Vimentin, GFAP, MAP2, 02, p75NGFR, GFAP, S-100, Synaptosis, Vimentin, Tau (Tau is only positive in cocultureof HNSCs+HOECs) could be found;(6)The supernatant fluid triturated from adult rat spinal cord stimulates NSCs to differentiate into neurons, but do not terminally differentiate;(7)Fibroblasts and O4 oligodendrocytes are not supported to grow under this culture medium.Part II: Isolation, culture and identification of rat and human olfactory ensheathing cellsOlfactory ensheathing cells/glials are the most powerful cells to enable the regeneration of axons in the central nervous system.

结果表明：①在适宜的浓度体外培养条件下，NSCs能形成典型的神经干细胞克隆球，Vimentin免疫荧光染色阳性，单靠丝裂原刺激或NSCs自我调节和分化诱导，不会产生典型的终末分化的成熟神经细胞；②海马源性的NSCs维持干细胞特性的时间更长，生物学特性更优；③传至第2～3代的NSCs尚未分化时移植最佳；④体外培养的NSCs能自我调控分化为神经元、星形胶质细胞、O2少突胶质细胞、雪旺氏细胞染色阳性克隆球和前体细胞；⑤各种子代克隆球和细胞存在广泛的突触联系；⑥NSCs与OECs联合培养时，不需丝裂原刺激即能形成克隆球，获得大量Vimentin、GFAP、MAP2、O2、p75NGFR、GFAP、S-100、Synaptosis、Vimentin、Tau(Tau只有人HNSCs+HOECs联合培养时出现阳染)染色阳性的克隆球和细胞；⑦脊髓研磨后的上清液刺激神经干细胞向神经元方向分化，但并不出现终末分化；⑧本研究培养条件不利于成纤维细胞、O4生长。

Results (1) The forward and reverse subtracted cDNA libraries of different metastastic potential large cell lung cancer cell lines were successfully constructed;(2) With blue-white colony and dot blot, 307 positive clones in the forward subtracted library and 78 positive clones in the reverse subtracted library were obtained;(3) 55 clones were successfully sequenced in the forward subtracted library. Homolog analysis confirmed 23 differential expression segments, which were similar to the human genes already known, including NME2, NPM1, MT 2A, HSPE1, TAFIA, EPRS, PX19 and EIF3S9 et al;(4) 31 clones were successfully sequenced in the reverse subtracted library. Homolog analysis confirmed 16 differentially expressed segments. 15 of them were similar to the human genes already known, including ANXA2, TUBB, PKN2, GNAS, EEF1A1, SSR2 and RPLPO et al. Only one segment had partial homology to known human genes. This segment was supposed to be the new EST segments which might not have been cloned.

结果(1)成功构建人大细胞肺癌高低转移株差异表达基因正向消减cDNA文库和反向消减cDNA文库；(2)经蓝白菌落筛选和斑点杂交，正向消减文库获得307个阳性克隆，反向消减文库获得78个阳性克隆；(3)正向消减文库挑选55个克隆成功测序，经同源性分析最终确定差异表达基因片段23个与已知人类全长基因具有很高相似性(95％～100％)，这些基因包括NME2、NPM1、MT2A、HSPE1、TAFlA、EPRS、PX19和EIF3S9等；(4)反向消减文库挑选31个克隆成功测序，经同源性检索和比对最终确定差异表达基因片段16个，其中15个与人类全长基因具有很高相似性(95％～100％)，包括ANXA2、TUBB、PKN2、GNAS、EEF1A1、SSR2和RPLPO等基因。1个片段和己知人类基因仅有部分相似性，表明它可能是未被克隆的人类基因EST片段。

Two field investigations were conducted to study clonal architecture and ramet population characteristics of Lysimachia congestiflora growing in different light conditions and the variation of Fragaria vesca ramet population characteristics along the elevation gradient in the transitional zone between Qinghai-Tibet plateau and Sichuan basin.

本文以青藏高原与四川盆地过渡带四种匍匐茎草本克隆植物为对象，应用野外调查和实验生态学方法研究克隆植物对异质性环境的适应对策以及克隆整合、克隆内分工行为对克隆植物在不同海拔生境中生长的贡献。

In order to disclose the adaptive strategies and capacity of Potaninia mongolica Maxim, acquired during its evolution history, the whole response system, which was composed of individual morphology plasticity response, organs anatomy structure response, physiological function response of anti-arid and anti-hotness, activities of protective enzymes response, endogenous hormone adjustment response, sexual reproduction strategies response of seeding, dispreading, sprouting and renewing, asexual reproduction strategies response of clonal growth patterns, clonal growth architectures, clonal growth architectures plasticity, heterogeneous resources utilization strategies, endogenous hormone distribution in clonal organs, foraging behavior, risk-spreading, and resource sharing, individual density allocation patterns response, niche differentiation response, species connection response, allelopathy response and biodiversity components response of the plant was profoundly explored in this paper.The studies come to at least four important results:(1) Taking the sensitive response and evading strategy to adapt to environment stress, Potaninia mongolica Maxim is a successful species has lived through the long-term evolution. Nevertheless, it is narrow climate and soil niche and characteristic of vegetation reproduction which has made the plant a rare and endangered species.

本论文通过对绵刺环境胁迫下个体形态可塑性响应、器官解剖结构响应、抗旱性和抗热性生理功能响应、保护酶系统激活响应、内源激素调控响应，有性繁殖对策中结实、扩散、萌发、更新等生活史过程响应，无性繁殖对策中克隆生长格局响应、克隆生长构型响应、克隆生长构型可塑性响应、克隆生长构型与资源异质性响应、内源激素调控克隆生长构型响应、克隆器官结构与功能响应、觅食行为响应、风险分摊响应、资源共享响应，种群中格局配置响应、生态位分异响应，群落中种间关联、异株克生响应、生物多样性组成响应等内容进行研究，揭示绵刺在进化过程中的适应对策和适应能力，为绵刺保护提供理论依据。

The transient transfection efficiency was measured by flow cytometry. The stable transfected cells were screened by G418. Results Electrotransfection had the highest transient transfection efficiency (56.8%), with the most masc clones of stably transfection and the least time taken to form big masc clones (20 days). Cationic polymer mediated transient transfection had the lowest efficiency (1.7%), with the least masc clones and the longest time taken to form the big masc clones (30 days). Cationic liposome transfection had a transient transfection efficiency of 39.9% and took 25 days to form the big masc clones.

结果 电转染有最高的瞬时转染效率(56.8%)，稳定转染得到的阳性克隆最多，且从细胞筛选到扩增至得到阳性大克隆所用的时间也最少（约20d）；用阳离子聚合物瞬时转染率低(1.7%)，稳定筛选同样可以得到阳性大克隆，但是数量很少且从细胞筛选到扩增至阳性大克隆所用的时间也最多（约30d）；阳离子脂质体转染效果居中，瞬时转染率为39.9%，从细胞筛选到扩增至得到阳性大克隆所用的时间约25d。

With Death guitarist Schuldiner adopting vocal duties, the band made a major impact on the scene.

随着死亡的吉他手Schuldiner接受主唱的职务，乐队在现实中树立了重要的影响。

But he could still end up breakfasting on Swiss-government issue muesli because all six are accused of nicking around 45 million pounds they should have paid to FIFA.

不过他最后仍有可能沦为瑞士政府&议事餐桌&上的一道早餐，因为这所有六个人都被指控把本应支付给国际足联的大约4500万英镑骗了个精光。