SATURATION MUTAGENESIS OF ZEBRAFISH COAGULATION PATHWAY

描述：(研究人员摘要)血液凝固的开始 通过外在途径是受以下因素影响的阈值中介事件 因子VIIa、组织因子和TFPI活性的相对水平。尽管 深入研究，关于启动和监管的重要问题仍然存在 凝血反应。申请者假设新的基因， 参与启动和调节外在的凝血途径， 仍有待确认。目前的提案使用斑马鱼作为一种 用于识别这一途径中新基因的基因工具。新的基因将是 通过斑马鱼基因组的化学诱变和随后的 对凝血酶产生缺陷突变株的全面筛查 外在途径。凝血酶缺陷斑马鱼表型的筛选 这一代将产生大量已知和 参与外源性凝血途径的未知基因。斑马鱼 显示有缺陷的凝血酶生成将由两者快速分类 生化和连锁分析，以消除已知基因的突变，以及 重点将放在影响表达、激活或 因子VIIa的活性。斑马鱼新基因Will的鉴定 最终允许鉴定与止血有关的人类同源物 血栓形成。人类同源基因的鉴定将加深我们对 凝血级联如何在体内发挥作用，提出了新的靶点 抗血栓治疗和为血栓形成提供候选遗传标记物 疾病。

DESCRIPTION: (Investigator's abstract) The initiation of blood coagulation through the extrinsic pathway is a threshold-mediated event influenced by relative levels of factor VIIa, tissue factor and TFPI activity. Despite intensive study, important questions remain regarding initiation and regulation of the coagulation response. The applicant hypothesizes that novel genes, involved in initiation and regulation of the extrinsic pathway of coagulation, remain to be identified. The current proposal employs the zebrafish as a genetic tool to identify novel genes in this pathway. The novel genes will be identified by chemical mutagenesis of the zebrafish genome followed by a comprehensive screen for mutants with defective thrombin generation by the extrinsic pathway. Screening for zebrafish phenotypes with defective thrombin generation will yield a significant number of mutations in both known and unknown genes involved in the extrinsic pathway of coagulation. Zebrafish that display defective thrombin generation will be rapidly sorted by both biochemical and linkage analyses to eliminate mutations in known genes, and focus will be placed on novel defects affecting the expression, activation or activity of factor VIIa. Identification of novel zebrafish genes will ultimately allow identification of human homologues involved in hemostasis and thrombosis. Identification of human homologues will further our understanding of how the coagulation cascade functions in vivo, suggest novel targets for antithrombotic therapy and provide candidate genetic markers for thrombotic disease.