BAC TransgeneOmics: A high-throughput method for exploration of protein function in mammals

Ina Poser, Mihail Sarov, James R A Hutchins, Jean Karim Hériché, Yusuke Toyoda, Andrei Pozniakovsky, Daniela Weigl, Anja Nitzsche, Björn Hegemann, Alexander W. Bird, Laurence Pelletier, Ralf Kittler, Sujun Hua, Ronald Naumann, Martina Augsburg, Martina M. Sykora, Helmut Hofemeister, Youming Zhang, Kim Nasmyth, Kevin P. WhiteSteffen Dietzel, Karl Mechtler, Richard Durbin, A. Francis Stewart, Jan Michael Peters, Frank Buchholz, Anthony A. Hyman

Research output: Contribution to journalArticlepeer-review

418 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)409-415
Number of pages7
JournalNature methods
Volume5
Issue number5
DOIs
StatePublished - May 2008

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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