GENERATION OF MOUSE MODELS FOR HUMAN GENETIC DISEASESTHROUGH GENE TARGETING

1)以小鼠转甲状腺视黄嘌呤编码基因（ttr）和小鼠胚胎癌F9细胞为模型系统，建立了一种易于将细微突变引入多种小鼠基因的替代载体。该载体由部分在其第二外显子上携带一个微妙突变的ttr和一盒编码新霉素抗性（neo）和单纯疱疹病毒胸苷激酶（HSV-tk）的基因组成，其两侧是ttr第二内含子的3 kb复制。2)为了研究未分化小鼠胚胎癌F9细胞的基因表达，我们制备了2132个表达序列标签（est），发现1416个与已知的基因和/或蛋白序列相匹配。我们试图开发一个系统来描述不匹配已知基因的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mrna相对应的cDNA克隆，并对其中两个克隆进行了鉴定，命名为rael和rae28。3)为了研究rae28基因在小鼠发育中的作用，我们在胚胎干细胞中通过基因靶向培养了rae28缺陷小鼠。令我们惊讶的是，纯合的rae28敲除小鼠携带了人类先天性疾病CATCH-22综合征的所有表型。我们发现，在rae28-/-纯合子胚胎的近轴中胚层中，Hoxb-3、a-4、a-5、b-3、b-4和d-4的表达前边界在吻侧方向发生了移位，Hoxb-3和b-4的表达前边界在斜形体和/或咽弓中也发生了类似的改变。这些改变的Hox编码被认为与在rae28-/-纯合子小鼠中观察到的后骨骼转化和神经嵴缺陷有关。这些结果表明，rae28基因参与了近轴中胚层和神经嵴发育过程中Hox基因表达和片段规范的调控。4)继续建立家族性淀粉样变性多发性神经病小鼠模型。

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.