Genetic Analysis of Snail Superfamily Genes in Mice

描述(申请人提供)：这项提案的长期目标是了解蜗牛超家族基因在哺乳动物胚胎发育过程中所扮演的角色。蜗牛超家族基因编码含锌指的DNA结合蛋白，作为转录抑制因子。这个超级家族有两个主要分支：蜗牛家族和Scratch家族。脊椎动物中的蜗牛超家族的每个分支都有两个成员：蜗牛和蜗牛；Scratch家族的Seratch1和Scratch2。我们一直在研究Snail基因家族成员SnA和Slug，并构建并分析了这两个基因的靶向零突变。SNA纯合子突变胚胎在原肠发育过程中死亡，在中胚层生成所需的上皮间充质过渡中表现出缺陷。鼻涕虫纯合子突变小鼠是存活的，但表现出生长迟缓和各种其他缺陷。然而，关于SNA基因在原肠形成后发生的发育事件中可能发挥的作用，或者蜗牛超家族基因Scratch1和Scratch2是否在胚胎发育过程中发挥重要作用，仍然存在许多问题。在这项建议中，将利用分子和遗传方法来研究蜗牛超家族基因在小鼠胚胎发育过程中所起的作用。这一建议的具体目的是：1)构建SNA基因功能等位基因的条件性丢失和获得，以分析其在原肠后发育阶段中的作用；2)通过构建SNA基因座的Slug基因的敲入等位基因，检验脊椎动物蜗牛和鼻涕虫基因在从鱼、两栖动物和鸟类向哺乳动物进化过程中角色互换的假设；3)构建并分析蜗牛超家族基因Scratch1和Scratch2的靶向性零突变；4)检测SNA基因在滋养层细胞谱系发育中的作用；5)利用SNA突变细胞系和胚胎的微阵列分析、DNA结合位点的电子分析和DNA结合研究，确定SNA蛋白的下游转录靶点。这些研究将进一步加深我们对蜗牛超家族基因在哺乳动物发育中的作用的理解，并将对研究人类正常和异常发育具有重要意义。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand the roles that Snail superfamily genes play during embryonic development in mammals. Snail superfamily genes encode zinc finger-containing DNA binding proteins that act as transcriptional repressors. This superfamily has two main branches: the Snail family and the Scratch family. Each branch of the Snail superfamily in vertebrates has two members: Snail and Slug for the Snail family, and Seratchl and Scratch2 for the Scratch family. We have been studying the Snail gene family members Sna and Slug, and have constructed and analyzed targeted null mutations of both of these genes. Sna homozygous mutant embryos die during gastrulation, exhibiting defects in the epithelialmesenchymal transition required for generation of the mesoderm. Slug homozygous mutant mice are viable, but show growth retardation and various other defects. However, many questions remain about the possible roles played by the Sna gene in developmental events occurring after gastrulation, or whether the Snail superfamily genes Scratchl and Scratch2 play essential roles during embryogenesis. In this proposal, both molecular and genetic approaches will be utilized to examine the roles that Snail superfamily genes play during embryogenesis in mice. The specific aims of this proposal are: 1) Construct conditional loss and gain of function alleles of the Sna gene to analyze its role during postgastrulation developmental stages; 2) Test the hypothesis that the roles of the vertebrate Snail and Slug genes have been interchanged during evolution from fish, amphibians and birds to mammals by constructing a knock-in allele of the Slug cDNA into the Sna locus; 3) Construct and analyze targeted null mutations of the Snail superfamily genes Scratch1 and Scratch2; 4) Examine the role of the Sna gene in development of the trophoblast cell lineage; 5) Identify downstream transcriptional targets of the Sna protein using microarray analysis of Sna mutant cell lines and embryos, in silico analysis of DNA binding sites, and DNA binding studies. These studies will further our understanding of the roles played by Snail superfamily genes during mammalian development, and will be relevant to the study of both normal and abnormal human development.