TARGETING MUTATIONS OF TYROSINE KINASE GENES IN THE MOUSE

这项建议描述了使用小鼠遗传学工具来确定c-abl原癌基因在体内的功能的实验。C-abl基因产物是一种胞质酪氨酸激酶，对信号转导和细胞周期控制非常重要。C-ABL缺陷基因敲除小鼠表现出许多表型：围产期死亡、跑动、骨骼异常和早期淋巴细胞谱系缺陷。有人提议对这些小鼠进行几项遗传学研究。首先，c-abl的新等位基因将通过基因打靶(“敲入”实验)产生。这些等位基因中的一个将允许在发育过程中的特定时间和选定的组织中有条件地删除基因；其他等位基因将表达改变形式的c-abl，缺乏负责蛋白质定位的特定结构域。对这些小鼠的检查应该有助于确定从c-ABL发出的每一条途径的功能；我们将特别感兴趣地检查B细胞的发展过程和可变区利用的全套。其次，将通过基因打靶产生名为PSTPIP1的Abi家族新成员的胚系突变，PSTPIP1是酵母CDC15基因的哺乳动物同源物，并将单独和其他敲除突变的影响进行表征。 我们的预期是，PSTPIP1的丢失可能会抑制Abl的丢失所引起的一些表型。第三，我们将产生并分析UV-DDB蛋白的胚系突变，该蛋白是人类xpe基因座的产物，我们已经证明它受Abl激酶的结合和调节。最后，我们将直接对氧化应激诱导的细胞系和组织中的基因进行分类，将ABL突变体的反应与野生型对照进行比较，从而确定Abl依赖的下游靶标。这些研究应该有助于确定c-ABL在哺乳动物发育和生理中的许多不同功能。

This proposal describes experiments using the tools of mouse genetics to determine the in vivo functions of the c-abl proto-oncogene. The c-abl gene product is a cytoplasmic tyrosine kinase important for signal transduction and control of the cell cycle. Knock-out mice deficient in c-abl exhibit a number of phenotypes: perinatal lethality, runting, bone abnormalities, and defects in early lymphoid cell lineages. Several genetic studies of these mice are proposed. First, new alleles of c-abl will be generated by gene targeting ("knock-in" experiments). One of these alleles will permit the conditional deletion of the gene at selected times in development and in selected tissues; others will express altered forms of c-abl lacking particular domains responsible for protein localization. Examination of these mice should help determine the functions of each pathway emanating from c-abl; we will be particularly interested to examine the course of B cell development and the repertoire of variable region utilization. Secondly, a germ-line mutation of a new member of the Abi family termed PSTPIP1, a mammalian homologue of the yeast Cdc15 gene, will be generated by gene targeting, and the effects of the mutation alone and with other knock-out mutations will be characterized. Our expectation is that the loss of PSTPIP1 may suppress some of the phenotypes caused by loss of Abl. Thirdly, we will generate and analyze germ line mutations of the UV-DDB proteins, products of the xpe locus in man, and which we have shown are bound and regulated by the Abl kinase. Finally, we will directly catalogue the genes induced in cell lines and in tissues by oxidative stress, comparing the responses in abl mutants with wild-type controls, and thereby identify the Abl-dependent downstream targets. These studies should help define the many diverse functions of c-abl in mammalian development and physiology.