人类基因组 3' UTR靶标表达克隆

人类基因组miRNA靶标表达克隆的特点

美国GeneCopoeia（复能基因）提供基于全人类基因组miRNA作用的靶标表达克隆，涵盖了公共数据库。miRNA靶标表达克隆包含SV40启动子驱动的报告基因（萤火虫荧光素酶）的下游紧接3’－UTR（即miRNA作用位点）序列。 第二个报告基因renilla luciferase，可用于监测转染，表达效率，并可以作内参。

通过萤火虫荧光素酶的活性分析，可获得miRNA对潜在靶标基因调控作用的结果。表达克隆转录后形成萤火虫荧光素酶ORF与3’UTR的嵌合体mRNA，如果有特异的miRNA可与3’－UTR某段序列互补结合，嵌合体中荧光素酶ORF所编码蛋白的活性也相应受到调控， 因此通过检测荧光素酶的发光强度就可以定量地测定miRNA对应靶标的mRNA调控的效果。

miRNA 前体和靶标表达克隆的应用

• 基因表达调控：通过转染或病毒颗粒感染的方式将单个或整套的miRNA导入培养细胞，进行单个miRNA或多个miRNA对基因的表达调控。对miRNA的调控效应和功能进行特定条件或体统化的研究
• miRNA靶标基因的确认和鉴定：利用miRNA靶标验证表达克隆，进行大规模的miRNA作用靶标基因的筛选和确证。一个miRNA可作用的所有靶标的基因，和可作用于一个mRNA的3’－UTR所有miRNA

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