Cavaney D M, Rakoczy P E, Constable I J
Lions Eye Institute, Centre for Ophthalmology and Visual Science, University of Western Australia, Nedlands.
Aust N Z J Ophthalmol. 1995 May;23(2):139-44. doi: 10.1111/j.1442-9071.1995.tb00143.x.
To study genes expressed by retinal pigment epithelial (RPE) cells during phagocytosis and digestion of rod outer segments (ROS), a complementary (c)DNA library was produced using an in-vitro model. The cDNA library can be used to study molecular changes which contribute to the development of diseases due to a failure in outer segment phagocytosis and digestion by RPE cells. Here we demonstrate a way to study genes and their functions using a molecular biological approach and describing the first step involved in this process, the construction of a cDNA library.
Human RPE cells obtained from the eyes of a seven-year-old donor were cultured and challenged with bovine ROS. The culture was harvested and total RNA was extracted. Complementary DNA was transcribed from the messenger (m)RNA and was directionally cloned into the LambdaGEM-4 bacteriophage vector successfully. Some clones were picked and the DNA extracted, to determine the size of the inserts as a measure of the quality of the library.
Molecular biology and cell culture are important tools to be used in eye research, especially in areas where tissue is limiting and animal models are not available. We now have a ROS challenged RPE cDNA library which will be used to identify genes responsible for degrading phagocytosed debris within the retinal pigment epithelium.
为了研究视网膜色素上皮（RPE）细胞在吞噬和消化视杆细胞外节（ROS）过程中表达的基因，我们利用体外模型构建了一个互补（c）DNA文库。该cDNA文库可用于研究由于RPE细胞对外节的吞噬和消化失败而导致疾病发生发展过程中的分子变化。在此，我们展示了一种利用分子生物学方法研究基因及其功能的途径，并描述了这一过程的第一步，即cDNA文库的构建。
从一名7岁供体的眼睛中获取人RPE细胞并进行培养，然后用牛ROS进行刺激。收集培养物并提取总RNA。从信使（m）RNA转录得到互补DNA，并成功地将其定向克隆到LambdaGEM - 4噬菌体载体中。挑选了一些克隆并提取DNA，以确定插入片段的大小，作为文库质量的一个指标。
分子生物学和细胞培养是眼科研究中重要的工具，特别是在组织样本有限且无法使用动物模型的领域。我们现在有了一个经ROS刺激的RPE cDNA文库，它将用于鉴定负责在视网膜色素上皮细胞内降解吞噬碎片的基因。
