生物学家开发出一种可对人类胚胎干细胞进行基因修饰的有效方法.研究团队指出,这种方法利用细菌人工染色体植入缺陷基因,使干细胞系快速发育成为可能,而这既可以作为人类基因疾病的研究模型,也可以作为筛选潜在疗法的试验平台.
    Yang Xu说：“这将使整个人类胚胎干细胞研究领域受益,毕竟,目前还几乎没有研究干细胞遗传学的有效方法.”这位位于圣地亚哥的加利福尼亚大学的生物学教授领到了这项研究.Xu和他的合作者Hoseok Song 和Sun-Ku(这两人都是Xu的研究小组博士后研究人员)在2010年1月8号的干细胞杂志(the journal Cell Stem Cell)上阐述了他们的技术成果.
    Professor Xu认为,大多数试图改变细胞基因组成的方法都被证明过于低效,而他的团队利用细菌人工染色体,即BAC,大大提高了这种效率.
    BAC是人工合成的环状人类DNA,这样细菌就可以像复制自己的基因组一样复制这些BAC.从市场上购买的BAC可在细菌胞内经修饰并插入某一基因的突变体.一旦经修饰的BAC被导入人类细胞,就可以和人类染色体上的某些片段配对,并将目标DNA片段置换入人类染色体,这个过程被称为同源重组.
    使用BAC改变人类细胞编码基因的优点是可以利用待修饰基因两端的长侧翼序列,这可以增加BAC与目标序列配对并完成置换重组的概率.而其他方法则受侧翼序列较短所限.
    Professor Xu说,通过BAC的应用,该团队将待处理细胞中完成基因修饰的比例提高到了20%,而传统方法则无法达到1%.
    他的团队已成功地将一个有缺陷的p53基因—一种抑癌基因—导入一个人类胚胎干细胞系,并通过重复这一操作,他们开发出一个p53的两个等位基因均有缺陷的细胞系.
    同时,他们也在另一个基因上取得了成功：ATM基因,该基因的突变可引起共济失调-毛细管扩张症,其病症是一系列的系统性缺陷,包括癌症风险的增加、某些脑细胞的退化和端粒(染色体端上的保护性结构)降解.
    携带两个缺陷ATM基因的基因修饰小鼠存在一些和人类相同的症状,但并非完成相同,如小鼠中的神经元并不退化,端粒也不降解.Professor Xu说：“如果你试图研究端粒的加速降解过程,就不能用小鼠做试验,而必须用人类细胞.”
    上述不同之处迫使Professor Xu的研究团队转而开发人类细胞系,期望能在实验室中研究这些发生于患者身上的病例过程.可喜的是,他们的实验结果表明ATM缺陷的胚胎干细胞系已经发生端粒降解,而其他病症,如特定脑细胞的退化,则是下一步研究的重点,Professor Xu说.
    据称这种基因修饰方法可以很容易的被应用于其他干细胞系的基因修饰.在项目开始阶段的工作中,他们使用了一种很容易由单个细胞发育成新细胞群的细胞系,而同样的方法也在另一个称为H9的细胞系上获得了成功,该细胞系已被证明很难进行复制增殖.
    直到2009年12月中旬,新细胞系被批准使用之前,H9细胞系是联邦政府所资助的研究项目中被允许使用的少数几个细胞系之一,所以很多研究者在诱导该细胞分化成某一特定组织方面积累了相当多的经验,这将使对H9细胞的基因修饰变得更有研究价值.
    该项目由加利福尼亚再生医学研究所(州立干细胞研究机构)资助.


Biologists Develop Efficient Genetic Modification OfHuman Embryonic Stem Cells
    Biologists have developed an efficient way to genetically modify human embryonic stem cells. Their approach, which uses bacterial artificial chromosomes to swap in defective copies of genes, will make possible the rapid development of stem cell lines that can both serve as models for human genetic diseases and as testbeds on which to screen potential treatments, they say.
    "This will help to open up the whole human embryonic stem cell field. Otherwise, there\'s really few efficient ways you can study genetics with them," said Yang Xu, professor of biology at the University of California, San Diego who directed the research. Xu and co-authors Hoseok Song and Sun-Ku Chung, both postdoctoral fellows in Xu\'s research group, describe their technique in the January 8 issue of the journal Cell Stem Cell.
    Most attempts to alter the genetic makeup of the cells have proved too inefficient, Xu said. His group used bacterial artificial chromosomes, or BACs, to improve the yield.
    BACs are synthesized circles of human DNA, which bacteria will replicate just like their own native chromosomes. Commercially available BACs can be modified within bacterial cells to insert altered copies of specific genes. Once the modified BACs are introduced into human cells, they will sometimes pair up with a matching segment of a human chromosome and swap segments of DNA, a process called homologous recombination.
    The advantage of using BACS to alter the genetic code in human cells comes from the long flanking sequences on either side of the modified gene, which increases the chance that the BAC with line up with native DNA in position for a swap. Other genetic approaches have been limited by shorter segments of DNA.
    Using BACs, the team was able to substitute modified genes in 20 percent of treated cells. Standard methods of genetic modification typically achieve modification in fewer than one percent of cells, Xu said.
    His group successfully transferred a defective copy of the gene p53, which suppresses cancer, into a human embryonic stem cell line. By repeating the process in a second round, they developed a line of cells in which both copies of the genes were disrupted.
    They also report success with a different gene, ATM, which when mutated in humans causes Ataxia-telangiectasia, a disease characterized by a host of systemic defects including increased cancer risk, degeneration of specific types of brain cells and degraded telomeres, the protective caps at the end of each chromosome.
    Genetically engineered mice with two bad copies of the ATM gene share some of these traits with human patients, but not all. Neurons don\'t degenerate in ATM mice, for example, and the telomeres are long. "If you want to study accelerated shortening of telomeres, you can\'t do it in the mouse. You can only do it in human cells," Xu said.
    Those differences propelled Xu\'s group to develop human cell lines instead, with the hope that some of the processes that go wrong in human patients could be studied in the lab. Already, they have demonstrated that their ATM-deficient embryonic stem cell line has damaged telomeres. Other characteristics, such as the degeneration of specific types of neurons, will be the subject of future experiments, Xu said. 


    The authors say their approach can easily be adapted to modify other human genes within other stem cells lines. For their initial work, Xu\'s group used a cell line that easily forms new colonies from single cells, but they also repeated the procedure in a cell line called H9, which has proved difficult to manipulate.
    Because H9 was among the few cells lines approved for use by researchers funded by the federal government before new lines began to be approved in mid-December 2009, many researchers already have considerable experience with coaxing the cells into differentiating into specific types of tissues, for example, which would make the ability to genetically modify them particularly valuable.
    The California Institute for Regenerative Medicine, the state\'s stem cell research agency, funded this project.
Source:
Yang Xu
University of California - San Diego 

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