In vivo biosensor screen for mutants in lipid metabolism

描述：（申请人的摘要）虽然基于形态学的遗传筛选 已经确定了影响斑马鱼的规格和模式的突变 胚胎结构，很少有试剂已被应用于评估生理 在体内发挥作用。我们生产了荧光脂质， 磷脂酶A2（PLA2），一种重要的信号和消化酶，存在于 肠上皮细胞的细胞质和刷状缘。这些裂缝 脂质通过PLA2增加或移动它们的荧光发射波长， 从而揭示活动物中的局部酶活性。当 给光学透明的斑马鱼幼虫施用这些脂质， PLA2活性的实时视觉测定。 受精后第5天斑马鱼幼虫摄入的荧光脂质 产生强烈的胆囊荧光，我们的发现反映了 脂质通过肠道PLA2加工，随后通过 肝胆系统由于胆囊荧光的速率和程度 这些试剂很容易在斑马鱼幼体中观察到， 在诱变筛选的背景下， 识别影响脂质加工的基因。 我们给受精后5天的诱变小鼠注射荧光脂质， 斑马鱼幼虫，并确定了一个突变，扰乱肠道 磷脂酶活性本建议书所述的工作概述了我们的计划 用于使用这些试剂进行大规模诱变筛选。通过识别 我们希望恢复调节脂质代谢的基因， 肠道和肝胆发育以及PLA2，一个基因， 印记作用于炎症、止血和细胞增殖。遗传 这些发育和生理过程的表征 对儿童先天性疾病相关研究的重要意义 肠和肝、癌症以及心血管和炎性疾病。

DESCRIPTION: (Applicant's Abstract) Although morphology-based genetic screens have identified mutations that affect specification and patterning of zebrafish embryonic structures, few reagents have been applied to assess physiological functions in vivo. We produced fluorescent lipids that are substrates for phospholipase A2 (PLA2), an important signaling and digestive enzyme present in the cytoplasm and brush border of the intestinal epithelium. Cleavage of these lipids by PLA2 increases or shifts their wavelength of fluorescent emission, thereby revealing localized enzymatic activity in live animals. When administered to optically transparent zebrafish larvae, these lipids provide a real-time visual assay of PLA2 activity. Fluorescent lipids ingested by day 5 post-fertilization zebrafish larvae produce intense gallbladder fluorescence, a finding we have shown reflects lipid processing by intestinal PLA2 and subsequent transport through the hepatobiliary system. Since the rate and degree of gallbladder fluorescence are easily observed in zebrafish larvae, these reagents provide a sensitive readout of digestive physiology that in the context of a mutagenesis screen can identify genes that influence lipid processing. We administered fluorescent lipids to mutagenized 5-day post-fertilization zebrafish larvae and have identified one mutation that perturbs intestinal phospholipase activity. The work described in this proposal outlines our plan for a large scale mutagenesis screen using these reagents. By identifying mutations that perturb lipid metabolism, we hope to recover genes that regulate intestinal and hepatobiliary development as well as PLA2, a gene with an imprint role in inflammation, hemostasis and cell proliferation. Genetic characterization of these developmental and physiological processes has important implications for research related to congenital diseases of the intestine and liver, cancer, and cardiovascular and inflammatory diseases.