The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.

通过Ago2交联免疫沉淀（CLIP）方法鉴定microRNA（miRNA）靶标，已为这类重要的非编码RNA生物学提供了重要见识。然而，这些方法在技术上具有挑战性，并且不容易应用于体内环境。为了克服这些局限性并促进体内miRNA功能的研究，我们已经开发了一种基于基因工程小鼠的方法，该小鼠具有从内源性Ago2基因座表达的条件Halo-Ago2等位基因。通过使用与HaloTag配体偶联的树脂，可以从表达内源Halo-Ago2等位基因的细胞和组织中纯化Ago2-miRNA-mRNA复合物。我们证明了该方法在小鼠胚胎干细胞，发育中的胚胎，成年组织以及人脑和肺癌的本地小鼠模型中的可重复性和敏感性。这种方法和我们生成的数据集将有助于在生理和病理条件下体内表征miRNA-mRNA网络。