title: Combining competition assays with genetic complementation strategies to dissect mouse embryonic stem cell self-renewal and pluripotency.
abstract: 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.
authors.affiliation: Department of Developmental and Regenerative Biology, The Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, New York, USA. dung-fang.lee@mssm.edu
authors.forename: Dung-Fang
authors.initials: DF
authors.lastname: Lee
authors.forename: Jie
authors.initials: J
authors.lastname: Su
authors.forename: Ana
authors.initials: A
authors.lastname: Sevilla
authors.forename: Julian
authors.initials: J
authors.lastname: Gingold
authors.forename: Christoph
authors.initials: C
authors.lastname: Schaniel
authors.forename: Ihor R
authors.initials: IR
authors.lastname: Lemischka
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D019070:Cell Lineage / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology*; D055785:Gene Knockdown Techniques; D005816:Genetic Complementation Test / Q000379:methods*; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology*; D034622:RNA Interference