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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)以小鼠反式甲状腺激素编码基因(TTR)和小鼠胚胎癌F9细胞为模型系统，建立了便于在多种小鼠基因中引入细微突变的替换载体。该载体由携带第二外显子微小突变的部分TTR和编码新霉素抗性(Neo)和单纯疱疹病毒胸苷激酶(HSV-tk)的基因的盒组成，其两侧主要是Ttr2第二内含子的3kb重复。2)为了研究未分化小鼠胚胎癌细胞F9的基因表达，我们制备了2,132个表达序列标签(EST)，发现1,416个与已知基因和/或蛋白质序列匹配。我们试图开发一种系统来鉴定与未知基因匹配的EST。我们还分离了17个与维甲酸介导的F9细胞分化过程中快速诱导的mRNAs相对应的cDNA克隆，并对其中的两个进行了鉴定，分别命名为RAEL和RAE28。3)为了研究rae28基因在小鼠发育中的作用，我们通过胚胎干细胞基因打靶的方法获得了rae28缺陷小鼠。令我们惊讶的是，纯合子rae28基因敲除的小鼠携带了人类先天性疾病Catch-22综合征中记录的所有表型。我们发现，在rae28纯合子胚胎的中胚轴旁，Hoxa-3、a-4、a-5、b-3、b-4和d-4表达的前边界向嘴方向移动，而在菱形核和/或咽弓中，Hoxb-3和b-4的表达也发生了类似的变化。这些改变的HOX密码被推测与在RAE28/纯合子小鼠中观察到的后部骨骼变形和神经脊缺陷有关。这些结果表明，rae28基因参与了HOX基因的表达和节段的调控。4)我们继续对家族性淀粉样变性多发性神经病小鼠模型进行研究。