利用Rosa26-Lox-Stop-Lox系统构建条件性过表达微小RNA的小鼠品系。

Generation of mouse lines conditionally over-expressing microRNA using the Rosa26-Lox-Stop-Lox system.

作者信息

Piovan Claudia, Amari Foued, Lovat Francesca, Chen Qun, Coppola Vincenzo

机构信息

Department of Molecular Virology, Immunology and Medical Genetics, Wexner Medical Center, Comprehensive Cancer Center, The Ohio State University, Biomedical Research Tower, Room 988, 460W. 12th Avenue, Columbus, OH, 43210, USA.

出版信息

Methods Mol Biol. 2014;1194:203-24. doi: 10.1007/978-1-4939-1215-5_11.

DOI:10.1007/978-1-4939-1215-5_11

PMID:25064105

Abstract

MicroRNAs are currently the object of intensive investigation due to their role in a myriad of physiological processes and pathological conditions, such as gene regulation and tumorigenesis. To better understand microRNA function, numerous laboratories have already taken advantage of the available techniques of genome editing in mouse. Here, we describe how to generate genetically engineered mouse lines using the popular Rosa-26 Lox-Stop-Lox Knock-In (Rosa-LSL-KI) targeting. This strategy allows for the selective overexpression of microRNAs of interest when coupled to a tissue-specific Cre-expressing line. The present protocol illustrates in detail both the engineering of the targeting vector and the generation of mutated ES clones ready for injection into mouse blastocysts.

摘要

由于微小RNA在众多生理过程和病理状况（如基因调控和肿瘤发生）中发挥作用，目前它们是深入研究的对象。为了更好地理解微小RNA的功能，许多实验室已经利用了小鼠基因组编辑的现有技术。在这里，我们描述了如何使用流行的Rosa-26 Lox-Stop-Lox敲入（Rosa-LSL-KI）靶向技术生成基因工程小鼠品系。当与组织特异性表达Cre的品系结合时，该策略允许选择性过表达感兴趣的微小RNA。本方案详细说明了靶向载体的构建以及用于注射到小鼠囊胚中的突变ES克隆的生成。