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克隆”是从英文“clone”音译而来,在生物学领域有3个不同层次的含义。
1.在分子水平,克隆一般指DNA克隆(也叫分子克隆)。含义是将某一特定DNA片断通过重组DNA技术插入到一个载体(如质粒和病毒等)中,然后在宿主细胞中进行自我复制所得到的大量完全相同的该DNA片断的“群体”。
2.在细胞水平,克隆实质由一个单一的共同祖先细胞分裂所形成的一个细胞群体。其中每个细胞的基因都相同。比如,使一个细胞在体外的培养液中分裂若干代所形成的一个遗传背景完全相同的细胞集体即为一个细胞克隆。又如,在脊椎动物体内,当有外源物(如细菌或病毒)侵入时,会通过免疫反应产生特异的识别抗体。产生某一特定抗体的所有浆细胞都是由一个B细胞分裂而成,这样的一个浆细胞群体也是一个细胞克隆。细胞克隆是一种低级的生殖方式-无性繁殖,即不经过两性结合,子代和亲代具有相同的遗传性。生物进化的层次越低,越有可能采取这种繁殖方式。
3.在个体水平,克隆是指基因型完全相同的两个或更多的个体组成的一个群体。比如,两个同卵双胞胎即为一个克隆!因为他(她)们来自同一个卵细胞,所以遗传背景完全一样。按此定义,“多利”并不能说成是一个克隆!因为“多利”只是孤单的一个。只有当那些英国胚胎学家能将两个以上完全相同的细胞核移植到两个以上完全相同的去核卵细胞中,得到两个以上遗传背景完全相同的“多利”时才能用克隆这个词来描述。所以在那篇发表于1997年2月出版在《Nature》杂志上的轰动性论文中,作者并没有把“多利”说成是一个克隆。
另外,克隆也可以做动词用,意思是指获得以上所言DNA、细胞或个体群体的过程。
二、克隆技术
1.DNA克隆
现在进行DNA克隆的方法多种多样,其基本过程如下图所示(未按比例)
可见,这样得到的DNA可以应用于生物学研究的很多方面,包括对特异DNA的碱基顺序的分析和处理,以及生物技术工业中有价值蛋白质的大量生产等等。
2.生物个体的克隆
(1)植物个体的克隆
在20世纪50年代,植物学家用胡萝卜为模型材料,研究了分化的植物细胞中遗传物质是否丢失问题,他们惊奇地发现,从一个单一已经高度分化的胡萝卜细胞
可以发育形成一棵完整的植株!由此,他们认为植物细胞具有全能性。从一棵胡萝卜中的两个以上的体细胞发育而成的胡萝卜群体的遗传背景完全一样,故为一个克隆。如此的植物的克隆过程是一个完全的无性繁殖过程!
(2)动物个体的克隆
① “多利”的诞生
1997年2月27日英国爱丁堡罗斯林(Roslin)研究所的伊恩·维尔莫特科学研究小组向世界宣布,世界上第一头克隆绵羊“多利”(Dolly)诞生,这一消息立刻轰动了全世界。
“多莉”的产生与三只母羊有关。一只是怀孕三个月的芬兰多塞特母绵羊,两只是苏格兰黑面母绵羊。芬兰多塞特母绵羊提供了全套遗传信息,即提供了细胞核(称之为供体);一只苏格兰黑面母绵羊提供无细胞核的卵细胞;另一只苏格兰黑面母绵羊提供羊胚胎的发育环境——子宫,是“多莉”羊的“生”母。其整个克隆过程简述如下:
从芬兰多塞特母绵羊的乳腺中取出乳腺细胞,将其放入低浓度的营养培养液中,细胞逐渐停止了分裂,此细胞称之为供体细胞;给一头苏格兰黑面母绵羊注射促性腺素,促使它排卵,取出未受精的卵细胞,并立即将其细胞核除去,留下一个无核的卵细胞,此细胞称之为受体细胞;利用电脉冲的方法,使供体细胞和受体细胞发生融合,最后形成了融合细胞,由于电脉冲还可以产生类似于自然受精过程中的一系列反应,使融合细胞也能象受精卵一样进行细胞分裂、分化,从而形成胚胎细胞;将胚胎细胞转移到另一只苏格兰黑面母绵羊的子宫内,胚胎细胞进一步分化和发育,最后形成一只小绵羊。出生的“多莉”小绵羊与多塞特母绵羊具有完全相同的外貌。
一年以后,另一组科学家报道了将小鼠卵丘细胞(围绕在卵母细胞外周的高度分化细胞)的细胞核移植到去除了细胞核的卵母细胞中得到20多只发育完全的小鼠。如呆“多利”因为只有一只,还不够叫做克隆羊的话,这些小鼠
就是名副其实的克隆鼠了。
② 通过细胞核移植克隆小鼠的基本过程
在本实验中,卵丘细胞是经如下过程得到的:通过连续几次注射绒毛膜促性腺激素,使雌鼠诱导成高产卵量状态。然后从雌鼠输卵管中收集卵丘细胞与卵母细胞的复合体。经透明质酸处理使卵丘细胞散开。选择直径为10-12微米的卵丘细胞用作细胞核供体(前期实验表明,若用直径更小或更大的卵丘细胞的细胞核,经过细胞核移植的卵母细胞很少发育到8细胞期)。所选择的卵丘细胞保持在一定的溶液环境中,在3小时内进行细胞核移植(与此不同的是,在获得“多利”时用作细胞核供体的乳腺细胞先在培养液中传代了3-6次)
卵母细胞(一般处于减数分裂中期 II )通过与上面描述类似的方法,从不同种的雌鼠中收集。在显微镜下小心地用直径大约7微米的细管取出卵母细胞的细胞核,尽量不取出细胞质。同样小心取出卵丘细胞的细胞核,也尽量去除所带的细胞质(通过使取出的细胞核在玻璃管中往复运动数次,以去除所带的少量的细胞质)。在细胞核被取出后5分钟之内,直接注射到已经去除了细胞核的卵母细胞中。进行了细胞核移植的卵母细胞先放在一种特制的溶液中1-6小时,然后加入二价的锶离子(Sr2+)和细胞分裂抑素B。前者使卵母细胞激活,后者抑制极体的形成和染色体的排除。再取出处理过的卵母细胞,放在没有锶和细胞分裂抑素B的特制的溶液中使细胞分裂形成胚胎。
不同阶段的胚胎(从2细胞期到胚泡期)被分别植入几天前与已经结扎雄鼠交配过的假孕母鼠的输卵管或子宫中发育。发育完全的胎儿鼠在大约19天后通过手术取出。
目前胚胎细胞核移植克隆的动物有小鼠、兔、山羊、绵羊、猪、牛和猴子等。在中国,除猴子以外,其他克隆动物都有,也能连续核移植克隆山羊,该技术比胚胎分割技术更进一步,将克隆出更多的动物。因胚胎分割次数越多,每份细胞越少,发育成的个体的能力越差。体细胞核移植克隆的动物只有一个,就是“多利”羊。
三、克隆技术的福音
1. 克隆技术与遗传育种
在农业方面,人们利用“克隆”技术培育出大量具有抗旱、抗倒伏、抗病虫害的优质高产品种,大大提高了粮食产量。在这方面我国已迈入世界最先进的前列。
2. 克隆技术与濒危生物保护
克隆技术对保护物种特别是珍稀、濒危物种来讲是一个福音,具有很大的应用前景。从生物学的角度看,这也是克隆技术最有价值的地方之一。
3. 克隆技术与医学
在当代,医生几乎能在所有人类器官和组织上施行移植手术。但就科学技术而言,器官移植中的排斥反应仍是最为头痛的事。排斥反应的原因是组织不配型导致相容性差。如果把“克隆人”的器官提供给“原版人”,作器官移植之用,则绝对没有排斥反应之虑,因为二者基因相配,组织也相配。问题是,利用“克隆人”作为器官供体合不合乎人道?是否合法?经济是否合算?
