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，但它们的最终靶基因 大部分仍是未知的。 在本建议中，我们将重点关注 鉴定血小板衍生生长因子（PDGF）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ES细胞中使用基因陷阱方法来识别和克隆基因 其表达被PDGF诱导或抑制，以研究它们的表达。 表达模式，并评估功能丧失的后果 在突变小鼠中。 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受体突变小鼠。 我们将研究 从体节分离的细胞中PDGF依赖的基因转录， 前体中胚层，其中PDGF已被牵连适当的 使用消减抑制PCR和杂交进行模式化 到“DNA芯片”微阵列。 为了找出基因上 在PDGF的上游，我们将使用新的基因陷阱载体， 本质上。 PDGF和PDGF受体基因将被靶向， 无启动子报告基因，允许鉴定PDGF上游 通过增加报告基因的表达。 表达 在Cre重组后，被捕获的基因将被破坏， 功能缺失突变。 这种方法将使我们能够 鉴定PDGF上游因子以及它们正常的生理活性 作用这些研究应该有助于我们了解生长因子调节 机制，并提供信息， 在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.