Combining competition assays with genetic complementation strategies to dissect mouse embryonic stem cell...

Substantial scientific interest has been dedicated recently to the crucial factors that control the pluripotent state of stem cells. To gain a comprehensive understanding of the molecular mechanisms regulating mouse embryonic stem cell (mESC) self-renewal and lineage differentiation, we have developed a robust method for studying the role of a particular gene in these processes. This protocol describes detailed procedures for the design and generation of the complementation rescue system and its application in dissecting the network of pluripotency-associated factors, using mESCs as a model. Specifically, three main procedures are described: (i) screening pluripotency-associated factors by competition assay; (ii) setting up an inducible complementation rescue system; and (iii) dynamically studying the pluripotency network response to target depletion. Completion of the competition assay and complementation rescue system takes 35 and 30 d, respectively, and an additional 16 d to study the dynamic molecular effects of a gene of interest in the pluripotency network.