ROLE OF WNT PROTEINS IN MYOGENIC SPECIFICATION IN DEVELOPING CHICK EMBRYOS

WNT 蛋白在鸡胚胎发育中肌生成规范中的作用

• 批准号: 6395897
• 负责人: Laura W. Burrus
• 金额: $ 12.7万
• 依托单位: SAN FRANCISCO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6395897
• 关键词: SDS polyacrylamide gel electrophoresis; biological signal transduction; cell growth regulation; chick embryo; cytogenetics; developmental genetics; enzyme linked immunosorbent assay; histogenesis; immunoaffinity chromatography; laboratory mouse; monoclonal antibody; myogenesis; nucleic acid sequence; polymerase chain reaction; protein purification; protein signal sequence; protein structure function; recombinant proteins; regulatory gene; striated muscles; transfection; vertebrate embryology

The development of a vertebrate organism from a single fertilized egg is a complex series of events involving both cell proliferation and specialization. Many of these events have been shown to be mediated by intercellular signals. One such family of signaling factors is the Wnt gene family, which is comprised of at least 20 related vertebrate genes (Adamson et el., 1994; Bouillet et al., 1996; Christian et al., 1991; Christiansen et al., 1995; Gavin et al., 1990; Hume and Dodd, 1993; Kispert et al., 1996; Ku and Melton, 1993; Nusse and Varmus, 1982; Roelink and Nusse, 1991; Roelink et al., 1990; Sidow, 1992; Tanda et el., 1995; Wainwright et al., 1988; Wolda and Moon, 1992). Wnt genes encode cysteine- rick polypeptides possessing 50-60% amino acid identify which are though to be secreted signaling molecules involved in cell growth, survival, and, or, differentiation (McMahon et al., 1992; Nusse and Varmus, 1992). My laboratory is particularly interested in the role(s) of directing pluripotential chick somite cells to become skeletal muscle. Although several Wnt family members have been shown to be sufficient and necessary for the induction of myogenesis in vertebrates (Leyns et al., 1997; Munsterberg et el., 1995; Stern et al., 1995; Wang et al., 1997), we are lacking important information about how Wnt proteins act at a cellular level. Despite the identification of the Wnt-1 cDNA more than thirteen years ago (Fung et al., 1985), studies pertaining to the cellular roles of Wnt proteins have been severely hampered by the lack of soluble active ligand. For reasons that are poorly understood, Wnt genes are extremely refractory to over-expression. In this grant, I propose to utilize a COS cell expression system to generate WNT fusion proteins which can easily be purified and used as immunogens for the generation of anti-Wnt monoclonal antibodies. By comparing the biochemical properties of recombinant Wnt proteins and endogenous Wnt proteins, we hope to identify the differences that will help us pinpoint the reason it has been so difficult to generate active soluble recombinant Wnt protein. Furthermore, Wnt protein purified from the COS cell expression system and/or from chick embryos (using immunoaffinity columns) will be used to directly assess the role of Wnt proteins in myogenic specifications. The development of these key reagents will open the door to numerous future studies pertaining to both development and cancer. Another goal of this project is to give students (with particular emphasis on under-represented minorities) an opportunity to do "hands-on" research. The intellectual and technical aspects of this project are quite accessible to both undergraduate and graduate students. This project will provide students with an excellent opportunity to utilize the techniques of biochemistry, molecular biology, cell biology, and developmental biology to address a scientific problem. Students will also be trained to keep good lab notebooks, critically analyze their data, orally present their data, prepare posters for meetings, and prepare manuscripts.

脊椎动物从一个受精卵发育成一个有机体， 一系列复杂的事件，包括细胞增殖和 专业化这些事件中的许多已被证明是由 细胞间信号一个这样的信号传导因子家族是Wnt 基因家族，其由至少20个相关脊椎动物基因组成 （Adamson等人，1994年; Bouillet等人，1996; Christian等人，一九九一年; Christiansen等人，1995; Gavin等人，1990; Hume and Dodd，1993; Kispert等人，1996; Ku和Melton，1993; Nusse和Varmus，1982; Roelink 和Nusse，1991; Roelink等人，1990; Sidow，1992; Tanda等人，一九九五年; 温赖特等人，1988年; Wolda和Moon，1992年）。Wnt基因编码半胱氨酸- 具有50-60%氨基酸同一性的Rick多肽， 是参与细胞生长、存活的分泌信号分子， 或分化（McMahon等人，1992; Nusse和Varmus，1992）。我 实验室特别感兴趣的是指导的作用（S） 多能性鸡体节细胞变成骨骼肌。虽然 几个Wnt家族成员已被证明是足够的和必要的 用于在脊椎动物中诱导肌肉发生（Leyns等，一九九七年; Munsterberg等人，1995; Stern等人，1995; Wang等人，（1997年），我们 缺乏关于Wnt蛋白如何作用于细胞的重要信息。 水平尽管Wnt-1 cDNA的鉴定超过13个 多年前（Fung等人，1985年），研究有关的细胞作用， Wnt蛋白由于缺乏可溶性活性物质而受到严重阻碍， 配体。由于人们知之甚少的原因，Wnt基因是非常重要的。 对过度表达不敏感。在这项拨款中，我建议利用一个COS 细胞表达系统来产生WNT融合蛋白， 纯化并用作产生抗Wnt单克隆抗体的免疫原 抗体的通过比较重组Wnt的生化特性， 蛋白质和内源性Wnt蛋白质，我们希望确定差异 这将帮助我们找出它难以产生的原因， 活性可溶性重组Wnt蛋白。此外，纯化的Wnt蛋白 来自COS细胞表达系统和/或来自鸡胚（使用 免疫亲和柱）将用于直接评估Wnt的作用 肌原性蛋白质。这些关键试剂的开发 将为未来的许多研究打开大门， 发展和癌症。 这个项目的另一个目标是让学生（特别强调 关于代表性不足的少数群体）的一个机会，做“动手”的研究。 该项目的智力和技术方面相当 本科生和研究生都可以使用。该项目将 为学生提供了一个很好的机会，利用技术 生物化学，分子生物学，细胞生物学， 生物学来解决一个科学问题学生还将接受培训， 保持良好的实验笔记本，批判性地分析他们的数据，口头报告 他们的数据，准备会议海报，并准备手稿。