克隆技术还可用来大量繁殖有价值的基因,例如,在医学方面,人们正是通过“克隆”技术生产出治疗糖尿病的胰岛素、使侏儒症患者重新长高的生长激素和能抗多种病毒感染的干挠素,等等。
1.在分子水平,克隆一般指DNA克隆(也叫分子克隆)。含义是将某一特定DNA片断通过重组DNA技术插入到一个载体(如质粒和病毒等)中,然后在宿主细胞中进行自我复制所得到的大量完全相同的该DNA片断的“群体”。
2.在细胞水平,克隆实质由一个单一的共同祖先细胞分裂所形成的一个细胞群体。其中每个细胞的基因都相同。比如,使一个细胞在体外的培养液中分裂若干代所形成的一个遗传背景完全相同的细胞集体即为一个细胞克隆。又如,在脊椎动物体内,当有外源物(如细菌或病毒)侵入时,会通过免疫反应产生特异的识别抗体。产生某一特定抗体的所有浆细胞都是由一个B细胞分裂而成,这样的一个浆细胞群体也是一个细胞克隆。细胞克隆是一种低级的生殖方式-无性繁殖,即不经过两性结合,子代和亲代具有相同的遗传性。生物进化的层次越低,越有可能采取这种繁殖方式。
3.在个体水平,克隆是指基因型完全相同的两个或更多的个体组成的一个群体。比如,两个同卵双胞胎即为一个克隆!因为他(她)们来自同一个卵细胞,所以遗传背景完全一样。按此定义,“多利”并不能说成是一个克隆!因为“多利”只是孤单的一个。只有当那些英国胚胎学家能将两个以上完全相同的细胞核移植到两个以上完全相同的去核卵细胞中,得到两个以上遗传背景完全相同的“多利”时才能用克隆这个词来描述。所以在那篇发表于1997年2月出版在《Nature》杂志上的轰动性论文中,作者并没有把“多利”说成是一个克隆。
另外,克隆也可以做动词用,意思是指获得以上所言DNA、细胞或个体群体的过程。
二、克隆技术
1.DNA克隆
现在进行DNA克隆的方法多种多样,其基本过程如下图所示(未按比例)
可见,这样得到的DNA可以应用于生物学研究的很多方面,包括对特异DNA的碱基顺序的分析和处理,以及生物技术工业中有价值蛋白质的大量生产等等。
2.生物个体的克隆
(1)植物个体的克隆
在20世纪50年代,植物学家用胡萝卜为模型材料,研究了分化的植物细胞中遗传物质是否丢失问题,他们惊奇地发现,从一个单一已经高度分化的胡萝卜细胞
可以发育形成一棵完整的植株!由此,他们认为植物细胞具有全能性。从一棵胡萝卜中的两个以上的体细胞发育而成的胡萝卜群体的遗传背景完全一样,故为一个克隆。如此的植物的克隆过程是一个完全的无性繁殖过程!
