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

Publikation: Beitrag in FachzeitschriftForschungsartikelBeigetragenBegutachtung

Beitragende

  • Ina Poser - , Max Planck Institute of Molecular Cell Biology and Genetics (Autor:in)
  • Mihail Sarov - , Technische Universität Dresden, Max Planck Institute of Molecular Cell Biology and Genetics (Autor:in)
  • James R A Hutchins - (Autor:in)
  • Jean-Karim Hériché - (Autor:in)
  • Yusuke Toyoda - (Autor:in)
  • Andrei Pozniakovsky - (Autor:in)
  • Daniela Weigl - (Autor:in)
  • Anja Nitzsche - (Autor:in)
  • Björn Hegemann - (Autor:in)
  • Alexander W Bird - (Autor:in)
  • Laurence Pelletier - (Autor:in)
  • Ralf Kittler - (Autor:in)
  • Sujun Hua - (Autor:in)
  • Ronald Naumann - , Max Planck Institute of Molecular Cell Biology and Genetics (Autor:in)
  • Martina Augsburg - (Autor:in)
  • Martina M Sykora - (Autor:in)
  • Helmut Hofemeister - , Professur für Biotechnologische Genomik (Autor:in)
  • Youming Zhang - (Autor:in)
  • Kim Nasmyth - (Autor:in)
  • Kevin P White - (Autor:in)
  • Steffen Dietzel - (Autor:in)
  • Karl Mechtler - (Autor:in)
  • Richard Durbin - (Autor:in)
  • A Francis Stewart - , Professur für Biotechnologische Genomik (Autor:in)
  • Jan-Michael Peters - (Autor:in)
  • Frank Buchholz - , Max Planck Institute of Molecular Cell Biology and Genetics (Autor:in)
  • Anthony A Hyman - (Autor:in)

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.

Details

OriginalspracheEnglisch
Seiten (von - bis)409-15
Seitenumfang7
FachzeitschriftNature Methods
Jahrgang5
Ausgabenummer5
PublikationsstatusVeröffentlicht - Mai 2008
Peer-Review-StatusJa

Externe IDs

Scopus 42949095911
PubMed 18391959
PubMedCentral PMC2871289
ORCID /0000-0002-4754-1707/work/142248061

Schlagworte

Schlagwörter

  • Animals, Anti-Bacterial Agents/pharmacology, Cell Line, Chromosomes, Artificial, Bacterial/genetics, Drug Resistance, Gene Expression Regulation, Gene Library, Genetic Engineering, Genome, Genomics/methods, Mammals/genetics, Protein Array Analysis, Protein Binding, Protein Transport, Proteins/genetics, Transgenes/genetics