TRANSGENIC MODULATION OF GAP JUNCTION INTERACTIONS

各种研究表明，连接蛋白43(Cx43)缝隙连接基因 在圆锥干心脏发育中起重要作用。这很有可能 涉及调控心脏神经脊(NC)细胞的发育。 这一发现表明，圆锥干心脏缺陷和 Cx43基因敲除(KO)的新生儿致死性可以挽救[部分， 至少]通过以下途径恢复NC细胞亚群的Cx43表达 一个CMV43转基因。此外，在表达Cx43的转基因小鼠中 构建对NC中的缝隙连接通讯进行上下调节的结构 细胞、圆锥干心脏也会出现缺陷。鉴于这些， 发现，这项拟议的研究试图了解Cx43在 神经脊发育。罗博士将使用这些现有的转基因 小鼠品系(和其他待制作的品系)开展的研究集中在 三个主要目标。首先，确定Cx43中的更改如何发挥作用 影响心肌NC细胞的出现和迁移行为。 第二，确定Cx43功能的变化是否会扰乱NC 差异化。第三，确定NC细胞中的缺陷是否单独存在 解释了Cx43 KO的致命性。在这些研究中，结合了 将使用体外(AIMS 1-3)和体内(AIMS 4，5)方法。 目的1是描述神经峰开始出现的时间。 迁移，并确定细胞间的黏附和细胞信号转导 调控NC迁移开始的重要途径可能是 被更改了。目标2是量化神经的速度和方向性。 顶峰迁移，并表征了NC细胞的运动反应 以适应不同的基质环境和不同的趋化剂。目标 3是通过量化表达来表征NC分化 平滑肌细胞、软骨和 黑素细胞。目的4是通过检查NC来确认体外结果 使用6.5驱动的绿色荧光蛋白标签在体内迁移 Kb Cx43启动子序列指定转基因表达 神经脊细胞谱系(Lo等人)1997年)。上午5点将确定 Cx43 KO小鼠能否长期存活 Cx43的表达通过Cx43恢复到所有神经脊谱系 Cx43启动子驱动的表达载体。

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.