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

通过染色体和发育工程方法创建具有人类 MHC 的小鼠

• 批准号: 12554036
• 负责人: IKEMURA Toshimichi
• 金额: $ 8.32万
• 依托单位: National Institute of Genetics
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12554036/
• 关键词: Artificial Chromosomes; MHC; Chromosome Engineering; DT40; Centromere; Developmental Engineering; Genome Engineering; Chromosome Vector; DT-40; 発光工学; DT-40細胞; 複製タイミング

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

遗传、染色体工程和转基因动物技术对包括医学、农业和制药在内的生命科学各个领域的进步作出了巨大贡献。我们发展了染色体工程技术，通过将人类染色体的几到几十Mb片段导入鸡DT40细胞，使其同源重组频率比哺乳动物细胞高几十到几百倍，从而可以有效地操纵人类染色体的大区域。在人工染色体的产生过程中，着丝粒具有重要的作用，其功能得到了适当的调控（Fukagawa et al., 2001; Nishihashi et al., 2002）。人类MHC基因座（4mb）由三个功能域（I、II和III类）组成。我们发现单个结构域在S期的不同时期复制，复制时间的转变发生在功能结构域的边界。French group报道，在猪MHC位点的…More病例中，着丝粒存在于II类和III类的边界。在人类MHC位点的这个边界上，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，预测调节复制时间的三联体结构也在细胞核内形成，并与着丝粒序列特别相关（Ohno et al., 2002）。人造染色体上存在的着丝粒DNA是在S期后期发现的，在融入宿主染色体后复制时间发生了变化。为了使功能正常的人工染色体得到准确调控，明确着丝粒的结构和功能是十分重要的。我们利用鸡DT40的基因敲除方法分析了着丝粒蛋白，并利用DNA芯片技术测量了人类带有MHC位点的第6染色体的复制时间。然后，我们尝试将人染色体从DT40细胞转移到小鼠胚胎干细胞。本研究获得的知识为创建具有长功能基因组区域（如人类MHC位点）的转基因小鼠提供了基础知识。少

项目成果

T. Fukagawa: "CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells"The EMBO Journal. 20. 4603-4617 (2001)

T. Fukakawa：“CENP-H 是一种组成性着丝粒成分，是脊椎动物细胞中 CENP-C 着丝粒靶向所必需的”EMBO 杂志。

A. Okamura: "Gene Structure, chromosomal localization and immunolocalization of chicken centromere proteins CENP-C and ZW10"Gene. 262. 283-290 (2001)

A. Okamura：“鸡着丝粒蛋白CENP-C和ZW10的基因结构、染色体定位和免疫定位”基因。

S.Kanaya: "Analysis of codon usage diversity of bacterial genes with a self-organizing map (SOM) : characterization of horizontally transferred genes with emphasis on the E. coli O157 genome."Gene. 276. 89-99 (2001)

S.Kanaya：“用自组织图谱 (SOM) 分析细菌基因的密码子使用多样性：水平转移基因的表征，重点是大肠杆菌 O157 基因组。”基因。

T.Hayashi: "Ubc9 is essential for viability of higher eukaryotic cells"Experimental Cell Research. 280. 212-221 (2002)

T.Hayashi：“Ubc9 对于高等真核细胞的活力至关重要”实验细胞研究。

Shiina T, Ando A, Suto Y, Kasai F, Shigenari A, Takishima N, Kikkawa E, Iwata K, Kuwano Y, Kitamura Y, Matsuzawa Y, Sano K, Nogami M, Kawata H, Li S, Fukuzumi Y, Yamazaki M, Tashiro H, Tamiya G, Kohd A, Okumura K, Ikemura T, Soeda E, Mizuki H, Kimura M, B

Shiina T、Ando A、Suto Y、Kasai F、Shigenari A、Takishima N、Kikkawa E、Iwata K、Kuwano Y、Kitamura Y、Matsuzawa Y、Sano K、Nogami M、Kawata H、Li S、Fukuzumi Y、Yamazaki M