Functional Analysis of Novel Genes in Eye Development an

通过使用基因组学和生物信息学的方法，我们已经确定了许多在人类和动物模型眼组织中表达的新基因和替代基因产物。其中几个已经被选择进行功能分析，使用广泛的功能和结构方法来确定它们在眼睛中的作用及其对正常视力和疾病的影响。这些措施包括： 1：血小板衍生生长因子D(PDGFD)属于PDGF/VEGF家族。在前段PDGFD中，它定位于虹膜和睫状体，而在视网膜中，它局限于外丛状层。我们发现PDGFD在控制晶状体生长中起关键作用，并且反义PDGFD强烈抑制晶状体上皮细胞的增殖。这一发现表明PDGFD在眼组织协调生长的机制中具有重要的体内作用。对眼内PDGFD通路的干预，也许是通过抗体或封闭肽，可能对某些白内障的治疗有用，包括手术后的继发性白内障。PDGFD也在光感受器和RPE中表达，提示可能在控制光感受器存活和控制视网膜血管形成中发挥作用。已经确定了PDGFD基因的功能启动子，并正确地将GFP报告基因的表达定向到眼睛组织。小鼠PDGFD基因的条件性敲除已接近完成。 2：Lengsin是谷氨酰胺合成酶(GS)超家族中的一个新成员，该超家族是脊椎动物眼晶状体特有的。它具有ATP结合活性，但到目前为止还没有明显的酶活性。Lengsin在终末分化的次级纤维细胞层中特异表达，在该层中，细胞核和其他细胞器丢失，细胞骨架和细胞内连接重新组织。长度蛋白的敲除模型已经完成，目前正在培育纯合子。 3：最近的研究表明补体因子H(CFH)在老年性黄斑变性(AMD)的发生发展中起着重要作用。我们已经为老年人的RPE/脉络膜和视网膜构建了酵母2-杂交体文库。利用CFH的AMD相关结构域7的构建物作为诱饵，我们已经确定了CFH在人RPE/脉络膜中潜在的重要结合靶点。这些技术正在通过包括表面等离子体共振光谱在内的技术进行进一步的评估。CFH与RPE/脉络膜蛋白的相互作用可能对其在补体系统中的作用产生局部影响，并可能为治疗干预提供机会。

Through use of genomics and bioinformatics approaches we have identified many novel genes and alternative gene products that are expressed in human and animal model eye tissues. Several of these have been selected for functional analysis using a broad range of functional and structural approaches to determine their roles in the eye and their implications for normal vision and for disease. These include: 1: Platelet-derived growth factor D (PDGFD) belongs to the PDGF/VEGF family. In the anterior segment PDGFD, it is localized to iris and ciliary body, whereas in the retina, it is restricted to the outer plexiform layer. We have shown that PDGFD has a key role in control of lens growth and that anti-PDGFD strongly inhibits lens epithelial cell proliferation This finding suggests a major in vivo role for PDGFD in the mechanisms of coordinated growth of eye tissues. Intervention in the PDGFD pathway in the eye, perhaps by antibody or blocking peptide, could be useful in the treatment of certain cataracts, including post-operative secondary cataract. PDGFD is also expressed in photoreceptors and in RPE, suggesting possible roles in controls of photoreceptors survival and control of retinal vascularization. A functional promoter of the PDGFD gene has been identified and correctly directs expression of a GFP reporter to eye tissues. A conditional knock out of PDGFD in mouse is nearing completion. 2: Lengsin is a novel member of the glutamine synthetase (GS) superfamily that is specific for the vertebrate eye lens. It has ATP-binding activity but, so far, no demonstrable enzyme activity. Lengsin is expressed specifically in the layer of terminally differentiating secondary fiber cells in which nuclei and other organelles are lost and cytoskeleton and intracellular junctions are reorganized. A knock out model of lengsin is completed and is currently in breeding for homozygosity. 3: Recent publications have shown an important role for complement factor H (CFH) in the development of age-related macular degeneration (AMD). We have constructed yeast 2-hybrod libraries for aged human RPE/choroid and retina. Using constructs for the AMD-related domain 7 of CFH as bait we have identified potentially important targets for CFH binding in human RPE/choroid. These are being further evaluated by techniques including surface plasmon resonance spectroscopy. Interaction of CFH with RPE/choroid proteins could have local effects on its role in the complement system and might provide opportunities for therapeutic intervention.