(2)动物个体的克隆
① “多利”的诞生
1997年2月27日英国爱丁堡罗斯林(Roslin)研究所的伊恩·维尔莫特科学研究小组向世界宣布,世界上第一头克隆绵羊“多利”(Dolly)诞生,这一消息立刻轰动了全世界。
“多莉”的产生与三只母羊有关。一只是怀孕三个月的芬兰多塞特母绵羊,两只是苏格兰黑面母绵羊。芬兰多塞特母绵羊提供了全套遗传信息,即提供了细胞核(称之为供体);一只苏格兰黑面母绵羊提供无细胞核的卵细胞;另一只苏格兰黑面母绵羊提供羊胚胎的发育环境——子宫,是“多莉”羊的“生”母。其整个克隆过程简述如下:
从芬兰多塞特母绵羊的乳腺中取出乳腺细胞,将其放入低浓度的营养培养液中,细胞逐渐停止了分裂,此细胞称之为供体细胞;给一头苏格兰黑面母绵羊注射促性腺素,促使它排卵,取出未受精的卵细胞,并立即将其细胞核除去,留下一个无核的卵细胞,此细胞称之为受体细胞;利用电脉冲的方法,使供体细胞和受体细胞发生融合,最后形成了融合细胞,由于电脉冲还可以产生类似于自然受精过程中的一系列反应,使融合细胞也能象受精卵一样进行细胞分裂、分化,从而形成胚胎细胞;将胚胎细胞转移到另一只苏格兰黑面母绵羊的子宫内,胚胎细胞进一步分化和发育,最后形成一只小绵羊。出生的“多莉”小绵羊与多塞特母绵羊具有完全相同的外貌。
一年以后,另一组科学家报道了将小鼠卵丘细胞(围绕在卵母细胞外周的高度分化细胞)的细胞核移植到去除了细胞核的卵母细胞中得到20多只发育完全的小鼠。如呆“多利”因为只有一只,还不够叫做克隆羊的话,这些小鼠
就是名副其实的克隆鼠了。
② 通过细胞核移植克隆小鼠的基本过程
在本实验中,卵丘细胞是经如下过程得到的:通过连续几次注射绒毛膜促性腺激素,使雌鼠诱导成高产卵量状态。然后从雌鼠输卵管中收集卵丘细胞与卵母细胞的复合体。经透明质酸处理使卵丘细胞散开。选择直径为10-12微米的卵丘细胞用作细胞核供体(前期实验表明,若用直径更小或更大的卵丘细胞的细胞核,经过细胞核移植的卵母细胞很少发育到8细胞期)。所选择的卵丘细胞保持在一定的溶液环境中,在3小时内进行细胞核移植(与此不同的是,在获得“多利”时用作细胞核供体的乳腺细胞先在培养液中传代了3-6次)
卵母细胞(一般处于减数分裂中期 II )通过与上面描述类似的方法,从不同种的雌鼠中收集。在显微镜下小心地用直径大约7微米的细管取出卵母细胞的细胞核,尽量不取出细胞质。同样小心取出卵丘细胞的细胞核,也尽量去除所带的细胞质(通过使取出的细胞核在玻璃管中往复运动数次,以去除所带的少量的细胞质)。在细胞核被取出后5分钟之内,直接注射到已经去除了细胞核的卵母细胞中。进行了细胞核移植的卵母细胞先放在一种特制的溶液中1-6小时,然后加入二价的锶离子(Sr2+)和细胞分裂抑素B。前者使卵母细胞激活,后者抑制极体的形成和染色体的排除。再取出处理过的卵母细胞,放在没有锶和细胞分裂抑素B的特制的溶液中使细胞分裂形成胚胎。
不同阶段的胚胎(从2细胞期到胚泡期)被分别植入几天前与已经结扎雄鼠交配过的假孕母鼠的输卵管或子宫中发育。发育完全的胎儿鼠在大约19天后通过手术取出。
目前胚胎细胞核移植克隆的动物有小鼠、兔、山羊、绵羊、猪、牛和猴子等。在中国,除猴子以外,其他克隆动物都有,也能连续核移植克隆山羊,该技术比胚胎分割技术更进一步,将克隆出更多的动物。因胚胎分割次数越多,每份细胞越少,发育成的个体的能力越差。体细胞核移植克隆的动物只有一个,就是“多利”羊。
三、克隆技术的福音
1. 克隆技术与遗传育种
在农业方面,人们利用“克隆”技术培育出大量具有抗旱、抗倒伏、抗病虫害的优质高产品种,大大提高了粮食产量。在这方面我国已迈入世界最先进的前列。
2. 克隆技术与濒危生物保护
克隆技术对保护物种特别是珍稀、濒危物种来讲是一个福音,具有很大的应用前景。从生物学的角度看,这也是克隆技术最有价值的地方之一。
3. 克隆技术与医学
在当代,医生几乎能在所有人类器官和组织上施行移植手术。但就科学技术而言,器官移植中的排斥反应仍是最为头痛的事。排斥反应的原因是组织不配型导致相容性差。如果把“克隆人”的器官提供给“原版人”,作器官移植之用,则绝对没有排斥反应之虑,因为二者基因相配,组织也相配。问题是,利用“克隆人”作为器官供体合不合乎人道?是否合法?经济是否合算?
克隆技术还可用来大量繁殖有价值的基因,例如,在医学方面,人们正是通过“克隆”技术生产出治疗糖尿病的胰岛素、使侏儒症患者重新长高的生长激素和能抗多种病毒感染的干挠素,等等。
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Cloning
(genetics) The process of asexual reproduction observed, for example, in bacteria and other unicellular micro-organisms which divide by simple fission, so that the daughter cells are genetically identical to each other and to the parent (except when mutation occurs). In higher organisms, genetically identical individuals may be produced by cloning. A body (somatic) cell is taken from an embryo in an early stage of development or from an adult, the nucleus transferred to an unfertilized ovum from which the nucleus has been removed, and the product grown in culture; daughter cells from the earliest divisions are removed, and grown in culture or implanted into host mothers to give genetically identical offspring. The successful cloning of a sheep (named Dolly, 1996–2003) was reported by scientists from the Roslin Institute, Edinburgh, UK, in February 1997. There is considerable potential application in animal rearing, but its application to humans is extremely unlikely (except in some rare instances of in vitro fertilization). The term molecular cloning is used in recombinant DNA technology, where a section of foreign DNA is inserted into an artificial bacterial chromosome (plasmid) and divides with it, thus ‘cloning’ the DNA.
In 1998, a Council of Europe protocol banning the cloning of human beings was signed in Paris by 19 states - the first international treaty on the issue. However, in 1999, predictions were being made about the application of the technique in other areas, such as bone-marrow grafting in leukaemia, and transplant medicine in general, and the controversy surrounding the ethics of human cloning continued to exercise professional and public opinion. In 2004, Newcastle University received Britain's first licence to clone human embryos. In early 2006, Dr Hwang Woo-suk, a South Korean scientist working at Seoul National University, was disgraced when he admitted that his recently published research on human stem cells had been fabricated.
This page discusses genetic cloning. For the computer-related use of the term, see Disk cloning.
Cloning is the process of creating an identical copy of an original organism or thing. A cloning in the biological sense, therefore, is a molecule, single cell (like bacteria, lymphocytes etc.) or multi-cellular organism that has been directly copied from and is therefore genetically identical to another living organism. Sometimes this term can refer to "natural" clones made either when an organism asexual reproduced by chance (as with identical twins), but in common parlance, a clone is an identical copy created intentionally.
The term clone is derived from κλων, the Greek word for "twig". In horticulture, the spelling clon was used until the twentieth century; the final e came into use to indicate the vowel is a "long o" instead of a "short o". Since the term entered the popular lexicon in a more general context, the spelling clone has been used exclusively.
Molecular cloning
Molecular cloning refers to the procedure isolating a DNA sequence of interest and obtaining multiple copies of it in an organism. Cloning is frequently employed to amplify DNA fragments containing genes, an essential step in their subsequent analysis. Frequently, the term cloning is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest. In practice, localisation of the gene does not always enable one to amplify the relevant genomic sequence.
