Department of Rehabilitation Engineering, Research Institute, National Rehabilitation Center for Persons with Disabilities, Tokorozawa, Japan.
Invest Ophthalmol Vis Sci. 2011 Aug 22;52(9):6662-70. doi: 10.1167/iovs.11-7479.
PURPOSE. To collect an entire set of full-length cDNA clones derived from human retina-derived cell lines and to identify full-length transcripts for retinal preferentially expressed genes. METHODS. The full-length cDNA libraries were constructed from a retinoblastoma cell line, Y79, and a retinal pigment epithelium cell line, ARPE-19, using the vector-capping method, which generates a genuine full-length cDNA. By single-pass sequencing of the 5'-end of cDNA clones and subsequent mapping to the human genome, the authors determined their transcriptional start sites and annotated the cDNA clones. RESULTS. Of the 23,616 clones isolated from Y79-derived cDNA libraries, 19,229 full-length cDNA clones were identified and classified into 4808 genes, including genes of >10 kbp. Of the 7067 genes obtained from the Y79 and ARPE-19 libraries, the authors selected 72 genes that were preferentially expressed in the eye, of which 131 clones corresponding to 57 genes were fully sequenced. As a result, we discovered many variants that were produced by different transcriptional start sites, alternative splicing, and alternative polyadenylation. CONCLUSIONS. The bias-free, full-length cDNA libraries constructed using the vector-capping method were shown to be useful for collecting an entire set of full-length cDNA clones for these retinal cell lines. Full-length transcriptome analysis of these cDNA libraries revealed that there were, unexpectedly, many transcript variants for each gene, indicating that obtaining the full-length cDNA for each variant is indispensable for analyzing its function. The full-length cDNA clones (approximately 80,000 clones each for ARPE-19 and Y79) will be useful as a resource for investigating the human retina.
目的。收集整套源自人眼视网膜细胞系的全长 cDNA 克隆，并鉴定视网膜优先表达基因的全长转录本。方法。使用载体加帽法构建源自视网膜母细胞瘤细胞系 Y79 和人视网膜色素上皮细胞系 ARPE-19 的全长 cDNA 文库，该方法可生成真正的全长 cDNA。通过对 cDNA 克隆 5'端的单轮测序，并随后映射到人类基因组，作者确定了它们的转录起始位点并注释了 cDNA 克隆。结果。从 Y79 衍生的 cDNA 文库中分离出的 23616 个克隆中，鉴定出 19229 个全长 cDNA 克隆，并将其分类为 4808 个基因，包括长度超过 10 kbp 的基因。从 Y79 和 ARPE-19 文库中获得的 7067 个基因中，作者选择了 72 个在眼部优先表达的基因，其中 57 个基因的 131 个克隆进行了全长测序。结果，我们发现了许多由不同转录起始位点、选择性剪接和选择性多聚腺苷酸化产生的变体。结论。使用载体加帽法构建的无偏、全长 cDNA 文库被证明可用于收集这些视网膜细胞系的整套全长 cDNA 克隆。这些 cDNA 文库的全长转录组分析表明，每个基因都存在许多意想不到的转录变体，表明获得每个变体的全长 cDNA 对于分析其功能是必不可少的。全长 cDNA 克隆（每个 ARPE-19 和 Y79 约 80000 个克隆）将作为研究人类视网膜的资源非常有用。
