Nature Methods
- 5, 409 - 415 (2008)
Published online: 6 April 2008; Corrected online: 29 April 2008 | doi:10.1038/nmeth.1199
There is a Corrigendum (August 2008) associated with this Article.
BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammalsIna Poser1, 10, Mihail Sarov1, 2, 10, James R A Hutchins3, Jean-Karim Hériché4, Yusuke Toyoda1, Andrei Pozniakovsky1, Daniela Weigl7, Anja Nitzsche1, Björn Hegemann3, Alexander W Bird1, Laurence Pelletier1, 5, Ralf Kittler1, 6, Sujun Hua6, Ronald Naumann1, Martina Augsburg1, Martina M Sykora3, Helmut Hofemeister2, Youming Zhang8, Kim Nasmyth9, Kevin P White6, Steffen Dietzel7, Karl Mechtler3, Richard Durbin4, A Francis Stewart2, Jan-Michael Peters3, Frank Buchholz1 & Anthony A Hyman11
Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany. 2
Technische Universitaet Dresden, Genomics, BioInnovations Centre, Am Tatzberg 47, D-01307 Dresden, Germany. 3
Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria. 4
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1HH, UK. 5
Samuel Lunenfeld Research Institute, 600 University Avenue, Room 1075A, Toronto, Ontario M5G 1X5, Canada. 6
Institute for Genomics and Systems Biology and Department of Human Genetics, University of Chicago, 431 CLSB, 920 E. 58th Street, Chicago, Illinois 60637, USA. 7
Ludwig-Maximilians-Universität München, Germany. 8
Gene Bridges GmbH, BioInnovationsZentrum, Tatzberg 47/49, Dresden, Germany. 9
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. 10
These authors contributed equally to this work.
Correspondence should be addressed to A Francis Stewart francis.stewart@biotec.tu-dresden.de or Jan-Michael Peters Jan-Michael.Peters@imp.univie.ac.at or Frank Buchholz Buchholz@mpi-cbg.de or Anthony A Hyman hyman@mpi-cbg.de The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.NOTE: In the version of this article initially published online, the name of one individual was misspelled in the Acknowledgments. The second sentence of the Acknowledgments paragraph should read, "We thank I. Cheesman for helpful discussions." The error has been corrected for all versions of the article.
MORE ARTICLES LIKE THIS These links to content published by NPG are automatically generated.
|