Cloning of any DNA sequence involves the following four steps: amplification, ligation, transfection, and screening/selection. Initially, the DNA fragment of interest needs to be amplified (many copies need to be produced). Amplification is commonly achieved by means of PCR. Subsequently, a ligation procedure is employed whereby the amplified fragment is inserted into a vector. The vector (which is frequently circular) is linearised by means of restriction enzymes, and incubated with the fragment of interest under appropriate conditions that allow for ligation. The yield of the ligation is typically low and depends on the procedure employed. Following ligation the vector with the insert of interest is transfected to cells. Most commonly electroporation is employed, although a number of alternative techniques are available, such as chemical sensitivation of cells. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low yield, there is the need to identify the cell colonies that have been transfected with the construct of interest containing the desired insertion sequence. Modern cloning vectors include selectable antibiotic resistance markers, which allow only for cells in which the vector has been transfected to grow. However this selection step does not guarantee that the DNA insert is present in the vector. Further investigation of the resulting colonies is required to confirm that cloning was successful. This can be accomplished by means of blue/white screening (α-factor complimentation) on X-gal medium and/or PCR, possibly followed by DNA sequencing.
Genetic cloning
Cloning a cell means to derive a (clonal) population of cells from a single cell. This is an important in vitro procedure when the expansion of a single cell with certain characteristics is desired, for example in the production of gene-targeted ES cells. Most individuals began as a single cell and are therefore the result of clonal expansion in vivo.
Organism
Cloning means to create a new organism with the same genetic information as a cell from an existing one(identical). It is an asexual method of reproduction, where fertilization or inter-gamete contact does not take place. Asexual reproduction (also known as agamogenesis) is a form of reproduction which does not involve meiosis, gamete formation, or fertilization. In laymen's terms, there is only one "parent" involved. This form of reproduction is common among simple organisms such as amoeba and other single-celled organisms, although most plants reproduce asexually as well (see vegetative reproduction).
Horticultural
The term clone is used in horticulture to mean all descendants of a single plant, produced by vegetative reproduction or apomixis. Many horticultural plant cultivars are clones, having been derived from a single individual, multiplied by some process other than sexual reproduction. As an example, some European cultivars of grapes represent clones that have been propagated for over two millennia. Other examples are potato and banana. Grafting can be regarded as cloning, since all the shoots and branches coming from the graft are genetically a clone of a single individual, although the root systems may be genetically genuine examples of cloning in the broader biological sense, as they create genetically identical organisms by biological means, but this particular kind of cloning has not come under ethical scrutiny and is generally treated as an entirely different kind of operation.
Many trees, shrubs, vines, ferns and other herbaceous perennials form clonal colonies. Parts of a large clonal colony often become detached from the parent, termed fragmentation, to form separate individuals. Some plants also form seeds asexually, termed apomixis, e.g. dandelion.
Animals
Cloning exists in nature in some animal species and is referred to as parthenogenesis. An example is the "Little Fire Ant" (Wasmannia auropunctata), which is native to Central and South America but has spread throughout many tropical environments.
Reproductive Cloning
Reproductive cloning is a technology used to generate an animal that has the same nuclear DNA as another currently or previously existing animal. Dolly the sheep, was created by reproductive cloning technology. In a process called "somatic cell nuclear transfer" (SCNT), scientists transfer genetic material from the nucleus of a donor adult cell to an egg whose nucleus, and thus its genetic material, has been removed. The reconstructed egg containing the DNA from a donor cell must be treated with chemicals or electric current in order to stimulate cell division. Once the cloned embryo reaches a suitable stage, it is transferred to the uterus of a female host where it continues to develop until birth.
Dolly or any other animal created using nuclear transfer technology is not truly an identical clone of the donor animal. Only the clone's chromosomal or nuclear DNA is the same as the donor. Some of the clone's genetic materials come from the mitochondria in the cytoplasm of the enucleated egg. Mitochondria, which are organelles that serve as power sources to the cell, contain their own short segments of DNA, although this is only 0.01% of the total DNA. Acquired mutations in mitochondrial DNA are believed to play an important role in the aging process.
Also mutations occur with every cell division so no two cells in an individual are identical, nor are clones. Thus, nuclear transfer clones from different maternal lineages are not clones in the strictest sense because the mitochondrial genome is not the same as that of the nucleus donor cell from which it was produced. This may have important implications for cross-species nuclear transfer in which nuclear-mitochondrial incompatibilities may lead to inviability.
Species cloned
The modern cloning techniques involving nuclear transfer have been successfully performed on several species. Landmark experiments in chronological order:
Tadpole: (1952) Many scientists questioned whether cloning had actually occurred and unpublished experiments by other labs were not able to reproduce the reported results.
Carp: (1963) In China, embryologist Tong Dizhou cloned a fish. He published the findings in an obscure Chinese science journal which was never translated into English.
Mice: (1986) was the first successfully cloned mammal; Soviet scientists Chaylakhyan, Veprencev, Sviridova, Nikitin had mice "Masha" cloned. Research was published in the magazine "Biofizika" volume ХХХII, issue 5 of 1987.
Sheep: (1996) From early embryonic cells by Steen Willadsen. Megan and Morag cloned from differentiated embryonic cells in June 1995 and Dolly the sheep in 1997.
Rhesus Monkey: Tetra (female, January 2000) from embryo splitting
Cattle: Alpha and Beta (males, 2001) and (2005) Brazil
Cat: CopyCat "CC" (female, late 2001), Little Nicky, 2004, was the first cat cloned for commercial reasons
Mule: Idaho Gem, a john mule born 2003-05-04, was the first horse-family clone.
Horse: Prometea, a Halflinger female born 2003-05-28, was the first horse clone.
For a complete list see: List of animals that have been cloned. During the first several divisions of a fertilized egg, no differentiation occurs and the cells can be separated without harm, but each will grow into an identical individual. This process has been used on cattle for decades to produce hundreds of identical individuals in some cases. This process is not considered cloning, but is called budding. The new individual is not derived from a differentiated cell, but from an undifferentiated egg. There is no way to determine which are the clones and which is the original.
Health aspects
The success rate of cloning has been low: Dolly the sheep was born after 277 eggs were used to create 29 embryos, which only produced three lambs at birth, only one of which lived, Dolly. Seventy calves have been created from 9,000 attempts and one third of them died young; Prometea took 328 attempts, and, more recently, Paris Texas was created after 400 attempts. Notably, although the first clones were frogs, no adult cloned frog has yet been produced from a somatic adult nucleus donor cell.
