MOLECULAR BIOLOGY OF OUTER RETINA-SPECIFIC PROTEINS

视网膜色素上皮(RPE)在视网膜病变中起着关键作用。 外视网膜的发育和功能。我们感兴趣的是 RPE特定机制，在监管和功能上都是如此 级别。这将帮助我们更好地了解 正常和疾病状态，并为我们提供进一步的工具 RPE的分析。为此，我们一直在研究该函数 以及对RPE65的调控，RPE65是一种表达受限于 RPE。我们研究了RPE65是视黄醇异构酶的可能性， 一种视觉必需的酶，(如RPE65)仅限于RPE。 首先，我们发现异构酶活性与RPE65和RPE65共同纯化。 与视黄酸酯合成酶活性是可分离的。我们发现 RPE65的2个抗体抑制了异构酶活性，提示 RPE65在异构酶反应中起作用或可能是 异构酶本身。为了更好地了解RPE65的功能及其 在RPE/光感受器相互作用中的作用我们正在制造RPE65 基因敲除老鼠。小鼠胚胎干细胞含有靶向 RPE65基因座的破坏被用来产生嵌合小鼠。 对这些嵌合体的后代进行基因分型确认的种系 RPE65的中断。经证实杂合子的男性和女性 交配产生纯合子基因敲除小鼠。这些将是 测试确定了这种基因敲除的表型。RPE65启动子 用含有RPE65的转基因小鼠研究其功能 启动者/报告者构建。在所有构造中，活动都是 局限于眼后段/RPE。从我们的数据来看 结论：i)RPE65启动子是RPE特异性的；ii) 启动子活性的最小长度在-700到 -起点上游300个基点；iii)可能存在正值和 这一区域上游的负控制因素。给定 RPE65与视黄酸代谢及RPE65基因突变的关系 基因可能导致早期光感受器功能障碍。因此， Leber先天性黑色素(LCA)患者，一种异质性 对以出生时失明为特征的疾病进行筛查 RPE65基因突变。复合杂合突变 导致RPE65提前终止的兄弟姐妹有2例。 任何一种突变的杂合子携带者都没有明显的症状。 支持这两个突变共同导致了这一点 LCA的形式，指定为LCA2。

The retinal pigment epithelium (RPE) plays a pivotal role in the development and function of the outer retina. We are interested in RPE-specific mechanisms, at both the regulatory and functional levels. This will help us gain a better understanding of the RPE in normal and disease states and provide us with tools for further analysis of the RPE. To this end we have been studying the function and regulation of RPE65, a gene whose expression is restricted to the RPE. We studied the possiblity that RPE65 is the retinol isomerase, an enzyme essential to vision and (like RPE65) restricted to the RPE. Firstly, we found that isomerase activity co-purifies with RPE65 and is separable from retinyl ester synthase activity. We found that isomerase activity was inhibited by 2 antibodies to RPE65, suggesting that RPE65 plays a role in the isomerase reaction or may be the isomerase itself. To better understand the function of RPE65 and its role in RPE/photoreceptor interactions we are making an RPE65 knockout mouse. Mouse embryonic stem cells containing a targeted disruption of the RPE65 locus were used to generate chimeric mice. Genotyping of progeny from these chimeras confirmed germline disruption of RPE65. Verified heterozygous males and females are being mated to generate homozygous knockout mice. These will be tested to establish a phenotype for this knockout. RPE65 promoter function was studied with transgenic mice containing RPE65 promoter/reporter constructs. In all constructs, activity was restricted to the posterior segment of the eye/RPE. From our data we conclude that: i) the RPE65 promoter is RPE-specific; ii) the minimal length for promoter activity is found from between -700 and -300 bp upstream of the start site; iii) there may be positive and negative control elements upstream of this region. Given the implication of RPE65 in retinoid metabolism, mutations in the RPE65 gene might cause an early dysfunction of photoreceptors. Thus, patients with Leber's congenital amaurosis (LCA), a heterogeneous disorder characterized by blindness at birth, were screened for mutations in the RPE65 gene. Compound heterozygous mutations resulting in premature termination of RPE65 were found in 2 siblings. Heterozygous carriers of either mutation were asymptomatic, strongly supporting that these two mutations together are responsible for this form of LCA, assigned as LCA2.