TRANSGENIC MODULATION OF GAP JUNCTION INTERACTIONS

间隙连接相互作用的转基因调节

• 批准号: 6181735
• 负责人: CECILIA W. LO
• 金额: $ 31.95万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6181735
• 关键词: cell adhesion; cell cell interaction; cell differentiation; cell migration; developmental genetics; gap junctions; gene dosage; gene expression; genetically modified animals; green fluorescent proteins; heart cell; heart disorder; histogenesis; laboratory mouse; membrane channels; neural crest; protein structure function; transforming growth factors

Various studies suggest that the connexin 43 (Cx43) gap junction gene plays in important role in conotruncal heart development. This likely involves modulating the development of cardiac neural crest (NC) cells. This is indicated by the finding that the conotruncal heart defects and neonatal lethality of the Cx43 knockout (KO) can be rescued [partially, at least] by restoring Cx43 expression to subpopulations of NC cells via a CMV43 transgene. Furthermore, in transgenic mice expressing Cx43 constructs that up or down regulate gap junctional communication in NC cells, conotruncal heart defects also arise. In light of these findings, the proposed research seeks to understand the role of Cx43 in neural crest development. Dr. Lo will use these existing transgenic mouse lines (and others to be made) to carry out studies focused on three main objectives. First, determine how changes in Cx43 function affect the emergence and migratory behavior of cardiac NC cells. Second, determine whether changes in Cx43 function may perturb NC differentiation. Third, determine whether defects in NC cells alone account for the Cx43 KO lethality. For these studies, a combination of in vitro (Aims 1-3) and in vivo (Aims 4,5) approaches will be utilized. Aim 1 is to characterize the timing of the onset of neural crest migration, and determine whether cell-cell adhesion and cell signaling pathways important in regulating the onset of NC migration may be altered. Aim 2 is to quantitate the rate and directionality of neural crest migration, and characterize the locomotory responses of NC cells to different matrix environments and various chemotropic agents. Aim 3 is to characterize NC differentiation by quantitating the expression of differentiation markers for smooth muscle cells, cartilage, and melanocytes. Aim 4 is to confirm the in vitro results by examining NC migration in vivo using a green fluorescent protein tag driven by a 6.5 kb Cx43 promoter sequence known to specify transgene expression in neural crest cell lineages (Lo et al. 1997). Am 5 is to determine whether long term survival of the Cx43 KO mouse may be achieved when Cx43 expression is restored to all neural crest lineages via a Cx43 expression vector driven by the Cx43 promoter.

各种研究表明连接蛋白 43 (Cx43) 间隙连接基因 在圆锥干心脏发育中起重要作用。这很可能 涉及调节心脏神经嵴（NC）细胞的发育。 发现圆锥干心脏缺陷和 Cx43 敲除 (KO) 的新生儿致死率可以挽救[部分， 至少]通过恢复 NC 细胞亚群的 Cx43 表达 CMV43 转基因。 此外，在表达 Cx43 的转基因小鼠中 上调或下调 NC 间隙连接通讯的结构 细胞，圆锥干心脏也会出现缺陷。 鉴于这些 研究结果表明，拟议的研究旨在了解 Cx43 在 神经嵴发育。 罗博士将利用这些现有的转基因 小鼠品系（以及其他待制作的品系）进行重点研究 三个主要目标。 首先，确定Cx43功能如何变化 影响心脏 NC 细胞的出现和迁移行为。 其次，确定 Cx43 功能的变化是否会干扰 NC 差异化。 三、判断NC细胞是否单独存在缺陷 解释 Cx43 KO 致死率。 对于这些研究，结合了 将采用体外（目标 1-3）和体内（目标 4,5）方法。 目标 1 是表征神经嵴出现的时间 迁移，并确定细胞间粘附和细胞信号传导是否 调节 NC 迁移发生的重要途径可能是 改变了。 目标 2 是量化神经网络的速率和方向性 嵴迁移，并表征 NC 细胞的运动反应 适应不同的基质环境和各种趋化剂。 目的 图3是通过定量表达来表征NC分化 平滑肌细胞、软骨细胞的分化标记物 黑素细胞。 目标 4 是通过检查 NC 来确认体外结果 使用由 6.5 驱动的绿色荧光蛋白标签进行体内迁移 kb Cx43 启动子序列已知用于指定转基因表达 神经嵴细胞谱系（Lo 等人，1997）。 上午 5 点确定 Cx43 KO 小鼠是否可以实现长期存活 通过 Cx43 恢复所有神经嵴谱系的 Cx43 表达 由 Cx43 启动子驱动的表达载体。