There were early claims that Dolly the Sheep had accelerated aging. Aging of this type is thought to be due to shortening of telomeres, regions at the tips of chromosomes which prevent genetic threads fraying every time a cell divides. Over time telomeres get worn down until cell-division is no longer possible — this is thought to be a cause of aging. However, subsequent studies showed that, if anything, Dolly's telomere were longer than normal. Dolly died in the year of 2003. Ian Wilmut said that Dolly's early death had nothing to do with cloning but with a respiratory infection common to lambs raised indoors like Dolly.
Consistent with Dolly's telomeres being longer, analysis of the telomeres from cloned cows showed that they were also longer. This suggests clones could live longer life spans although many died young after excessive growth. Researchers think that this could eventually be developed to reverse aging in humans, provided that this is based chiefly on the shortening of telomeres. Although some work has been performed on telomeres and aging in nuclear transfer clones, the evidence is at an early stage.
Dolly the Sheep
Dolly (1996-07-05 – 2003-02-14), an ewe, was the first mammal to have been successfully cloned from an adult cell (while the mice in USSR was cloned from embryo cell back in 1986 ). She was cloned at the Roslin Institute in Scotland and lived there until her death when she was 6. Her birth was announced on 1997-02-22.
The name "Dolly" came from a suggestion by Jesse Haase who helped with her birth, in honor of Dolly Parton, because it was a mammary cell that was cloned. The technique that was made famous by her birth is somatic cell nuclear transfer, in which a non-reproductive cell containing a nucleus is placed in a de-nucleated ovum (which then develops into a fetus). When Dolly was cloned in 1996 from a cell taken from a six-year-old ewe, she became the center of much controversy that still exists today.
Dolly's success is truly remarkable because it proved that the genetic material from a specialized adult cell, such as an udder cell programmed to express only those genes needed by udder cells, could be reprogrammed to generate an entire new organism. Before this demonstration, scientists believed that once a cell became specialized as a liver, heart, udder, bone, or any other type of cell, the change was permanent and other unneeded genes in the cell would become inactive. Some scientists believe that errors or incompleteness in the reprogramming process cause the high rates of death, deformity, and disability observed among animal clones.
On 2003-04-09 her stuffed remains were placed at Edinburgh's Royal Museum, part of the National Museums of Scotland.
Ian Wilmut's role in cloning Dolly the sheep is in doubt. In 2006 he admitted under oath in a Scottish court that he did not clone Dolly the Sheep and was not responsible for the scientific breakthrough which made it all possible. He credited Keith Campbell as being the brains behind Dolly the Sheep.
In August 2006, Iranian scientists oversaw the birth of the Middle East’s first cloned animal – a lamb that died minutes after it was born. However, in September 2006, Iranian scientists successfully cloned a sheep, by somatic cell nuclear transfer, at the Royan research institute in Isfahan, Iran’s second cloned lamb is still alive.
Human cloning
Human cloning is the creation of a genetically identical copy of an existing, or previously existing human, by growing cloned tissue from that individual. The term is generally used to refer to artificial human cloning; human clones in the form of identical twins are commonplace, with their cloning occurring during the natural process of reproduction.
Human cloning is amongst the most controversial forms of the practice. There have been numerous demands for all progress in the human cloning fields to be halted. One of the most ethically questionable problems with human cloning is farming of organs from clones. For example, many believe it is unethical to use a human clone to save the life of another. In this scenario, the cloned human would be euthanized so that the vital organs could be harvested. This process of renewing the body's organs would potentially increase the life expectancy of a human by 50 years.
The cloning described above is reproductive cloning, not to be confused with research cloning in which only parts (such as an organ) are cloned using genetic material from a patient's tissues.
Ethical issues of human cloning
Roman Catholicism and many conservative Christian groups have opposed human cloning and the cloning of human embryos, believing that a human life begins the moment a human egg becomes fertilized. Other Christian denominations such as the United Church of Christ do not believe a fertilized egg constitutes a living being, but still they oppose the cloning of embryonic cells. The World Council of Churches, representing nearly 400 denominations worldwide, opposed cloning of both human embryos and whole humans in February 2006. The United Methodist Church opposed research and reproductive cloning in May 2000 and again in May 2004.
Libertarian views on the subject suggest that the federal government of the United States does not have the power to regulate cloning, as it is not given any such authority by the US constitution. (Similar to abortion rights.)
At present, the main objection to human cloning is that the cloned individual may be biologically damaged, due to the inherent unreliability of its origin: researchers currently are unable to safely and reliably clone non-human primates.
However, many believe that as cloning research and methods improve, concerns of safety and reliability will no longer be an issue. However, it must be pointed out that this has yet to occur, and may never occur. Rudolph Jaenisch, a professor at Harvard, has pointed out that we have become more efficient at producing clones which are still defective (Development Dynamics. Volume 235, pages 2460-2469. 2006). Other arguments against cloning come from various religious orders (believing cloning violates God's will or the natural order of life), and a general discomfort some have with the idea of "meddling" with the creation and basic function of life. This unease often manifests itself in contemporary novels, movies, and popular culture, much like numerous other scientific discoveries and inventions before. Various fictional scenarios portray clones being unhappy, soulless, or unable to integrate into society. Furthermore, clones are often depicted not as unique individuals but as "spare parts," providing organs for the clone's original (or any non-clone that requires replacement organs).
Needless to say, cloning is a poignant and important topic, reflected by its frequent discussion and debate among politicians, scientists, the media, religions, and the general public.
Cloning extinct and endangered species
Cloning, or more precisely, the reconstruction of functional DNA from extinct species has, for decades, been a dream of some scientists. The possible implications of this were dramatized in the best-selling novel by Michael Crichton and high budget Hollywood thriller Jurassic Park. In real life, one of the most anticipated targets for cloning was once the Woolly mammoth, but attempts to extract DNA from frozen mammoths have been unsuccessful, though a joint Russo-Japanese team is currently working toward this goal.
