GENERATION OF MOUSE MODELS FOR HUMAN GENETIC DISEASESTHROUGH GENE TARGETING

通过基因靶向生成人类遗传疾病小鼠模型

基本信息

批准号：
07457040
负责人：
SHIMADA Kazunori
金额：
$ 4.86万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

1) A replacement vector convenient for introducing subtle mutations into various mouse genes has been developed using as a model system, the mouse transthyretin-encoding gene (ttr) and mouse embryonal carcinoma F9 cells. The vector consists of part of ttr carrying a subtle mutation in its second exon, and a cassette of the neomycin-resistance (neo) - and herpes simplex virus thymidine kinase (HSV-tk) -encoding genes flanked with a 3-kb duplication of mostly the second intron of ttr.2) To study gene expression in undifferentiated mouse embryonal carcinoma F9 cell, we prepraed 2,132 expressed sequence tags (ESTs) and found that 1,416 match known gene and/or protein sequences. We tried to develop a system for characterizing ESTs matching no known genes. We also isolated 17 cDNA clones corresponding to mRNAs induced rapidly during retinoic acid-mediated F9 cell differentiation and characterized two of them, named rael and rae28, in this study.3) To study the role of the rae28gene in mouse development, we generated rae28-deficient mice by gene targeting in embryonic stem cells. To our surprise, the homozygous rae28-knock out mice carried all the phenotypes noted in the human congenital disorder CATCH-22 syndrome. We found that the anterior boundaries of Hoxa-3, a-4, a-5, b-3, b-4 and d-4 expression are shifted in the rostral direction in the paraxial mesoderms of the rae28-/- homozygous embryos, and those of Hoxb-3 and b-4 expression are also similarly altered in the rhombomeres and/or pharyngeal arches. These altered Hox codes were presumed to be correlated with the posterior skeletal transformations and neural crest defects observed in the rae28-/- homozygous mice. These results indicate that the rae28 gene is involved in the regulation of Hox gene expression and segment specification during paraxial mesoderm and neural crest development.4) We continued studies on mouse models for familial amyloidotic polyneuropathy.

1) 使用小鼠运甲状腺素蛋白编码基因（ttr）和小鼠胚胎癌F9细胞作为模型系统，开发了一种便于将细微突变引入各种小鼠基因的替代载体。该载体由在其第二个外显子中携带细微突变的 ttr 部分和新霉素抗性 (neo) 和单纯疱疹病毒胸苷激酶 (HSV-tk) 编码基因的盒组成，两侧带有 3 kb 重复的主要是ttr的第二个内含子。2）为了研究未分化小鼠胚胎癌F9细胞中的基因表达，我们准备了2,132个表达序列标签（EST）和发现 1,416 个匹配已知基因和/或蛋白质序列。我们试图开发一个系统来表征与未知基因匹配的 EST。我们还分离了 17 个与视黄酸介导的 F9 细胞分化过程中快速诱导的 mRNA 相对应的 cDNA 克隆，并在本研究中对其中两个克隆进行了表征，命名为 rael 和 rae28。3) 为了研究 rae28 基因在小鼠发育中的作用，我们生成了 rae28 -通过胚胎干细胞中的基因靶向来治疗缺陷小鼠。令我们惊讶的是，rae28 敲除纯合子小鼠携带人类先天性疾病 CATCH-22 综合征中的所有表型。我们发现在 rae28-/- 纯合胚胎的近轴中胚层中，Hoxa-3、a-4、a-5、b-3、b-4 和 d-4 表达的前边界沿吻侧方向移动， Hoxb-3和b-4的表达在菱形节和/或咽弓中也发生类似的改变。这些改变的 Hox 代码被推测与在 rae28-/- 纯合子小鼠中观察到的后骨骼转变和神经嵴缺陷相关。这些结果表明rae28基因参与轴旁中胚层和神经嵴发育过程中Hox基因表达和节段规范的调节。4)我们继续对家族性淀粉样多发性神经病的小鼠模型进行研究。