title: BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.
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.
authors.affiliation: Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany.
authors.forename: Ina
authors.initials: I
authors.lastname: Poser
authors.forename: Mihail
authors.initials: M
authors.lastname: Sarov
authors.forename: James R A
authors.initials: JR
authors.lastname: Hutchins
authors.forename: Jean-Karim
authors.initials: JK
authors.lastname: Hériché
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Toyoda
authors.forename: Andrei
authors.initials: A
authors.lastname: Pozniakovsky
authors.forename: Daniela
authors.initials: D
authors.lastname: Weigl
authors.forename: Anja
authors.initials: A
authors.lastname: Nitzsche
authors.forename: Björn
authors.initials: B
authors.lastname: Hegemann
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Bird
authors.forename: Laurence
authors.initials: L
authors.lastname: Pelletier
authors.forename: Ralf
authors.initials: R
authors.lastname: Kittler
authors.forename: Sujun
authors.initials: S
authors.lastname: Hua
authors.forename: Ronald
authors.initials: R
authors.lastname: Naumann
authors.forename: Martina
authors.initials: M
authors.lastname: Augsburg
authors.forename: Martina M
authors.initials: MM
authors.lastname: Sykora
authors.forename: Helmut
authors.initials: H
authors.lastname: Hofemeister
authors.forename: Youming
authors.initials: Y
authors.lastname: Zhang
authors.forename: Kim
authors.initials: K
authors.lastname: Nasmyth
authors.forename: Kevin P
authors.initials: KP
authors.lastname: White
authors.forename: Steffen
authors.initials: S
authors.lastname: Dietzel
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
authors.forename: Richard
authors.initials: R
authors.lastname: Durbin
authors.forename: A Francis
authors.initials: AF
authors.lastname: Stewart
authors.forename: Jan-Michael
authors.initials: JM
authors.lastname: Peters
authors.forename: Frank
authors.initials: F
authors.lastname: Buchholz
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Hyman
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D004351:Drug Resistance; D005786:Gene Expression Regulation; D015723:Gene Library; D005818:Genetic Engineering; D016678:Genome; D023281:Genomics / Q000379:methods*; D008322:Mammals / Q000235:genetics* / Q000378:metabolism*; D040081:Protein Array Analysis; D011485:Protein Binding; D021381:Protein Transport; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D011506:Proteins