In 2000, a cow named dendi Bessie gave birth to a cloned Asian gaur, an endangered species, but the calf died after two days. In 2003, a banteng was successfully cloned, followed by three African wildcats from a thawed frozen embryo. These successes provided hope that similar techniques (using surrogate mothers of another species) might be used to clone extinct species. Anticipating this possibility, tissue samples from the last bucardo (Pyrenean Ibex) were frozen immediately after it died. Researchers are also considering cloning endangered species such as the giant panda, ocelot, and cheetah. The "Froz
Cloning
(genetics) The process of asexual reproduction observed, for example, in bacteria and other unicellular micro-organisms which divide by simple fission, so that the daughter cells are genetically identical to each other and to the parent (except when mutation occurs). In higher organisms, genetically identical individuals may be produced by cloning. A body (somatic) cell is taken from an embryo in an early stage of development or from an adult, the nucleus transferred to an unfertilized ovum from which the nucleus has been removed, and the product grown in culture; daughter cells from the earliest divisions are removed, and grown in culture or implanted into host mothers to give genetically identical offspring. The successful cloning of a sheep (named Dolly, 1996–2003) was reported by scientists from the Roslin Institute, Edinburgh, UK, in February 1997. There is considerable potential application in animal rearing, but its application to humans is extremely unlikely (except in some rare instances of in vitro fertilization). The term molecular cloning is used in recombinant DNA technology, where a section of foreign DNA is inserted into an artificial bacterial chromosome (plasmid) and divides with it, thus ‘cloning’ the DNA.
In 1998, a Council of Europe protocol banning the cloning of human beings was signed in Paris by 19 states - the first international treaty on the issue. However, in 1999, predictions were being made about the application of the technique in other areas, such as bone-marrow grafting in leukaemia, and transplant medicine in general, and the controversy surrounding the ethics of human cloning continued to exercise professional and public opinion. In 2004, Newcastle University received Britain's first licence to clone human embryos. In early 2006, Dr Hwang Woo-suk, a South Korean scientist working at Seoul National University, was disgraced when he admitted that his recently published research on human stem cells had been fabricated.
This page discusses genetic cloning. For the computer-related use of the term, see Disk cloning.
Cloning is the process of creating an identical copy of an original organism or thing. A cloning in the biological sense, therefore, is a molecule, single cell (like bacteria, lymphocytes etc.) or multi-cellular organism that has been directly copied from and is therefore genetically identical to another living organism. Sometimes this term can refer to "natural" clones made either when an organism asexual reproduced by chance (as with identical twins), but in common parlance, a clone is an identical copy created intentionally.
The term clone is derived from κλων, the Greek word for "twig". In horticulture, the spelling clon was used until the twentieth century; the final e came into use to indicate the vowel is a "long o" instead of a "short o". Since the term entered the popular lexicon in a more general context, the spelling clone has been used exclusively.
Molecular cloning
Molecular cloning refers to the procedure isolating a DNA sequence of interest and obtaining multiple copies of it in an organism. Cloning is frequently employed to amplify DNA fragments containing genes, an essential step in their subsequent analysis. Frequently, the term cloning is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest. In practice, localisation of the gene does not always enable one to amplify the relevant genomic sequence.
Cloning of any DNA sequence involves the following four steps: amplification, ligation, transfection, and screening/selection. Initially, the DNA fragment of interest needs to be amplified (many copies need to be produced). Amplification is commonly achieved by means of PCR. Subsequently, a ligation procedure is employed whereby the amplified fragment is inserted into a vector. The vector (which is frequently circular) is linearised by means of restriction enzymes, and incubated with the fragment of interest under appropriate conditions that allow for ligation. The yield of the ligation is typically low and depends on the procedure employed. Following ligation the vector with the insert of interest is transfected to cells. Most commonly electroporation is employed, although a number of alternative techniques are available, such as chemical sensitivation of cells. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low yield, there is the need to identify the cell colonies that have been transfected with the construct of interest containing the desired insertion sequence. Modern cloning vectors include selectable antibiotic resistance markers, which allow only for cells in which the vector has been transfected to grow. However this selection step does not guarantee that the DNA insert is present in the vector. Further investigation of the resulting colonies is required to confirm that cloning was successful. This can be accomplished by means of blue/white screening (α-factor complimentation) on X-gal medium and/or PCR, possibly followed by DNA sequencing.
Genetic cloning
Cloning a cell means to derive a (clonal) population of cells from a single cell. This is an important in vitro procedure when the expansion of a single cell with certain characteristics is desired, for example in the production of gene-targeted ES cells. Most individuals began as a single cell and are therefore the result of clonal expansion in vivo.
Organism
Cloning means to create a new organism with the same genetic information as a cell from an existing one(identical). It is an asexual method of reproduction, where fertilization or inter-gamete contact does not take place. Asexual reproduction (also known as agamogenesis) is a form of reproduction which does not involve meiosis, gamete formation, or fertilization. In laymen's terms, there is only one "parent" involved. This form of reproduction is common among simple organisms such as amoeba and other single-celled organisms, although most plants reproduce asexually as well (see vegetative reproduction).
Horticultural
The term clone is used in horticulture to mean all descendants of a single plant, produced by vegetative reproduction or apomixis. Many horticultural plant cultivars are clones, having been derived from a single individual, multiplied by some process other than sexual reproduction. As an example, some European cultivars of grapes represent clones that have been propagated for over two millennia. Other examples are potato and banana. Grafting can be regarded as cloning, since all the shoots and branches coming from the graft are genetically a clone of a single individual, although the root systems may be genetically genuine examples of cloning in the broader biological sense, as they create genetically identical organisms by biological means, but this particular kind of cloning has not come under ethical scrutiny and is generally treated as an entirely different kind of operation.
Many trees, shrubs, vines, ferns and other herbaceous perennials form clonal colonies. Parts of a large clonal colony often become detached from the parent, termed fragmentation, to form separate individuals. Some plants also form seeds asexually, termed apomixis, e.g. dandelion.
Animals
Cloning exists in nature in some animal species and is referred to as parthenogenesis. An example is the "Little Fire Ant" (Wasmannia auropunctata), which is native to Central and South America but has spread throughout many tropical environments.
Reproductive Cloning
Reproductive cloning is a technology used to generate an animal that has the same nuclear DNA as another currently or previously existing animal. Dolly the sheep, was created by reproductive cloning technology. In a process called "somatic cell nuclear transfer" (SCNT), scientists transfer genetic material from the nucleus of a donor adult cell to an egg whose nucleus, and thus its genetic material, has been removed. The reconstructed egg containing the DNA from a donor cell must be treated with chemicals or electric current in order to stimulate cell division. Once the cloned embryo reaches a suitable stage, it is transferred to the uterus of a female host where it continues to develop until birth.
