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）最近被描述为一类新的调节分子，控制了无脊椎动物中分化和模式的方面。在脊椎动物中也发现了类似的miRNA，但是它们在较高生物体中的功能仍然很大未知。该提案是针对理解miRNA在脊椎动物发育中的作用的。我们发现，某些miRNA在肢体和心脏的形态发生过程中差异表达。我们将使用微阵列和迷你阶段方法来识别这些发育结构中表达的所有miRNA，包括鉴定在发育中的肢体芽中表达的miRNA，包括在前前和后limb原始中差异表达的miRNA，以及那些在心脏原则中产生的左右右手。这些miRNA的空间分布将由北印迹和携带“传感器”构建体的瞬态转基因小鼠的生产确定，这些小鼠旨在表达lacz，除非存在给定的miRNA。将制作其他传感器来检查单个miRNA家族的所有旁系同源物的差异表达模式（IET7）；以及在基因组中的簇中编码的几个不同的miRNA。基于这些表达模式，将使用逆转录病毒膜片在雏鸡中解决miRNA的功能。最后，我们将研究1个miRNA家族miR196在调节小鼠中HOX基因功能中的作用。将单独淘汰3个MIR196基因座，并将构建三重突变体。这些miR调节HOXB8 mRNA的稳定性和调节HOXA7 mRNA的翻译。 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.