Development of Human Artificial Chromosome

人类人工染色体的研制

• 批准号: 06044273
• 负责人: MASUMOTO Hiroshi
• 金额: --
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Overseas Scientific Survey.
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06044273/
• 关键词: centromere; telomere; artificial chromosome; YAC; alphoid DNA; minor satellite DNA

We have been interested in the structures and the functions of the essential elements required for the maintenance of linear chromosomes in mammalian cells. The centromere and the telomere structures are the elements necessary for such chromosome functions. We have been studying the DNA sequences and the DNA-protein interaction at the centromere regions of human (alphoid DNA and CENP-B) and mouse chromosomes. Through this international collaboration with Dr.Howard Cooke, we hope to construct mammalian artificial chromosome (MAC) in yeast and transfer them to mammalian cells with the advantage that MAC can be rescued back into the yeast host.Recently we have developed cloning methods for several hundred kb fragments of alphoid DNA and mouse minor satellite DNA into Yeast Artificial Chromosomes (YACs) without the rearrangement and succeeded in obtaining several stable YAC clones containing alphoid DNA or mouse minor satellite DNA.Dr.Cooke's group developed methods for retrofitting the te … More lomere sequences of YAC clones with YAC vectors modified with human telomere sequences in the yeast cells. During the retrofitting process the insert DNA of YAC also rearranged with very high rate. Therefore, in parallel with the retrofitting we began to apply in vitro digestion and ligation methods to exchange the telomere sequence of YACs. They also developed the YAC DNA amplification and isolation methods and introduced several telomere modified YACs into mammalian cells using microinjection or lipofection. Both the methods for YAC introduction turned out to be efficient and be able to use for assay systems. It was necessary for the progress of our study to choose a recipient cell with high telomerase activity because the stability of the modified YACs greatly depended on the telomerase activity in the cells. As the next step towards the development of MAC, we are now retrofitting alphoid YACs and mouse minor satellite YACs and going to introduce these modified YACs into mammalian cells. Less

我们对维持哺乳动物细胞线性染色体所需的结构和功能感兴趣。丝粒和端粒结构是此类染色体功能所需的要素。我们一直在研究人（Alphoid DNA和CENP-B）和小鼠染色体的中心区域的DNA序列和DNA蛋白相互作用。通过与Howard Cooke博士的这一国际合作，我们希望在酵母中构建哺乳动物人造染色体（MAC），并将其转移到哺乳动物细胞中，并具有将Mac回收回酵母宿主的优势，我们已经开发了克隆方法。我们已经开发了几百个hove tna和小鼠小卫星DNA的数百kb碎片（Yeast offerage offersect of chrome offerage of chrome offermoss offerage offersy）。稳定的YAC克隆含有字母DNA或小鼠次要卫星DNA.DR.COOKE的组开发了改造TE的方法……更多的YAC克隆的Lomere序列，其YAC载体的YAC载体在酵母细胞中用人端粒序列进行了修饰。在改装过程中，YAC的插入DNA也以非常高的速率重新排列。因此，与改造并行，我们开始应用体外消化和连接方法来交换YAC的端粒序列。他们还开发了YAC DNA扩增和分离方法，并使用显微注射或LiPofection将几个端粒修饰的YAC引入了哺乳动物细胞中。 YAC简介的两种方法都非常有效，并且能够用于测定系统。我们研究的进展有必要选择具有较高端粒活性的受体细胞，因为改进的YAC的稳定性在很大程度上取决于细胞中的端粒活性。作为迈向MAC开发的下一步，我们现在正在改造字母YAC和小鼠次要卫星YAC，并将这些改良的YAC引入哺乳动物细胞。较少的