Dolly or any other animal created using nuclear transfer technology is not truly an identical clone of the donor animal. Only the clone's chromosomal or nuclear DNA is the same as the donor. Some of the clone's genetic materials come from the mitochondria in the cytoplasm of the enucleated egg. Mitochondria, which are organelles that serve as power sources to the cell, contain their own short segments of DNA, although this is only 0.01% of the total DNA. Acquired mutations in mitochondrial DNA are believed to play an important role in the aging process.
Also mutations occur with every cell division so no two cells in an individual are identical, nor are clones. Thus, nuclear transfer clones from different maternal lineages are not clones in the strictest sense because the mitochondrial genome is not the same as that of the nucleus donor cell from which it was produced. This may have important implications for cross-species nuclear transfer in which nuclear-mitochondrial incompatibilities may lead to inviability.
Species cloned
The modern cloning techniques involving nuclear transfer have been successfully performed on several species. Landmark experiments in chronological order:
Tadpole: (1952) Many scientists questioned whether cloning had actually occurred and unpublished experiments by other labs were not able to reproduce the reported results.
Carp: (1963) In China, embryologist Tong Dizhou cloned a fish. He published the findings in an obscure Chinese science journal which was never translated into English.
Mice: (1986) was the first successfully cloned mammal; Soviet scientists Chaylakhyan, Veprencev, Sviridova, Nikitin had mice "Masha" cloned. Research was published in the magazine "Biofizika" volume ХХХII, issue 5 of 1987.
Sheep: (1996) From early embryonic cells by Steen Willadsen. Megan and Morag cloned from differentiated embryonic cells in June 1995 and Dolly the sheep in 1997.
Rhesus Monkey: Tetra (female, January 2000) from embryo splitting
Cattle: Alpha and Beta (males, 2001) and (2005) Brazil
Cat: CopyCat "CC" (female, late 2001), Little Nicky, 2004, was the first cat cloned for commercial reasons
Mule: Idaho Gem, a john mule born 2003-05-04, was the first horse-family clone.
Horse: Prometea, a Halflinger female born 2003-05-28, was the first horse clone.
For a complete list see: List of animals that have been cloned. During the first several divisions of a fertilized egg, no differentiation occurs and the cells can be separated without harm, but each will grow into an identical individual. This process has been used on cattle for decades to produce hundreds of identical individuals in some cases. This process is not considered cloning, but is called budding. The new individual is not derived from a differentiated cell, but from an undifferentiated egg. There is no way to determine which are the clones and which is the original.
Health aspects
The success rate of cloning has been low: Dolly the sheep was born after 277 eggs were used to create 29 embryos, which only produced three lambs at birth, only one of which lived, Dolly. Seventy calves have been created from 9,000 attempts and one third of them died young; Prometea took 328 attempts, and, more recently, Paris Texas was created after 400 attempts. Notably, although the first clones were frogs, no adult cloned frog has yet been produced from a somatic adult nucleus donor cell.
There were early claims that Dolly the Sheep had accelerated aging. Aging of this type is thought to be due to shortening of telomeres, regions at the tips of chromosomes which prevent genetic threads fraying every time a cell divides. Over time telomeres get worn down until cell-division is no longer possible — this is thought to be a cause of aging. However, subsequent studies showed that, if anything, Dolly's telomere were longer than normal. Dolly died in the year of 2003. Ian Wilmut said that Dolly's early death had nothing to do with cloning but with a respiratory infection common to lambs raised indoors like Dolly.
Consistent with Dolly's telomeres being longer, analysis of the telomeres from cloned cows showed that they were also longer. This suggests clones could live longer life spans although many died young after excessive growth. Researchers think that this could eventually be developed to reverse aging in humans, provided that this is based chiefly on the shortening of telomeres. Although some work has been performed on telomeres and aging in nuclear transfer clones, the evidence is at an early stage.
Dolly the Sheep
Dolly (1996-07-05 – 2003-02-14), an ewe, was the first mammal to have been successfully cloned from an adult cell (while the mice in USSR was cloned from embryo cell back in 1986 ). She was cloned at the Roslin Institute in Scotland and lived there until her death when she was 6. Her birth was announced on 1997-02-22.
The name "Dolly" came from a suggestion by Jesse Haase who helped with her birth, in honor of Dolly Parton, because it was a mammary cell that was cloned. The technique that was made famous by her birth is somatic cell nuclear transfer, in which a non-reproductive cell containing a nucleus is placed in a de-nucleated ovum (which then develops into a fetus). When Dolly was cloned in 1996 from a cell taken from a six-year-old ewe, she became the center of much controversy that still exists today.
Dolly's success is truly remarkable because it proved that the genetic material from a specialized adult cell, such as an udder cell programmed to express only those genes needed by udder cells, could be reprogrammed to generate an entire new organism. Before this demonstration, scientists believed that once a cell became specialized as a liver, heart, udder, bone, or any other type of cell, the change was permanent and other unneeded genes in the cell would become inactive. Some scientists believe that errors or incompleteness in the reprogramming process cause the high rates of death, deformity, and disability observed among animal clones.
On 2003-04-09 her stuffed remains were placed at Edinburgh's Royal Museum, part of the National Museums of Scotland.
Ian Wilmut's role in cloning Dolly the sheep is in doubt. In 2006 he admitted under oath in a Scottish court that he did not clone Dolly the Sheep and was not responsible for the scientific breakthrough which made it all possible. He credited Keith Campbell as being the brains behind Dolly the Sheep.
In August 2006, Iranian scientists oversaw the birth of the Middle East’s first cloned animal – a lamb that died minutes after it was born. However, in September 2006, Iranian scientists successfully cloned a sheep, by somatic cell nuclear transfer, at the Royan research institute in Isfahan, Iran’s second cloned lamb is still alive.
Human cloning
Human cloning is the creation of a genetically identical copy of an existing, or previously existing human, by growing cloned tissue from that individual. The term is generally used to refer to artificial human cloning; human clones in the form of identical twins are commonplace, with their cloning occurring during the natural process of reproduction.
