Transgenic rat overexpressing miR-15/16 cluster:functional implications in develo

描述(申请人提供)：microRNAs是一类新的非编码小RNA，它通过降解mRNA或抑制蛋白质翻译来负向调节基因的表达。MiRNA簇miR-15/16与多种人类疾病有关，如癌症和胚胎发育。特别是，miR-15/16被发现以受体Acvr2a为靶点，Acvr2a是Nodal信号通路的核心组成部分，众所周知，该信号通路控制胚胎发育。为了更深入地了解这个簇在发育和人类疾病中的作用，研究人员建议建立两个转基因大鼠模型：一个是利用慢病毒载体中的人泛素C启动子(UBC)在所有组织中表达miR-15/16的普遍存在的转基因大鼠(UBC-miR-15/16)；第二个是四环素(Tet)诱导的转基因大鼠(Tet-miR-15/16)，其中miR-15/16簇将由Tet调控的慢病毒载体驱动。将Tet-miR-15/16转基因大鼠与反向反式启动子RTTA-M2驱动的转基因大鼠rosa26-RTTA-M2杂交，获得同时表达miR-15/16和RTTA-M2的条件双转基因大鼠。在这个双转基因大鼠中miR-15/16簇的表达将受到强力霉素的严密调控。如果UBC-miR-15/16转基因大鼠模型表现为胚胎致死表型，该双转基因大鼠模型将为研究miR-15/16的生物学功能提供一种替代方法。研究人员将利用分子生物学、组织学和免疫组织化学方法对这些转基因大鼠进行基因分型，并对不同发育阶段的胚胎、出生后和成年大鼠进行表型分析。这两个模型对于研究miR-15/16簇如何参与发育和其他人类疾病的多个信号通路的分子机制具有重要价值。 项目简介：将产生表达miR-15/16簇的无处不在的四环素诱导的转基因大鼠，以研究它们在发育和其他人类疾病中的作用。

DESCRIPTION (Provided by Applicant): MicroRNAs are a new class of non-coding small RNAs that negatively regulate gene expression by either degrading mRNA or suppressing protein translation. A miRNA cluster, miR-15/16, has been implicated in a variety of human diseases, such as cancer, and embryonic development. In particular, miR-15/16 was found to target the receptor Acvr2a, a core component of the Nodal signaling pathway, which is well-known to control embryogenesis. To gain more insight into this cluster's role in development and human diseases, the investigator proposes to generate two transgenic rat models: one is a ubiquitous transgenic rat (UBC-miR-15/16) that expresses miR-15/16 in all tissues using a human ubiquitin C promoter (UBC) in a lentiviral vector; Second is a tetracycline(Tet) inducible transgenic rat (Tet-miR-15/16), in which miR-15/16 cluster will be driven by Tet regulated lentiviral vector. The Tet-miR-15/16 transgenic rat will be crossed with a transgenic rat, Rosa26-rtTA-M2, that the reverse transactivator rtTA-M2 was driven by a ubiquitous promoter Rosa 26 the investigator generated in his laboratory recently to obtain a conditional double transgenic rat that expresses both the miR-15/16 and rtTA-M2. The expression of miR-15/16 cluster in this double transgenic rat will be tightly controlled by the doxycycline. This double transgenic rat model will provide an alternative approach to study the biological functions of miR-15/16 if the UBC-miR-15/16 transgenic rat model displays an embryonic lethal phenotype. The investigator will characterize these transgenic rats by genotyping, phenotyping of embryos, postnatals, and adults at different developmental stages using molecular biological, histological, and immunohistochemical approaches. Those two models will be highly valuable to address the molecular mechanisms by how miR-15/16 cluster is involved in multiple signaling pathways in development and other human diseases. PROJECT NARRATIVE: The ubiquitous and tetracycline inducible transgenic rats expressing miR-15/16 cluster will be generated to investigate their roles in development and other human diseases.