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

为了建立一种分析高阶核结构的新方法，我们先前开发了一种人类人工(HAC)载体，该载体表现为一个独立的小染色体，并允许通过HAC上的LACO串联来分析其上加载的基因组。我们关注HoxB基因簇，因为这些基因在发育过程中随着转录的开启和关闭而显著改变其核区域，而且这种现象在ES细胞的神经元分化过程中是可以重现的。我们获得了该基因座的基于BAC的基因组，并设法标记了一个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基因簇被不必要的方式部分缺失/突变，特别是在一株用于BAC重组工程的细菌中，很可能是由于其高度重复的序列，并且不能以想要的方式进行loxP标记。因此，我们计划通过染色体转移的方法，将携带人HoxB基因簇的人chr17直接导入小鼠ES细胞，然后进行同源重组介导的序列标记，以便进行可视化。我们建立了一种将人CHL17基因导入小鼠ES细胞的方法，获得了携带人CHL17基因的小鼠ES细胞克隆，并用维甲酸观察了其向神经元分化的情况。然而，我们观察到了与上述相同的现象--意外缺失专门发生在小鼠RS细胞中。因此，我们提出了在精通重组的细菌和动物工厂中处理高度重复的序列是极其困难的，并提出了分析这些基因的替代方法是标记一个序列，以便在ES细胞中关注的内源基因旁边进行可视化

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