Developmental regulation by miRNAs

miRNA 的发育调控

• 批准号: 7046164
• 负责人: CLIFFORD J. TABIN
• 金额: $ 32.28万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7046164
• 关键词: bone development; cardiogenesis; chick embryo; gene expression; genetic regulation; genetically modified animals; laboratory mouse; microRNAs; microarray technology; northern blottings; vertebrate embryology

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) have recently been described as an important new class of regulatory molecules controlling aspects of differentiation and patterning in invertebrates. Similar miRNAs have been discovered in vertebrates, but their function in higher organisms remains largely unknown. This proposal is directed towards understanding the role of miRNAs during vertebrate development. We have found that certain miRNAs are differentially expressed during morphogenesis of the limb and heart. We will use microarray and mini-Sage approaches to identify all miRNAs expressed in these developing structures, including the identification of miRNAs expressed in the developing limb buds, including those differentially expressed in the fore- and hind-limb primordia as well as those left-right asymetrically produced in the heart primordium. The spatial distribution of these miRNAs will be determined by Northern blot and by production of transient transgenic mice carrying "sensor" constructs which are designed to express lacZ everywhere except where a given miRNA is present. Additional sensors will be made to examine the differential expression patterns of all paralogues of a single miRNA family (Iet7); and also of several distinct miRNAs encoded in a cluster in the genome. Based on these expression patterns, the function of miRNAs will be addressed in the chick using retroviral misexpression. Finally, we will study the roles of 1 family of miRNAs, miR196, in regulating Hox gene function in mice. The 3 miR196 loci will be knocked-out individually and the triple mutant will be constructed. These miRs regulate stability of the HoxB8 mRNA and modulate translation of the HoxA7 mRNA. The miR196 target sequence in the HoxB8 3'UTR will be deleted by a knock-in approach to determine the developmental significance of this regulation and the miR196 target sequence in the HoxA7 3'UTR will be replaced with the HoxB8 miR198 target sequence to test whether the mode of regulation (RNA cleavage versus translational control) functionally matters.

描述（由申请人提供）：MicroRNA（miRNA）最近被描述为一类重要的新型调节分子，控制无脊椎动物的分化和模式化方面。在脊椎动物中也发现了类似的 miRNA，但它们在高等生物中的功能仍然很大程度上未知。该提案旨在了解 miRNA 在脊椎动物发育过程中的作用。我们发现某些 miRNA 在肢体和心脏的形态发生过程中存在差异表达。我们将使用微阵列和mini-Sage方法来鉴定在这些发育结构中表达的所有miRNA，包括鉴定在发育中的肢芽中表达的miRNA，包括在前肢和后肢原基以及左肢原基中差异表达的miRNA。右侧不对称地产生于心脏原基。这些miRNA的空间分布将通过Northern印迹和通过产生携带“传感器”构建体的瞬时转基因小鼠来确定，该构建体被设计为在除给定miRNA存在的地方之外的各处表达lacZ。将制作额外的传感器来检查单个 miRNA 家族 (Iet7) 的所有旁系同源物的差异表达模式；以及基因组中编码的几个不同的 miRNA。根据这些表达模式，将使用逆转录病毒错误表达来解决小鸡中 miRNA 的功能。最后，我们将研究 1 个 miRNA 家族 miR196 在调节小鼠 Hox 基因功能中的作用。 3 个 miR196 位点将被单独敲除并构建三重突变体。这些 miR 调节 HoxB8 mRNA 的稳定性并调节 HoxA7 mRNA 的翻译。通过敲入的方法删除HoxB8 3'UTR中的miR196靶序列以确定这种调节的发育意义，并将HoxA7 3'UTR中的miR196靶序列替换为HoxB8 miR198靶序列以测试是否调节模式（RNA 切割与翻译控制）在功能上很重要。