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: 6107755
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107755
• 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.

脊椎动物有机体是从一个受精卵发育而来的 一系列复杂的事件涉及细胞增殖和 专业化。这些事件中的许多已经被证明是由 细胞间信号。一个这样的信号因子家族就是WNT 基因家族，由至少20个相关脊椎动物基因组成 (Adamson等人，1994；Bouillet等人，1996；Christian等人，1991； 克里斯汀森等人，1995年；加文等人，1990年；休谟和多德，1993年； 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，1995； Wainwright等人，1988；Wolda和Moon，1992)。WNT基因编码半胱氨酸- 含有50%-60%氨基酸的瑞克多肽可以识别哪些是 是分泌的信号分子，参与细胞的生长、存活和， 或者，差异化(McMahon等人，1992；Nusse和Varmus，1992)。我的 实验室对导演(S)的角色特别感兴趣 具有多潜能的鸡体节细胞可转化为骨骼肌。虽然 事实证明，WNT的几个家庭成员是足够和必要的 用于脊椎动物肌肉发生的诱导(Leyns等人，1997； Munsterberg等人，1995；Stern等人，1995；Wang等人，1997)，我们是 缺乏关于Wnt蛋白如何在细胞内发挥作用的重要信息 水平。尽管Wnt-1的cDNA13个以上已被鉴定 几年前(冯等人，1985)，与细胞角色有关的研究 WNT蛋白因缺乏可溶性活性而严重受阻。 莱兰德。出于一些鲜为人知的原因，Wnt基因 不易过度表达。在这笔赠款中，我建议使用COS 细胞表达系统产生的WNT融合蛋白可以很容易地 纯化的抗Wnt单抗的制备及其免疫原性 抗体。通过比较重组Wnt的生化性质 蛋白质和内源性Wnt蛋白，我们希望找出差异 这将帮助我们准确地找出为什么很难产生 活性可溶性重组Wnt蛋白。此外，还纯化了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 生肌规格中的蛋白质。这些关键试剂的研究进展 将为未来与这两个领域相关的众多研究打开大门 发展和癌症。 这个项目的另一个目标是给学生(特别强调 关于代表不足的少数群体)有机会进行“实践”研究。 这个项目的智力和技术方面相当 本科生和研究生都可以使用。这个项目将 为学生提供利用这些技术的绝佳机会 生物化学、分子生物学、细胞生物学和发育 生物学来解决一个科学问题。学生还将接受培训，以 保存好实验室笔记本，批判性地分析数据，口头演示 他们的数据，准备会议海报，准备手稿。