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

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.