克隆和动物克隆

１９９７年２月２３日,英国苏格兰爱丁堡罗斯林研究所Ｗｉｌｍｕｔ和Ｃａｍｐｂｅｌ领导的研究小组,用高度分化的乳腺细胞成功克隆绵羊的消息在英国《Ｎａｔｕｒｅ》杂志发表后,在全世界引起轰动［１］。由于哺乳动物的克隆成功,人的克隆已出现成功的可能,因此已引起...     

克隆

1977年2月23日,英国《Nature》发表Wilwut等用羊乳腺上皮细胞成功克隆出绵羊多利的消息后,在全世界引起了很大的反响,克隆问题引起了人们的普遍关注。由于历史     

克隆问世

经过许多年的失败之后,它终于诞生了。2005年8月,一组韩国科学家宣布他们首次成功地克隆出一只阿富汗猎犬。这只小狗终于加入了由绵羊、山羊、小鼠、大鼠、猫、牛、猪、马、兔子、鹿和骡子组成的克隆动物俱乐部。但是,对于一些爱狗者而言,这并不意味着让他们的宠物再生已经为期不远。     

克隆与克隆动物基因相同吗

美籍华人牛满江教授,著名生物学家,１９１２年生于中国河北省。１９３２年考入北京大学,１９３６年毕业留校任助教,１９３７年在昆明西南联大任教。１９４４年由北大派出去美国斯坦福大学深造,１９４６年获博士学位。自此,先后在斯坦福大学,洛克菲勒医学研究所,后改名为洛克菲勒大学和坦普尔大学从事教学和研究工作。并获得坦普尔大学终身教授,名字被载入美国科学家名人录。从１９５３年起,他潜心于“核糖核酸在发育中独特功能”的研究,是这一研究领域的先行者。他创立了“外基因学说”,开创了人工培育新物种的新思路。获得“利利学术”及“古根海姆”奖。１９７０年被选为台湾中央研究院院士。牛教授身居海外,对祖国科学教育事业极为关注,１９７８年,首先接收中科院派去美国的访问学者,打破中美学术交流的禁令,１９８０年中科院授予他科学顾问,中国各地的大学及科研机构授予他名誉教授、顾问头衔者３０多个。近来,为促进中国生命科学的研究和教育事业的发展,在他主持下在北京成立了“牛满江基金会”,将为培养优秀人才,促进国际学术交流方面作更多的贡献。     

动物克隆

简要介绍了动物克隆技术的发展历史和现状,以及该项技术在医学、畜牧业、濒危动物保护等研究领域的应用前景。     

克隆动物研究进展

本文介绍了上世界上中国条用细胞核移植技术克隆动物的研究历史。综述了细胞核移植的程序、方法和影响因素,包括受体卵母细胞的去核、供体细胞核的制备、核移植、激活、受体细胞与供体细胞的融合、重组胚的体内和体外培养以及胚胎移植产生克隆动物。对克隆动物研究和应用前景进行了讨论。近期的研究结果表明,多代克隆可产生在量遗传性相同的动物,不久的将来克隆技术在商业上的应用将成为现实。     

克隆凝血因子Ⅷ

生物技术业最富雄心且令人振奋的目标之一已经达到。在本期发表的四篇论文中,Genentech公司(在伦敦皇家自由医院Tuddenham等帮助下)和遗传研究所公司(在罗彻斯特Mayo诊所帮助下)报告了指导合成人凝血因子Ⅷ的:C的DNA的克隆,以及克隆DNA在哺乳动物细胞系中表达以生产这种凝血因子的情况。     

治疗性克隆

治疗性克隆是利用核移植技术将病人的体细胞核移植到去核的卵母细胞中 ,使其重编程并发育成囊胚 ,然后再用胚胎干细胞分离技术从克隆囊胚的ICM分离出多能胚胎干细胞 (ES)。这种干细胞在遗传学上和病人完全一致 ,再定向诱导其分化成病人所需要的体细胞进行移植 ,以取代和修复患者已丧失功能的细胞、组织或器官 ,而达到完全治愈。治疗性克隆不仅解决移植物与受者间的免疫排斥反应问题 ,而且可以解决移植物的来源问题。     

