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PMID: 18391959 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

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

Nature methods ·Vol. 5 ·No. 5 ·2008-05-00 ·Pages 409-15

Poser I, Sarov M, Hutchins JR, Hériché JK, Toyoda Y, Pozniakovsky A, Weigl D, Nitzsche A, Hegemann B, Bird AW, Pelletier L, Kittler R, Hua S, Naumann R, Augsburg M, Sykora MM, Hofemeister H, Zhang Y, Nasmyth K, White KP, Dietzel S, Mechtler K, Durbin R, Stewart AF, Peters JM, Buchholz F, Hyman AA

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.

MeSH Terms
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,metabolism Protein Array Analysis Protein Binding Protein Transport Proteins/genetics,metabolism Transgenes/genetics
Chemicals
Anti-Bacterial Agents Proteins
Authors & Affiliations
27 authors, click to expand affiliations / ORCID
Poser Ina
Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany.
Sarov Mihail
Hutchins James R A
Hériché Jean-Karim
Toyoda Yusuke
Pozniakovsky Andrei
Weigl Daniela
Nitzsche Anja
Hegemann Björn
Bird Alexander W
Pelletier Laurence
Kittler Ralf
Hua Sujun
Naumann Ronald
Augsburg Martina
Sykora Martina M
Hofemeister Helmut
Zhang Youming
Nasmyth Kim
White Kevin P
Dietzel Steffen
Mechtler Karl
Durbin Richard
Stewart A Francis
Peters Jan-Michael
Buchholz Frank
Hyman Anthony A
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Article Info
Journal
Nature methods
Abbr.
Nat Methods
ISSN
1548-7105
DOI
10.1038/nmeth.1199
Published
2008-05-00
Epub
2008-00-06
Pages
409-15
Language
English
Region
United States
NLM ID
101215604
PMCID
PMC2871289
Subset
IM
Grants
Wellcome Trust · 077192 · United Kingdom
NHGRI NIH HHS · R01 HG004428 · United States
NHGRI NIH HHS · 1R01HG004428-01 · United States
Databases
GEO
GSE10845
Corrections
ErratumIn
-
CommentIn
PMID: 18446155
CommentIn
PMID: 23193563
External Links
PubMed source DOI: 10.1038/nmeth.1199 PMC Full Text View journal details
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