Human cloning is amongst the most controversial forms of the practice. There have been numerous demands for all progress in the human cloning fields to be halted. One of the most ethically questionable problems with human cloning is farming of organs from clones. For example, many believe it is unethical to use a human clone to save the life of another. In this scenario, the cloned human would be euthanized so that the vital organs could be harvested. This process of renewing the body's organs would potentially increase the life expectancy of a human by 50 years.
The cloning described above is reproductive cloning, not to be confused with research cloning in which only parts (such as an organ) are cloned using genetic material from a patient's tissues.
Ethical issues of human cloning
Roman Catholicism and many conservative Christian groups have opposed human cloning and the cloning of human embryos, believing that a human life begins the moment a human egg becomes fertilized. Other Christian denominations such as the United Church of Christ do not believe a fertilized egg constitutes a living being, but still they oppose the cloning of embryonic cells. The World Council of Churches, representing nearly 400 denominations worldwide, opposed cloning of both human embryos and whole humans in February 2006. The United Methodist Church opposed research and reproductive cloning in May 2000 and again in May 2004.
Libertarian views on the subject suggest that the federal government of the United States does not have the power to regulate cloning, as it is not given any such authority by the US constitution. (Similar to abortion rights.)
At present, the main objection to human cloning is that the cloned individual may be biologically damaged, due to the inherent unreliability of its origin: researchers currently are unable to safely and reliably clone non-human primates.
However, many believe that as cloning research and methods improve, concerns of safety and reliability will no longer be an issue. However, it must be pointed out that this has yet to occur, and may never occur. Rudolph Jaenisch, a professor at Harvard, has pointed out that we have become more efficient at producing clones which are still defective (Development Dynamics. Volume 235, pages 2460-2469. 2006). Other arguments against cloning come from various religious orders (believing cloning violates God's will or the natural order of life), and a general discomfort some have with the idea of "meddling" with the creation and basic function of life. This unease often manifests itself in contemporary novels, movies, and popular culture, much like numerous other scientific discoveries and inventions before. Various fictional scenarios portray clones being unhappy, soulless, or unable to integrate into society. Furthermore, clones are often depicted not as unique individuals but as "spare parts," providing organs for the clone's original (or any non-clone that requires replacement organs).
Needless to say, cloning is a poignant and important topic, reflected by its frequent discussion and debate among politicians, scientists, the media, religions, and the general public.
Cloning extinct and endangered species
Cloning, or more precisely, the reconstruction of functional DNA from extinct species has, for decades, been a dream of some scientists. The possible implications of this were dramatized in the best-selling novel by Michael Crichton and high budget Hollywood thriller Jurassic Park. In real life, one of the most anticipated targets for cloning was once the Woolly mammoth, but attempts to extract DNA from frozen mammoths have been unsuccessful, though a joint Russo-Japanese team is currently working toward this goal.
In 2000, a cow named dendi Bessie gave birth to a cloned Asian gaur, an endangered species, but the calf died after two days. In 2003, a banteng was successfully cloned, followed by three African wildcats from a thawed frozen embryo. These successes provided hope that similar techniques (using surrogate mothers of another species) might be used to clone extinct species. Anticipating this possibility, tissue samples from the last bucardo (Pyrenean Ibex) were frozen immediately after it died. Researchers are also considering cloning endangered species such as the giant panda, ocelot, and cheetah. The "Froz
参考资料: http://www.reference.com/search?q=cloning
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Cloning
(genetics) The process of asexual reproduction observed, for example, in bacteria and other unicellular micro-organisms which divide by simple fission, so that the daughter cells are genetically identical to each other and to the parent (except when mutation occurs). In higher organisms, genetically identical individuals may be produced by cloning. A body (somatic) cell is taken from an embryo in an early stage of development or from an adult, the nucleus transferred to an unfertilized ovum from which the nucleus has been removed, and the product grown in culture; daughter cells from the earliest divisions are removed, and grown in culture or implanted into host mothers to give genetically identical offspring. The successful cloning of a sheep (named Dolly, 1996–2003) was reported by scientists from the Roslin Institute, Edinburgh, UK, in February 1997. There is considerable potential application in animal rearing, but its application to humans is extremely unlikely (except in some rare instances of in vitro fertilization). The term molecular cloning is used in recombinant DNA technology, where a section of foreign DNA is inserted into an artificial bacterial chromosome (plasmid) and divides with it, thus ‘cloning’ the DNA.
In 1998, a Council of Europe protocol banning the cloning of human beings was signed in Paris by 19 states - the first international treaty on the issue. However, in 1999, predictions were being made about the application of the technique in other areas, such as bone-marrow grafting in leukaemia, and transplant medicine in general, and the controversy surrounding the ethics of human cloning continued to exercise professional and public opinion. In 2004, Newcastle University received Britain's first licence to clone human embryos. In early 2006, Dr Hwang Woo-suk, a South Korean scientist working at Seoul National University, was disgraced when he admitted that his recently published research on human stem cells had been fabricated.
(genetics) The process of asexual reproduction observed, for example, in bacteria and other unicellular micro-organisms which divide by simple fission, so that the daughter cells are genetically identical to each other and to the parent (except when mutation occurs). In higher organisms, genetically identical individuals may be produced by cloning. A body (somatic) cell is taken from an embryo in an early stage of development or from an adult, the nucleus transferred to an unfertilized ovum from which the nucleus has been removed, and the product grown in culture; daughter cells from the earliest divisions are removed, and grown in culture or implanted into host mothers to give genetically identical offspring. The successful cloning of a sheep (named Dolly, 1996–2003) was reported by scientists from the Roslin Institute, Edinburgh, UK, in February 1997. There is considerable potential application in animal rearing, but its application to humans is extremely unlikely (except in some rare instances of in vitro fertilization). The term molecular cloning is used in recombinant DNA technology, where a section of foreign DNA is inserted into an artificial bacterial chromosome (plasmid) and divides with it, thus ‘cloning’ the DNA.
In 1998, a Council of Europe protocol banning the cloning of human beings was signed in Paris by 19 states - the first international treaty on the issue. However, in 1999, predictions were being made about the application of the technique in other areas, such as bone-marrow grafting in leukaemia, and transplant medicine in general, and the controversy surrounding the ethics of human cloning continued to exercise professional and public opinion. In 2004, Newcastle University received Britain's first licence to clone human embryos. In early 2006, Dr Hwang Woo-suk, a South Korean scientist working at Seoul National University, was disgraced when he admitted that his recently published research on human stem cells had been fabricated.
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