克隆动物研究进展

本文介绍了世界上和中国采用细胞核移植技术克隆动物的研究历史。综述了细胞核移植的程序、方法和影响因素,包括受体卵母细胞的去核、供体细胞核的制备、核移植、激活、受体细胞与供体细胞的融合、重组胚的体内和体外培养以及胚胎移植产生克隆动物。对克隆动物研究和应用前景进行了讨论。近期的研究结果表明,多代克隆可产生大量遗传性相同的动物,不久的将来克隆技术在商业上的应用将成为现实。     

定位候选克隆

当前,人类基因组研究的重心正在由“结构”向“功能”转移,一个以基因组功能研究为主要内容的所谓“后基因组时代”（ｐｏｓｔ－ｇｅ－ｎｏｍｉｃｓ）,也即功能基因组（ｆｕｎｃｔｉｏｎａｌｇｅ－ｎｏｍｉｃｓ）时代,即将到来。如何获取基因的功能信息,即与人类重大...     

用差异显示PCR技术克隆冬小麦春化设定cDNA克隆实验系统的建立

差异显示PCR技术是一种新近发展起来的在真核细胞中检测与克隆特异表达基因的手段。本文建立了用此技术克隆春化相关基因的研究系统,并提供了诸如总RNA的提取、污染DNA的去除、sscDNA的逆转录、PCR参数、扩增cDNA的电泳、特异cDNA的收集与重扩增等在方法上的细节。用这种技术分离了一个仅在春化20天这一特异时期表达的春化设定cDNA克隆VPC28。这些结果也适用于更加广泛的类似研究。     

In situ PCR technique based on pricking microinjection for cDNA cloning in single cells of barley coleoptile and powdery mildew pathogen

To clone cDNAs of mRNA specifically expressed at the infection sites, we applied the polymerase chain reaction (PCR) combined with pricking microinjection to barley coleoptile epidermis inoculated with powdery mildew pathogen. In essence, first-strand cDNAs were synthesized in situ the needle-pricked epidermal cells in which fungal haustoria had formed, and were subsequently amplified by PCR with synthetic primers. The amplified DNAs were subcloned into a plasmid vector for the construction of a cDNA library. The antisense RNAs were in vitro-transcribed from subcloned DNAs, labelled, and introduced into pathogen-invaded coleoptile epidermal cells by pricking microinjection. Target cell-specific cDNAs were identified by a specific in situ hybridization in the pathogen-invaded cells. This technique was also applied to the amplification and identification of cDNAs which were reverse-transcribed from mRNAs of targeted infection structures of the powdery mildew pathogens inoculated onto barley coleoptile epidermis.     

Cloning and characterization of a symbiosis-related gene from an ectomycorrhizal fungus Laccaria bicolor

An in vitro system for a Laccaria bicolor×Pinus resinosa interaction was used to identify and clone a symbiosis-regulated gene from L. bicolor employing the mRNA differential display technique (DDRT–PCR). The DDRT–PCR identified several cDNAs that are differentially expressed as early as 6 h into the interaction. One such cDNA was used to screen a L. bicolor cDNA library enriched for mRNAs expressed during early interaction with red pine seedlings. Characterization of a cDNA clone, PF6.2, showed that it contained a 1551 bp insert coding for a protein of 433 amino acids. Sequence analysis of the PF6.2 cDNA revealed the presence of several evolving repeats in the protein. To confirm this, the gene corresponding to PF6.2 was isolated and sequenced. The PF6.2 gene consisted of seven exons interrupted by six relatively small introns. Although the amino-acid sequence of the PF6.2 did not show significant overall similarity to any previously characterized proteins, of several direct repeats it contained a feature similar to other proteins involved in signal transduction through protein–protein interaction. Northern analysis showed that the PF6.2 mRNA was detectable in the fungus 6 h after interaction and continued to be expressed in established ectomycorrhizas, suggesting that it plays an important role in the formation and maintenance of the symbiosis.     

