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

Dung Fang Lee; Jie Su; Ana Sevilla; Julian Gingold; Christoph Schaniel; Ihor R. Lemischka

doi:10.1038/nprot.2012.018

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

Dung Fang Lee, Jie Su, Ana Sevilla, Julian Gingold, Christoph Schaniel, Ihor R. Lemischka

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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.

Original language	English
Pages (from-to)	729-748
Number of pages	20
Journal	Nature Protocols
Volume	7
Issue number	4
DOIs	https://doi.org/10.1038/nprot.2012.018
State	Published - Apr 2012

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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.",

author = "Lee, {Dung Fang} and Jie Su and Ana Sevilla and Julian Gingold and Christoph Schaniel and Lemischka, {Ihor R.}",

note = "Funding Information: acknowleDGMents 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{\textquoteright}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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AU - Schaniel, Christoph

AU - Lemischka, Ihor R.

N1 - Funding Information: acknowleDGMents 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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Combining competition assays with genetic complementation strategies to dissect mouse embryonic stem cell self-renewal and pluripotency

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