GENE EXPRESSION, FUNCTION AND MUTAGENESIS

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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.