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Deconstructing Survivin: comprehensive genetic analysis of Survivin function by conditional knockout in a vertebrate cell line

Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK

Correspondence to S. Ruchaud: s.ruchaud{at}ed.ac.uk or William C. Earnshaw: Bill.Earnshaw{at}ed.ac.uk

Survivin is a key cellular protein thought to function in apoptotic regulation, mitotic progression, or possibly both. In this study, we describe the isolation of two conditional knockouts of the survivin gene in chicken DT40 cells. DT40 cells lacking Survivin die in interphase after failing to complete cytokinesis. However, these cells show normal sensitivity to the chemotherapeutic agent etoposide. Expression of Survivin mutants against a null background to reassess the role of several key residues reveals that DT40 cells can grow normally if their sole Survivin is missing a widely studied cyclin-dependent kinase phosphorylation site or sites reportedly essential for binding to Smac or aurora B. Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth. As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.

Z. Yue's present address is Dept. of Cell and Developmental Biology, University of Dundee, Dundee DD1 5EH, Scotland, UK.

A. Carvalho and R. Gassmann's present address is Dept. of Cellular and Molecular Medicine, Ludwig Institute for Cancer Research, La Jolla, CA 92093.

F. Lai's present address is Centre for Genomic Regulation, Barcelona 08003, Spain.

E. Gudmundsdottir's present address is NimbleGen Systems of Iceland LLC, 113 Reykjavik, Iceland.

C.G. Morrison's present address is Dept. of Biochemistry, National University of Ireland Galway, Galway, Ireland.

Abbreviations used in this paper: BIR, baculovirus IAP repeat; CPC, chromosomal passenger protein complex; hSurvivin, human Survivin; IAP, inhibitor of apoptosis protein; INCENP, inner centromere protein; NES, nuclear export sequence; tTA, tetracycline transactivator.

© 2008 Yue et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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