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Nat Protoc. 2012 Mar 22;7(4):729-48. doi: 10.1038/nprot.2012.018.

Combining competition assays with genetic complementation strategies to dissect mouse embryonic stem cell self-renewal and pluripotency.

Author information

1
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

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.

PMID:
22441292
DOI:
10.1038/nprot.2012.018
[Indexed for MEDLINE]

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