DEVELOPEMNT OF TRANSGENIC TECHNOLOGY AND IT'S USE FOR CANCER RESEARCH

转基因技术的发展及其在癌症研究中的应用

• 批准号: 09253243
• 负责人: YAMAMURA Kenichi
• 金额: $ 15.36万
• 依托单位: KUMAMOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-09253243/
• 关键词: ES CELL; GENE TRAP; CRE-LOXP; RECOMBINATIO; KNOCKOUT MOUSE; CHIMERIC MOUSE; ARTIFICIAL CHROMOSOME; 人工細菌染色体; 酵母人工染色体; 胚葉体

Using the recombination system Cre-loxP derived from bacteriophage P1, we developed the gene insertion system. This system comprises of two mutant lox, lox71 and lox66. We constructed a new trap vector that carry this lox71. The method using this trap vector was termed as the exchangeable gene trap, because we can insert any gene of interest after gene trapping. We alaso demonstrated that the efficiency of gene integration was improved by combination of mutant lox71/66 and mutant lox511 that carry mutation in the spacer region. ES cells can be induced to differentiate to form embryoid bodies by removing from feder cells and maintaining in suspension culture. We used this embryoid body formation as a screening system to trap genes involved in cell proliferation and early stages of development. We established 24 trap lines. Among these half of them showed lethilty in development and growth. We isolated the genes trapped by trap vector and tne nucleotide sequences were determined. These s … More eqences were compared with DNA database using balst program. These include several classes of proteins involved in transcription, cell growth, cell division, singal transduction, translation and transportaiton. Among trap lines established, we analyzed two lines in which CBP (creb binding protein) and c-crk genes are disrupted. A heterozygous mouse for CBP mutation is a mouse model for human disease, Rubinstein-Taybi syndrome, and shows most of phenotypes found in human patient. A homozygous mouse is embryonic lethal due to failures of hematopoiesis and vasculogenesis. A homozygous mouse for c-crk mutation did not show any phenotype. However, they died within 24 hours after birth by backcrossing to C57BL/6 suggesting the presence of modifier gene. A method for microinjecting bacterial artificial chromosome has been established and 34 strains of mice that carry bacterial artificial chromosome were generated. Among these, 31 cases are found to have complete bacterial artificial chromosome without any deletion. BACs containing T gene and qkI gene were shown to rescue T mutant and qk mutant, respectively. Less

利用噬菌体P1的重组系统Cre-loxP，我们开发了基因插入系统。该系统包括两个突变lox，lox 71和lox 66。我们构建了一个新的陷阱载体，携带这个lox 71。使用这种诱捕载体的方法被称为可交换基因诱捕，因为我们可以在基因诱捕后插入任何感兴趣的基因。我们还证明了在间隔区携带突变的突变体lox 71/66和突变体lox 511的组合提高了基因整合的效率。ES细胞可以通过从饲养层细胞中取出并保持在悬浮培养中来诱导分化形成胚状体。我们使用这种胚状体形成作为筛选系统来捕获参与细胞增殖和发育早期阶段的基因。我们设置了24条陷阱线。其中半数以上的个体发育和生长表现为迟缓。我们分离了陷阱载体捕获的基因，并测定了核苷酸序列。这些s ...更多信息 用Balst程序将其与DNA数据库进行比较。其中包括参与转录、细胞生长、细胞分裂、信号转导、翻译和转运的几类蛋白质。在建立的诱捕系中，我们分析了CBP（creb结合蛋白）和c-crk基因被破坏的两个系。CBP突变的杂合子小鼠是人类疾病Rubinstein-Taybi综合征的小鼠模型，并显示出在人类患者中发现的大多数表型。纯合子小鼠由于造血和血管生成的失败而具有胚胎致死性。c-crk突变的纯合子小鼠没有表现出任何表型。然而，它们在出生后24小时内通过与C57 BL/6回交而死亡，表明存在修饰基因。建立了显微注射细菌人工染色体的方法，获得了34株携带细菌人工染色体的小鼠。其中31例具有完整的细菌人工染色体，无任何缺失。含有T基因和qkI基因的BAC分别显示出拯救T突变体和qk突变体。少

项目成果

Shimada,H. et al.: "Comparison of ES cell fate in sandwiched aggregates and co-cultured aggregates during blastocyst formation by monitored GFP"Mol.Reprod. De. 52. 376-382 (1999)

岛田，H.

Terauchi, Y.et al.: "Increased insulin sensitivity and hypoglycaemia in mice lackIng the p85a subunit of phosphoinositide 3-kinase."Nature Genetics. 21. 230-235 (1999)

Terauchi, Y. 等人：“缺乏磷酸肌醇 3-激酶 p85a 亚基的小鼠的胰岛素敏感性和低血糖增加。”《自然遗传学》。

Okamoto, T.et al: "The mouse interferon-g transgene chronic hepatitis model."Int.J.Mol.Med.. 3. 517-520 (1999)

Okamoto，T.等人：“小鼠干扰素-g 转基因慢性肝炎模型。”Int.J.Mol.Med.. 3. 517-520 (1999)

Akbar,S. et al.: "Molecular cloning of the human gene STK10 encoding LOK and comparative chromosomal mapping of the human, mouse and rat"Immunogenetics. 49. 369-375 (1999)

阿克巴，S.

Oike et al.: "Mice homozygous for a truncated form of a CREB-binding protein (CBP) exhibit defects in hematopoiesis and vasculo-angiogenesis."Blood. 93. 2771-2779 (1999)

Oike 等人：“CREB ​​结合蛋白 (CBP) 截短形式的纯合小鼠表现出造血和血管生成缺陷。”血液。