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Towards establishment of higher-order of nuclear architecture analysis method by using a human artificial vector

利用人类人工向量建立高阶核结构分析方法

基本信息

批准号：
18510174
负责人：
INOUE Toshiaki
金额：
$ 2.5万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

In order to establish a novel method to analyze a higher-order nuclear architecture, we previously developed a human artificial (HAC) vector which behaves as an independent mini-chromosome and allows the analysis of visualization of the loaded genome on it through LacO tandem on HAC. We focused on the HoxB gene cluster because the genes dramatically alter its nuclear territory during development with on-off of the transcription and this phenomena is known to be reproducible in neuronal differentiation from ES cells. We obtained BAC-based genome for the locus and managed to tag a loxP site to allow the loading of it onto HAC, by BAC-recomibineering. We observed that the HoxB gene cluster was partially deleted/mutated by an unwanted manner specifically in a bacterial strain used for BAC-recombineering most likely due to its highly repetitive sequence and could not carry out a LoxP-tagging in a wanted manner. Thus, we planned to directly introduce human Chr17 carrying human HoxB gene cluster into mouse ES cells by chromosome transfer method, following homologous recombination-mediated tagging of sequence to allow visualizing. We established a method to transfer human Chr17 into mouse ES cells method and obtained mouse ES cell clones carrying human Chr17 and observed neuronal differentiation of such cell clones by retinoic acid. However, we observed the same phenomena as above-unwanted deletion specifically occurred in mouse RS cells. Therefore, we presented that highly repetitive sequence is extremely difficult to deal in recombination-proficient bacterial and animal mills and propose that the alternative way to analyze such genes is to tag a sequence to allow visualization next to the endogenous gene of interest in ES cells

为了建立一种新的方法来分析高阶核结构，我们以前开发了一种人类人工（HAC）载体，其表现为一个独立的迷你染色体，并允许通过HAC上的LacO串联分析其上装载的基因组的可视化。我们专注于HoxB基因簇，因为这些基因在发育过程中随着转录的开关而显著改变其核区域，并且已知这种现象在ES细胞的神经元分化中是可再现的。我们获得了该基因座的基于BAC的基因组，并设法标记loxP位点以允许通过BAC重组工程将其装载到HAC上。我们观察到，HoxB基因簇在用于BAC重组工程的细菌菌株中以不需要的方式被部分缺失/突变，这最可能是由于其高度重复的序列，并且不能以所需的方式进行LoxP标记。因此，我们计划通过染色体转移方法将携带人HoxB基因簇的人Chr 17直接导入小鼠ES细胞，随后同源重组介导的序列标记以允许可视化。我们建立了将人Chr 17转染小鼠ES细胞的方法，获得了人Chr 17的小鼠ES细胞克隆，并观察了视黄酸对该细胞克隆的神经分化作用。然而，我们观察到与上述相同的现象-不需要的缺失特别发生在小鼠RS细胞中。因此，我们提出，高度重复的序列是非常难以处理的重组熟练的细菌和动物米尔斯，并提出了替代的方式来分析这样的基因是标签的序列，以允许可视化旁边的内源性基因的目的在ES细胞