ANALYSIS OF MOUSE DEVELOPMENT USING CELL LINES

这项研究的长期目标是描述遗传控制 小鼠胚胎发育过程中的复杂事件 在一个专门用来生成和传输随机 插入突变到小鼠中，ES细胞的培养物预先确定 因为携带高拷贝数的前病毒序列被用于 制造生殖细胞嵌合体 三种不同的突变 鉴定 这些包括（1）隐性致死突变， 在中期胚胎中，（2）影响尾的隐性突变 形态学和（2）影响初级性别的突变 XY胚胎的决定。 该提案的一个主要目标是 以鉴定与以下疾病相关的特异性逆转录病毒整合位点： 每一个突变 使用前病毒序列作为探针，我们将 分子克隆插入位点。 细胞缺陷 将描述与每个突变相关的基因。 我们将使 多能胚胎干细胞系（ES细胞）研究 发育失败的细胞生物学，并确定 突变表型是否可归因于以下缺陷： 特定的细胞谱系。 此外，构建的cDNA文库 从未分化的ES细胞和从诱导的ES细胞， 在培养中分化，已经筛选了转录本， 是受发育调控的 三个这样的成绩单已经被 已识别并部分表征。 的时空 这些基因的表达将在发育中进行检查。 胚胎 这些研究将使用原位杂交技术 和针对由以下蛋白质编码的蛋白质产物的抗体 这些基因。 为了测试特定基因的发育作用， 我们计划在实验上操纵转基因植物的表达， 使用ES细胞系构建嵌合体。 这些研究将 希望能找到负责调节 哺乳动物的发育

The long-term goal of this research is to describe genetic control of complex events underlying the development of the mouse embryo. In a program specifically designed to generate and transfer random insertional mutations into mice, cultures of ES cells predetermined as carrying high copy numbers of proviral sequences were used to make germ line chimeras. Three different mutations have been identified. These include (1) a recessive lethal mutation acting in the mid-term embryo, (2) a recessive mutation affecting tail morphology and (2) a mutation which affects primary sex determination in the XY embryo. A major goal of this proposal is to identify specific retroviral integration site(s) associated with each mutation. Using the proviral sequence as a probe, we will molecularly clone the locus of insertion. The cellular defect(s) associated with each mutation will be described. We will make of pluripotential embryonic stem cell lines (ES cells) to investigate the cell biology of the developmental failure, and to determine whether the mutant phenotype(s) is attributable to defects in specific cell lineages. In addition, cDNA libraries constructed from undifferentiated ES cells and from ES cells induced to differentiate in culture, have been screened for transcripts which are developmentally regulated. Three such transcripts have been identified and partially characterized. The temporal and spatial expression of these genes will be examined in the developing embryo. These studies will use in situ hybridisation techniques and antibodies directed against the protein products encoded by these genes. To test for a developmental role of specific genes, we plan to experimentally manipulate expression in transgenic chimeras constructed using ES cell lines. These studies will hopefully identify genes responsible for regulating processes of mammalian development.