Creation of a mouse with human MHC by Chromosomal- and Developmental-engineering methods

基因和染色体工程以及转基因动物技术极大地促进了生命科学各个领域的进步，包括医学、农业和制药。我们发展了染色体工程技术，通过将几个到几十个Mb的人类染色体片段导入鸡DT 40细胞中来操纵人类染色体的大结构域，其中同源重组的频率是哺乳动物细胞中同源重组频率的几十到几百倍，因此可以有效地操纵人类染色体。Centomere在具有适当调节的功能的人工染色体的产生中具有重要作用（Chuagawa等人，2001 ; Nishihashi等，2002）JHC基因座（4 Mb）由三个功能结构域（I类、II类和III类）组成。我们发现，在S期的不同时期复制的各个域和复制时间的过渡发生在功能域的边界。法国团体报道称， ...更多信息 在猪MHC基因座的情况下，着丝粒存在于II类和III类的边界中。在人MHC基因座的这一边界中，我们发现了具有形成三螺旋潜力的特征序列（Kanaya等人，）。我们还发现，预测调节复制定时的三链体结构也在细胞核内形成，并且特别与着丝粒序列相关（Ohno等人，2002年）。在S期后期发现了存在于人工染色体上的着丝粒DNA，并且在整合到宿主染色体中后改变了复制定时。为了获得精确调控的具有适当功能的人工染色体，阐明着丝粒的结构和功能是非常重要的。我们用基因敲除的方法分析了鸡DT 40的着丝粒蛋白，并用DNA芯片技术测定了人类第6号染色体MHC位点的复制时间。然后，我们尝试将人的染色体从DT 40细胞中转移到小鼠ES细胞中，本研究所获得的知识为建立具有长功能基因组区域如人MHC基因座的转基因小鼠提供了基础知识。少

Genetic and chromosomal engineering and transgenic animal technology have greatly contributed to progress of all fields of life sciences, including medicine, agriculture, and pharmacy. We developed chromosome engineering technique to manipulate a large domain of human chromosomes, by introducing several to several-tens Mb portions of human chromosome into chicken DT40 cells, in which frequencies of homologous recombination are several tens to hundreds times higher than those in mammals cell and therefore human chromosome can be efficiently manipulated. Centomere has important roles in production of artificial chromosome with functions properly regulated (Fukagawa et al., 2001 ; Nishihashi et al, 2002).The human MHC locus (4 Mb) consists of three functional domains (classes I, II, and III). We found individual domains to replicate in different periods during S phase and transition of replication timing to occurs at boundaries of the functional domains. French group reported that in the … More case of the pig MHC locus, centromere exists in the boundary of the classes II and III. In this boundary in the human MHC locus we found characteristic sequences that have potentiality to form triple helix (Kanaya et al.,). We also found the triplex structure, which is predicted to modulate replication timing, was formed also within the nucleus and specially associated with centromere sequences (Ohno et al., 2002). Centromeric DNA that exists on the artificial chromosome was found in the late period during S phase, and the replication timing changed after integrating into a host's chromosome. To get artificial chromosomes with proper functions accurately regulated, it is important to clarify the centromeric structures and functions. We analyzed centromeric proteins using gene knocking-out method developed in chicken DT40 and also measured replication timing of the human 6th chromosome with MHC locus using DNA chip techniques. Then, we attempted to transfer human chromosomes from DT40 cells to mouse ES cells.The knowledge acquired in the present study has given basic knowledge to create transgenic mice that hold the long functional genome regions such as human MHC locus. Less