GENE EXPRESSION, FUNCTION AND MUTAGENESIS

生长因子诱导多种细胞反应，包括 增殖、存活和迁移，但它们的最终目标基因 在很大程度上仍然是未知的。在这份提案中，我们将重点关注 识别血小板衍生生长因子(PDGFs)的靶点 参与肌肉和心血管的各个方面 发展，以及控制其表达的上游因素。 我们将在胚胎干细胞中使用基因陷阱方法来鉴定和克隆基因。 其表达受PDGF诱导或抑制，以研究其 表达模式，并评估功能丧失的后果 在突变的小鼠身上。我们将集中精力研究那些 在细胞类型中表达或导致突变表型 缺乏PDGF或PDGF受体突变的小鼠。我们会研究 体节细胞中依赖PDGF的基因转录 卵裂期前中胚层，其中PDGF已被正确地牵连 图案化，使用消减-抑制聚合酶链式反应和杂交 到“DNA芯片”微阵列。找出遗传因素 在PDGF的上游，我们将使用新的基因陷阱载体来激活基因 从本质上讲。PDGF和PDGF受体基因将被靶向 一个无启动子的报告基因，可以在上游识别PDGF 影响因素由记者增加表达。的表达 捕获的基因将在Cre重组后被破坏，从而导致 功能突变的丧失。这种方法将使我们能够 识别PDGF上游因素及其正常生理 角色。这些研究应该有助于我们理解生长因子的调节 机制，并提供有关特定和相互作用的信息 生理过程中的生长因子信号通路。

Growth factors induce a variety of cellular responses including proliferation, survival and migration but their final target genes remain for the most part unknown. In this proposal, we will focus on identifying targets for platelet derived growth factors (PDGFs) which are involved in various aspects of muscle and cardiovascular development, as well as upstream factors that control their expression. We will use a gene trap approach in ES cells to identify and clone genes whose expression is induced or repressed by PDGF, to study their expression pattern, and to assess the consequences of loss of function in mutant mice. We will concentrate our efforts on genes that are expressed or result in mutant phenotypes in cell types which are deficient in PDGF or PDGF receptor mutant mice. We will study PDGF-dependent gene transcription in cells isolated from somites and presomitic mesoderm, in which PDGF has been implicated for proper patterning, using subtraction-suppression PCR and hybridization to "DNA chip" microarrays. To identify factors that are genetically upstream of PDGF, we will use new gene trap vectors that activate a gene constitutively. The PDGF and PDGF receptor genes will be targeted with a promoterless reporter gene, allowing identification of PDGF upstream factors by increased expression of the reporter. Expression of the trapped genes will be disrupted following Cre recombination, resulting in loss of function mutations. This methodology will allow us to identify PDGF upstream factors as well as their normal physiological role. These studies should help us understand growth factor regulatory mechanisms, and provide information on the specificity and interplay of growth factor signaling pathways in physiological processes.