MOLECULAR BIOLOGY OF OUTER RETINA-SPECIFIC PROTEINS

视网膜色素上皮细胞（RPE）在视网膜色素变性中起着关键作用。 外视网膜的发育和功能。 我们有兴趣 RPE特异性机制，在调节和功能 程度. 这将有助于我们更好地了解RPE， 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 和RPE 65的调节，RPE 65是一种表达仅限于细胞内的基因。 RPE。我们研究了RPE 65是视黄醇异构酶的可能性， 一种对视觉必需的酶，（如RPE 65）仅限于RPE。 首先，我们发现异构酶活性与RPE 65共纯化， 可与视黄酯合酶活性分离。我们发现 异构酶活性被2种针对RPE 65的抗体抑制，表明 RPE 65在异构酶反应中发挥作用或可能是 异构酶本身。为了更好地了解RPE 65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RPE 65 敲除小鼠。 小鼠胚胎干细胞含有靶向 RPE 65基因座的破坏用于产生嵌合小鼠。 对这些嵌合体的后代进行基因分型证实了种系 RPE65的破坏。 经验证的杂合男性和女性 交配以产生纯合敲除小鼠。 这些将是 测试以建立这种敲除的表型。rpe65启动子 用含有RPE 65的转基因小鼠研究功能 启动子/报告基因构建体。 在所有构建体中，活性为 局限于眼睛的后段/RPE。根据我们的数据， 结论是：i）RPE 65启动子是RPE特异性的; ii）RPE 65启动子是RPE特异性的。 发现启动子活性的最小长度在-700和-800之间。 起始位点上游约300 bp; iii）可能存在阳性， 阴性对照元件位于该区域上游。鉴于 RPE 65在类维生素A代谢中的意义，RPE 65突变 基因可能导致光感受器的早期功能障碍。 因此，在本发明中， Leber先天性黑蒙（LCA）患者，一种异质性 以出生时失明为特征的疾病， RPE 65基因突变 复合杂合突变 导致RPE 65提前终止的有2例同胞。 任何一种突变的杂合子携带者都没有症状， 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.