LENS METABOLIC COOPERATION GAP JUNCTIONS AND CATARACT

描述(申请人描述)：由于其独特的结构和 功能，晶状体完全依赖于晶状体剩余的纤维。 通过缝隙连接细胞间与表面上皮细胞相互连接 沟通途径。离子动态平衡是必不可少的，以避免 白内障：高浓度的可溶性蛋白质在晶状体中沉淀 晶状体纤维细胞质。此应用程序的长期目标是 实验演示了通过GAP进行细胞间通信的功能 晶状体发育和动态平衡的结合点。缝隙连接细胞间 通道由连接蛋白组成，连接蛋白是一个完整的膜蛋白家族。 三种连接蛋白在晶状体中表达：连接蛋白43(Cx43)、Cx46和CX50。 现在可以使用带有这些连接蛋白的特定缺失的小鼠系 基因。Cx43基因敲除的动物出生时就死于心脏缺陷；另一只 两条挖空线是可行的和肥沃的，尽管是独特的和特定的镜头 每一种连接蛋白缺失都会导致病理改变。建议进行实验，以 建立Cx43在晶状体中被特定消融的小鼠系 Cre/loxP系统去除Cx43基因。这些动物将被用来繁殖 CX46和CX50敲击线可生产双重敲击和三重敲击 动物，允许深入分析晶状体中连接蛋白的功能 发展和动态平衡。由于CX50基因敲除导致了一个小镜头， 这种细胞周期下降的机制将在实验中得到证实 调查过了。首先，CX46的编码区将被“敲入”到CX50上 轨迹。这些动物表面上会产生正常数量的缝隙 连接通道，但通道将仅由Cx46组成，因此 缺乏野生型连接蛋白多样性。如果这些动物表现出正常的晶状体，那么 需要绝对数量的通道来确保正常的细胞周期计时。 如果不是，则需要唯一的CX50通道属性。细胞周期速率在 野生型和基因敲除的晶状体将用[3]H-胸腺嘧啶核苷和BrdU测量 标记，分裂细胞与连接蛋白的表达模式相关。 原代培养将被用来尝试重建改变的细胞周期 体外计时。最终确定了一个新的CX50突变体。 与野生型CX50共表达时显示显性负活性 非洲爪哇卵母细胞对。表达该突变体的转基因小鼠系由 将开发和研究αA-晶状体蛋白启动子。

DESCRIPTION (Applicant's Description): Due to its unique structure and function, the ocular lens depends absolutely on the lens fibers remaining interconnected to the surface epithelial cells by gap junctional intercellular communication pathways. Ionic homeostasis is essential in order to avoid cataract: precipitation of the high concentration of soluble proteins in the lens fiber cytoplasm. The long-term objectives of this application are to experimentally demonstrate the function of intercellular communication via gap junctions in lens development and homeostasis. Gap junctional intercellular channels are composed of connexins, a family of integral membrane proteins. Three connexins are expressed in the lens: connexin43 (Cx43), Cx46 and Cx50. Mouse lines are now available with specific deletions of each of these connexin genes. The Cx43 knock-out animals die at birth due to heart defects; the other two knock-out lines are viable and fertile, although unique and specific lens pathology results from each connexin deletion. Experiments are proposed to develop mouse lines in which Cx43 is specifically ablated in the lens by using the Cre/loxP system to remove the Cx43 gene. These animals will be bred with the Cx46 and Cx50 knock-out lines to produce double and triple knock-out animals, permitting an in-depth analysis of connexin function in lens development and homeostasis. As the Cx50 knock-out results in a small lens, the mechanisms underlying this decrease in the cell cycle will be experimentally investigated. First, the coding region of Cx46 will be "knocked in" to the Cx50 locus. These animals will ostensibly produce the normal numbers of gap junctional channels, but the channels will be composed of only Cx46, thus lacking wild-type connexin diversity. If these animals show normal lenses, then an absolute number of channels is required to insure normal cell cycle timing. If not, then unique Cx50 channel properties are required. Cell cycle rates in wild-type and knock-out lenses will be measured with [3]H-thymidine and BrdU labeling, and the dividing cells correlated with connexin expression patterns. Primary cultures will be used to attempt to reconstruct altered cell cycle timing in vitro. Finally, a novel mutant of Cx50 has been identified which reveals dominant-negative activity when co-expressed with wild-type Cx50 in Xenopus oocyte pairs. A transgenic mouse line expressing this mutant driven by the alphaA-crystallin promoter will be developed and studied.