TRANSGENIC MODULATION OF GAP JUNCTION INTERACTIONS

间隙连接相互作用的转基因调节

• 批准号: 2889514
• 负责人: CECILIA W. LO
• 金额: $ 31.24万
• 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/2889514
• 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。这可能 涉及调节心脏神经嵴（NC）细胞的发育。 这是由圆锥干心脏缺陷和 可以挽救Cx43敲除（KO）的新生儿致死性[部分地， 至少]通过恢复NC细胞亚群的Cx43表达， CMV 43转基因。 此外，在表达Cx43的转基因小鼠中， 在NC中上调或下调缝隙连接通讯的结构 细胞，圆锥干心脏缺陷也出现。 鉴于这些 研究发现，拟议的研究旨在了解Cx43在 神经嵴发育 卢博士将利用这些现有的转基因 小鼠线（和其他）进行研究，重点是 三大目标。 首先，确定Cx43功能的变化 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 分化 第三，确定NC单元是否单独存在缺陷 解释Cx43 KO致死率。 在这些研究中， 将采用体外（目标1-3）和体内（目标4、5）方法。 目的1是描述神经嵴出现的时间 迁移，并确定是否细胞间粘附和细胞信号转导 调节NC迁移的重要途径可能是 改变了 目的2是定量神经元的频率和方向性， 嵴迁移，并表征NC细胞的运动反应 不同的基质环境和不同的趋化因子。 目的 3是通过定量表达来表征NC分化 平滑肌细胞、软骨细胞和 黑素细胞 目的4通过检测NC来证实体外结果 使用由6.5 μ mol/L的荧光素驱动的绿色荧光蛋白标签进行体内迁移 kb Cx43启动子序列，已知其指定转基因在 神经嵴细胞谱系（Lo et al. 1997）。 Am 5是为了确定 Cx43 KO小鼠的长期存活是否可以实现， Cx43表达通过Cx43表达恢复到所有神经嵴谱系。 由Cx43启动子驱动的表达载体。