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包括医学、农业和制药在内的所有生命科学领域的进步。我们发展了染色体工程技术，通过将人类染色体的几个到几十个Mb片段导入鸡DT40细胞，使其同源重组的频率比哺乳动物细胞高出几十到数百倍，从而可以有效地操纵人类染色体。Centomere在合成具有适当调控功能的人工染色体中具有重要作用(Fukagawa等人，2001；Nishihashi等人，2002)。人类MHC基因座(4Mb)由三个功能结构域组成(I、II和III类)。我们发现，在S阶段，单个结构域在不同的时期进行复制，复制时间转变到功能结构域的边界。法国一家集团报道称，在…在更多猪MHC基因座的情况下，着丝粒存在于II和III类的边界上。在人类MHC基因座的这一边界上，我们发现了有可能形成三螺旋的特征序列(Kanaya等人，)。我们还发现，被预测为调节复制时间的三链结构也在核内形成，并与着丝粒序列特别相关(Ohno等人，2002年)。人工染色体上的着丝粒DNA存在于S时期的晚期，整合到寄主染色体后复制时序发生了变化。为了获得具有正确功能的人工染色体，阐明着丝粒的结构和功能是非常重要的。我们利用基因敲除技术分析了鸡DT40中的着丝粒蛋白，并用DNA芯片技术测定了人6号染色体MHC基因座的复制时序。然后，我们尝试将人的染色体从DT40细胞转移到小鼠的ES细胞中。本研究获得的知识为创造拥有人类MHC基因座等长功能基因组区域的转基因小鼠提供了基础知识。较少

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