Biological function microRNAs enriched in RPE: in vitro and in vivo models

MicroRNA(MiRNA)是一组短小的(1925个核苷酸)非编码RNA，能够通过与靶mRNAs的3个非翻译区(3UTRs)碱基配对来下调基因的表达。MiRNAs的结构在许多物种中高度保守，表明这些分子具有重要的功能。我们已经鉴定出几种miRNAs在人胚胎视网膜色素上皮(HfRPE)中的表达水平相对较高，而在视网膜和脉络膜中表达相对较高。这些潜在的调节富集性miRNAs miR-184、miR-187、miR-200、miR-221/222、miR-204、miR-211--被发现与维持RPE的总组织阻力有关，提示这些miRNAs在RPE屏障功能中的重要性。尽管它们相对丰富，但microRNAs在RPE中的详细功能仍有待确定。发展microRNA基因敲除小鼠有助于了解单个microRNA的体内功能。 作为最初的方法，我们生产了基因靶向构建体，被指定为从小鼠身上结构性地去除单个microRNA。通过同源重组，将编码单个microRNA的基因片段替换为新霉素抗性基因表达盒。由于neo基因两侧有一对Lox-p位点，因此携带在靶基因中的neo基因可以通过Cre-Lox-p机制消除。 我们可能会遇到以下情况： 由于单个miRNAs可以特定地与关键的发育途径联系在一起，我们将研究击倒特定的microRNA是否会导致RPE和其他组织中microRNA表达的任何结构异常。 如果microRNA的功能对发育至关重要，胚胎死亡可能发生，那么我们将产生条件基因敲除小鼠，其中microRNA将从RPE中消除。 如果miRNA的功能是微调基因表达的话，那就是环境压力引起的噪音。我们将确定相关的刺激和相应的障碍。 如果一个microRNA位于基因的内含子内，它的表达和功能往往与其宿主基因有关。因此，我们将结合宿主基因来研究microRNA的功能。 无论如何，基因敲除小鼠的RPE将被培养并用作体外模型系统，以测试功能丧失和相应的救援策略。

MicroRNA (miRNA) is a group of short (1925 nucleotide), non-coding RNAs that are capable of downregulate gene expression by base pairing with the 3 untranslated regions (3UTRs) of target mRNAs. The structure of miRNAs is highly conserved across many species, indicating important function of these molecules. We have identified several miRNAs that express in relative high levels in human fetal retinal pigment epithelium (hfRPE) compared to retina and choroids. These potential regulatory enriching miRNAs miR-184, miR-187, miR-200, miR-221/222, miR-204, miR-211 -- were found to be associated with the maintenance of total tissue resistance of the RPE, suggesting the importance of these miRNAs in RPE barrier function. Despite their relative enrichment, detailed functions of microRNAs in RPE remain to be determined. Developing microRNA knockout mice will facilitate the understanding invivo function of individual microRNA. As an initial approach, we produced gene-targeting constructs that are designated to ablate individual microRNA constitutively from mouse. The gene segment that encodes individual microRNA was replaced with a neomycin resistant gene expression cassette via homologous recombination. Since a pair of the Lox-p sites flanks neo gene, the neo gene that is bearing within targeted allele can be eliminated by Cre-Lox-p mechanism. We can encounter following possible scenarios: As individual miRNAs can be specifically linked to critical developmental pathways, we will examine if knocking down a particular microRNA will cause any structure abnormalities in RPE and other tissues where the microRNA has shown expression. If function of a microRNA is pivotal to development, embryonic lethality can happen, then we will produce conditional knockout mice where a microRNA will be eliminated from RPE. If function of a miRNA is to fine-tune gene expression "noise" elicited by the environmental stress. We will determine the relevant stimulations and corresponding disorders. If a microRNA is resided within intron of a gene, its expression and function are often associated with its host gene. Therefore, the function of a microRNA will be studied in association with its host gene. In any case, RPE from knockout mice will be cultured and used as invitro model system to test loss of function and corresponding rescue strategies.