Cloning of region-specific genetic markers of planaria using a new method--ordered differential display

A new method for finding differentially expressed genes, termed ordered differential display of mRNAs (ODD), was used in the search for region-specific molecular markers of freshwater planarian Dugesia tigrina. In this method, the effect of selective suppression of a polymerase chain reaction (PCR) is used for the differential amplification of a pool of 3'-terminal cDNA fragments generated by digestion of cDNAs with a restriction endonuclease. In the resulting amplified cDNAs, every mRNA is represented by a cDNA fragment whose length is determined by the position of the restriction site nearest to the 3'-terminus. Subsequent PCR with primers 3'-extended by two random nucleotides allowed the amplification of 1/192 part of all cDNA molecules present in the sample. The comparison of the generated pools of cDNA molecules separated by PAGE leads to the identification of differentially expressed sequences. The systematic study of the total mRNA pool is achieved by the successive use of all possible combinations of extended primers. Some sequences preferentially expressed along the anterior-posterior axis of planarian were identified using ODD.     

Global amplification of cDNA from limiting amounts of tissue. An improved method for gene cloning and analysis

In this study we present an improved polymerase chain reaction (PCR)-based methodology to generate large amounts of high-quality complementary DNA (cDNA) from small amounts of initial total RNA. Global amplification of cDNA makes it possible to simultaneously clone many cDNAs and to construct directional cDNA libraries from a sequence-abundance-normalized cDNA population, and also permits rapid amplification of cDNA ends (RACE), from a limited amount of starting material. The priming of cDNAs with an adapter oligo-deoxythymidine (oligo-dT) primer and the ligation of a modified oligonucleotide to the 3′ end of single-stranded cDNAs, through the use of T4 RNA ligase, generates known sequences on either end of the cDNA population. This helps in the global amplification of cDNAs and in the sequence-abundance normalization of the cDNA population through the use of PCR. Utilization of a long-range PCR enzyme mix to amplify the cDNA population helps to reduce bias toward the preferential amplification of shorter molecules. Incorporation of restriction sites in the PCR primers allows the amplified cDNAs to be directionally cloned into appropriate cloning vectors to generate cDNA libraries. RACE-PCR done with biotinylated primers and streptavidin-coated para-magnetic particles are used for the efficient isolation of either full-length coding or noncoding strands.     

Cloning of cancer-related genes of Rat6 fibroblasts by using an improved differential display method]

The p53 gene is the most frequently mutated gene identified so far in human cancers. When a mutant p53(135)-val gene was allowed to be over-expressed in Rat6(R6) cells, a high incidence of spontaneous transformation was observed in long-term culture of this cell line(R6 # 13-8). To identify genes involved in cell transformation, parental p53 over-expressing cell, R6 # 13-8, and its spontaneous transformant T2, were analyzed by an improved mRNA differential display technique, which was reproducible, simpler, and was able to clone cDNA longer than 500 bp, and was with less false positives. When 33 10-mer or 12-mer single primers with arbitrary but defined sequence were used for PCR, over 90 discrete cDNAs were obtained from R6 # 13-8 and T2 cells. Three differentially expressed cDNAs were identified, one of them is highly expressed in T2 cells, while the other two, 0.8 kb and 0.9 kb long, are highly expressed in R6 # 13-8 cells. The latter were cloned and confirmed by Northern hybridization. Both cloned fragment were not homologous with any published sequence. Our results suggest that the activation and inactivation of genes are involved in the process of the spontaneous transformation from R6 # 13-8 to T2.     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫(Eimeria tenella)孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交(SSH)技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。