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Nucleic Acids Research Advance Access published online on June 3, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp480
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Studying vertebrate topoisomerase 2 function using a conditional knockdown system in DT40 cells

Mark Johnson, Hui Hui Phua, Sophia C. Bennett, Jennifer M. Spence and Christine J. Farr*

Department of Genetics, University of Cambridge, Downing St, Cambridge CB2 3EH, UK

*To whom correspondence should be addressed. Tel: +44 1223 333972; Fax: +44 1223 333992; Email: c_farr{at}mole.bio.cam.ac.uk

Received March 11, 2009. Revised April 28, 2009. Accepted May 18, 2009.

DT40 is a B-cell lymphoma-derived avian cell line widely used to study cell autonomous gene function because of the high rates with which DNA constructs are homologously recombined into its genome. Here, we demonstrate that the power of the DT40 system can be extended yet further through the use of RNA interference as an alternative to gene targeting. We have generated and characterized stable DT40 transfectants in which both topo 2 genes have been in situ tagged using gene targeting, and from which the mRNA of both topoisomerase 2 isoforms can be conditionally depleted through the tetracycline-induced expression of short hairpin RNAs. The cell cycle phenotype of topo 2-depleted DT40 cells has been compared with that previously reported for other vertebrate cells depleted either of topo 2{alpha} through gene targeting, or depleted of both isoforms simultaneously by transient RNAi. In addition, the DT40 knockdown system has been used to explore whether excess catenation arising through topo 2 depletion is sufficient to trigger the G2 catenation (or decatenation) checkpoint, proposed to exist in differentiated vertebrate cells.

Present addresses: Mark Johnson, Molecular Signalling Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge, UK Hui Hui Phua, The Liggins Institute, The University of Auckland, Auckland, New Zealand Sophia Bennett, The Wellcome Trust Centre for Human Genetics, University Of Oxford, Oxford, UK Jennifer Spence, London School of Hygiene and Tropical Medicine, Infectious and Tropical Diseases, Parasite Molecular Biology Unit, Keppel St, London, UK


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