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

描述（由申请人提供）：MicroRNAs是一类新的非编码小rna，通过降解mRNA或抑制蛋白质翻译负性调节基因表达。miRNA簇miR-15/16与多种人类疾病，如癌症和胚胎发育有关。特别是，miR-15/16被发现靶向受体Acvr2a，这是众所周知的控制胚胎发生的节点信号通路的核心成分。为了更深入地了解这一簇在发育和人类疾病中的作用，研究者提出了两种转基因大鼠模型：一种是普遍存在的转基因大鼠（UBC-miR-15/16），它在慢病毒载体中使用人泛素C启动子（UBC）在所有组织中表达miR-15/16；第二种是四环素（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。Tet-miR-15/16转基因大鼠将与Rosa26-rtTA-M2转基因大鼠杂交，该转基因大鼠的反转录激活子rtTA-M2由研究者最近在其实验室生成的无所不在的启动子Rosa26驱动，从而获得同时表达miR-15/16和rtTA-M2的条件双转基因大鼠。在这种双转基因大鼠中miR-15/16簇的表达将受到强力霉素的严格控制。如果UBC-miR-15/16转基因大鼠模型显示出胚胎致死性表型，这种双转基因大鼠模型将为研究miR-15/16的生物学功能提供另一种方法。研究者将利用分子生物学、组织学和免疫组织化学方法，对这些转基因大鼠的胚胎、产后和不同发育阶段的成年大鼠进行基因分型、表型分析。这两个模型对于解决miR-15/16集群如何参与发育和其他人类疾病的多种信号通路的分子机制将具有很高的价值。

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.