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Nature Methods 5, 409–415 (1 May 2008) | doi:10.1038/nmeth.1199

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

Ina Poser , Mihail Sarov , James R A Hutchins , Jean-Karim H|[eacute]|rich|[eacute]| , Yusuke Toyoda , Andrei Pozniakovsky , Daniela Weigl , Anja Nitzsche , Bj|[ouml]|rn 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 White , Steffen Dietzel , Karl Mechtler , Richard Durbin , A Francis Stewart , Jan-Michael Peters , Frank Buchholz & Anthony A Hyman

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.