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

Nature Protocols volume 7, pages 729–748 (2012)Cite this article

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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.

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Figure 1: Fluorescence-based competition assay and mESC pluripotency characterization.
Figure 2: Genetic complementation system setup and overview of steps involved in screening and expanding rescue clones.
Figure 3: Dynamic knockdown of Nanog impairs mESC pluripotency network.

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Acknowledgements

We sincerely acknowledge N. Ivanova for her original development of the rescue strategy. We thank S. Ghaffari, X. Zhang and Y.S. Ang for sharing their results for use in this protocol. We gratefully acknowledge the members of the Lemischka and Moore laboratories for advice and discussions. We thank G. Daley (Children's Hospital Boston) for Ainv15 ESCs. This work was supported by the US National Institutes of Health grant no. 5R01GM078465 (to I.R.L.), and the Empire State Stem Cell Fund through the New York State Department of Health (NYSTEM) grant nos. C024176 (to I.R.L.) and C024410 (to C.S. and I.R.L.). D.-F.L. is a New York Stem Cell Foundation Stanley and Fiona Druckenmiller Fellow.

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Author notes

  1. Dung-Fang Lee, Jie Su and Ana Sevilla: These authors contributed equally to this work.

Authors and Affiliations

  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, Jie Su, Ana Sevilla, Julian Gingold, Christoph Schaniel & Ihor R Lemischka

  2. Department of Pharmacology and System Therapeutics, Mount Sinai School of Medicine, New York, New York, USA

    Ihor R Lemischka

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  1. Dung-Fang Lee
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Contributions

D.-F.L., J.S. and A.S. designed the experiments, carried out the work and wrote the protocol. J.G. edited and modified the protocol design. C.S. helped direct and coordinate research activity, edited and modified the protocol; I.R.L. directed the study and coordinated research activity.

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Correspondence to Dung-Fang Lee or Ihor R Lemischka.

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Lee, DF., Su, J., Sevilla, A. et al. Combining competition assays with genetic complementation strategies to dissect mouse embryonic stem cell self-renewal and pluripotency. Nat Protoc 7, 729–748 (2012). https://doi.org/10.1038/nprot.2012.018

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