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

利用噬菌体P1衍生的重组体系Cre-loxP，我们开发了基因插入系统。该系统由两个突变体lox71和lox66组成。我们构建了一个新的携带这种lox71的陷阱载体。使用这种诱捕载体的方法被称为可交换基因诱捕，因为我们可以在诱捕基因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lox71/66和突变体lox511组合可以提高基因整合的效率。将胚胎干细胞从供体细胞中分离出来，维持在悬浮培养中，可以诱导胚胎干细胞分化形成胚样体。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早期发育阶段的基因。我们建立了24条陷阱线。其中半数在发育和生长过程中表现出致命性。我们分离了诱捕载体捕获的基因，并测定了其中的核苷酸序列。并利用balst程序与DNA数据库进行比对。其中包括参与转录、细胞生长、细胞分裂、信号转导、翻译和运输的几类蛋白质。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突变的杂合小鼠是人类疾病Rubinstein-Taybi综合征的小鼠模型，它显示了人类患者的大多数表型。纯合子小鼠由于造血和血管发生的失败是胚胎致死的。c-crk突变纯合子小鼠未表现出任何表型。然而，通过回交C57BL/6，它们在出生后24小时内死亡，这表明存在修饰基因。建立了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例为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BACs分别可以拯救T突变体和qk突变体。少

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