NEIBank: Est Analysis & Bioinformatics For Ocular Genomi

眼睛是一个复杂的系统，由各种发育起源的高度分化的组织组成。许多对眼睛功能至关重要的基因是组织特异性的，其中许多已知的基因与遗传性眼病有关。大多数人类表达的基因仅通过表达序列标签（EST）已知。由于眼组织在cDNA文库中的代表性很差，因此开始了一个名为NEIBank的项目，以提高EST的覆盖率，并开发一个眼的分子百科全书。构建了几种人眼组织的cDNA文库，包括透镜、虹膜、RPE/脉络膜、视网膜、小梁网、泪腺和圆锥角膜，并通过深度测序进行了检测。使用定制软件GRIST对来自每个未标准化文库的2，000至10，000个克隆进行测序、分析和聚类。已建立了几个重要动物模型的文库，包括大鼠的角、视网膜和全眼，小鼠的全眼、泪腺、视网膜和RPE，狗和兔的前节和后节，以及斑马鱼的透镜和全眼。许多新的基因和遗传变异已被确定。总共约有40，000个眼部cDNA克隆已被测序并组织在基于网络的数据库（http：//www.example.com）中，该数据库包含关键词和染色体位置数据。neibank.nei.nih.gov这项工作产生的人类DNA资源已被用于创建用于眼部研究的微阵列。已经构建了具有超过13，000个非冗余人眼表达序列的cDNA微阵列，并正在进行测试。 通过NEIBank发现的几个新基因已被选择用于验证和进一步研究。其中包括一种与谷氨酰胺合成酶和虹膜表达生长因子相关的丰富的新型透镜特异性转录物。Lengsin表达与透镜中的终末分化和核破裂有关。重组小鼠和人蛋白质正被用于功能和结构研究，并且已经制备了使用拟南芥蛋白启动子的转基因小鼠。目前正在利用“重组工程”技术建立一种针对黑曲霉素的基因敲除小鼠模型。我们已经证明，IEGF/PDGFD在控制透镜生长和眼前节发育中具有关键作用。抗IEGF抗体阻断透镜细胞增殖，可能在继发性和囊下白内障中具有转化研究应用。该因子也在视网膜中特异性表达，并且可能在光感受器健康中起作用。对一个新的圆锥角膜文库的分析表明，该文库包含人类角膜表达基因的极好代表，但也揭示了参与这种疾病的重要候选基因。来自这些数据的线索正在被追踪，沿着的还有其他几种新的基因产物，包括retbindin和Mp 19 ins。Retbindin可能是视网膜的类胡萝卜素结合蛋白，而Mp 19 ins可能在人类透镜的正确形成中具有特定作用。

The eye is a complex system of highly differentiated tissues of various developmental origins. Many genes essential for eye function are tissue-specific and many of those known are associated with genetic eye diseases. The majority of human expressed genes are known only through expressed sequence tags (ESTs). Since eye tissues were poorly represented in the cDNA libraries initially contributed to dbEST a project called NEIBank was begun to improve the EST coverage and to develop a molecular encyclopedia for the eye. cDNA libraries for several human eye tissues, including lens, iris, RPE/choroid, retina, trabecular meshwork, lacrimal gland and keratoconus cornea have been constructed and examined by in-depth sequencing. Between 2,000 and 10,000 clones from each un-normalized library have been sequenced, analyzed and clustered using custom software, GRIST. Several libraries have been made for important animal models, including angle, retina and whole eye of rat, whole eye, lacrimal gland, retina and RPE of mouse, anterior and posterior segments for dog and rabbit and lens and whole eye for zebrafish . Many novel genes and genetic variants have been identified. In total, approximately 40,000 ocular cDNA clones have been sequenced and organized in a web-based database (http://neibank.nei.nih.gov) that contains keyword and chromosomal location data. The human DNA resource generated by this work have been used to create micro-arrays for ocular studies. A cDNA microarray with over 13,000 non-redundant human eye expressed sequences has been constructed and is being tested. Several novel genes discovered through NEIBank have been selected for verification and further study. These include lengsin an abundant novel lens specific transcript related to glutamine synthetase and iris-expressed growth factor. Lengsin expression is associated with terminal differentiation and nuclear breakdown in the lens. Recombinant mouse and human proteins are being used for function and structure studies and transgenic mice using the lengsin promoter have been made. A knock-mouse model for lengsin is under construction using "recombineering" technology. We have shown that IEGF/PDGFD has a key role in control of lens growth and development of the anterior segment. Antibody to IEGF blocks lens cell proliferation and may have translational research applications in secondary and subcapsular cataracts. This factor is also specifically expressed in the retina and may have a role in photoreceptor health. Analysis of a new keratoconus cornea library has shown that the library contains an excellent representation of human cornea expressed genes but has also revealed an important candidate gene for involvement in this condition. The leads from these data are being pursued, along with several other novel gene products including retbindin and Mp19ins. Retbindin may be the carotenoid binding protein of the retina, while Mp19ins may have a specific role in correct formation of the human lens.