LENS METABOLIC COOPERATION GAP JUNCTIONS AND CATARACT

描述（申请人的描述）：由于其独特的结构和 功能，目镜透镜完全取决于剩余的透镜纤维 通过细胞间隙连接与表面上皮细胞相互连接 通信路径。离子稳态是必不可少的，以避免 白内障：白内障中高浓度可溶性蛋白质的沉淀， 透镜纤维细胞质。本申请的长期目标是 实验证明了间隙的细胞间通讯功能 透镜发育和稳态中的连接。细胞间隙连接 通道由连接蛋白（connexins）组成，连接蛋白是一个完整的膜蛋白家族。 在透镜中表达三种连接蛋白：连接蛋白43（Cx 43）、Cx 46和Cx 50。 现在已经有了这些连接蛋白中每一种都有特定缺失的小鼠品系 基因. Cx43基因敲除的动物在出生时死于心脏缺陷; 两个敲除系是可行的和可育的，虽然独特和具体的透镜 病理结果来自每个连接蛋白缺失。实验被提议为 开发小鼠品系，其中Cx43在透镜中通过使用 Cre/loxP系统去除Cx43基因。这些动物将与 Cx46和Cx 50敲除系产生双重和三重敲除 动物，允许深入分析透镜中的连接蛋白功能 发育和体内平衡。由于Cx 50敲除导致小透镜， 细胞周期减少的潜在机制将通过实验来确定。 研究了首先，Cx46的编码区将被“敲入”Cx 50， 基因座这些动物表面上会产生正常数量的间隙 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。如果这些动物的晶状体正常， 需要绝对数量的通道以确保正常的细胞周期定时。 如果不是，则需要唯一的Cx 50通道属性。细胞周期速率 将使用[3] H-胸苷和BrdU测量野生型和敲除晶状体 标记，分裂细胞与连接蛋白表达模式相关。 原代培养物将用于尝试重建改变的细胞周期 体外定时。最后，已经鉴定了Cx 50的新突变体，其 当与野生型Cx 50共表达时， 爪蟾卵母细胞对。一种表达该突变体的转基因小鼠系， 将开发和研究α A-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.