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

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  • A Corrigendum to this article was published on 01 August 2008

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.

NOTE: In the version of this article initially published online, the name of one individual was misspelled in the Acknowledgments. The second sentence of the Acknowledgments paragraph should read, “We thank I. Cheesman for helpful discussions.” The error has been corrected for all versions of the article.

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Figure 1: BAC TransgeneOmics pipeline.
Figure 2: Tagging cassettes and 96-well recombineering pipeline.
Figure 3: Pipeline fidelity and efficiency.
Figure 4: Localization and purification of tagged proteins.
Figure 5: Identification of DNA-binding sites by chromatin immunopurification.
Figure 6: Generation of BAC-transgenic mice using mouse ES cells stably expressing mouse PCNA-LAP.

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Gene Expression Omnibus

Change history

  • 29 April 2008

    In the version of this article initially published online, the name of one individual was misspelled in the Acknowledgments. The second sentence of the Acknowledgments paragraph should read, "We thank I. Cheesman for helpful discussions." The error has been corrected for all versions of the article.

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Acknowledgements

We are grateful to J. Ellenberg and Z. Maliga for stimulating discussions, to K. Neugebauer for the help in establishing the ChIP protocol, and to O. Hudecz, C. Stingl and G. Mitulovic (Institute of Molecular Pathology) and A. Ssykor, M. Biesold, D. Richter, K. Kozak and D. Drechsel (Max Planck Institute for Molecular Cell Biology and Genetics) for excellent assistance. We thank I. Cheesman for helpful discussions. This work has been supported by the 6th Framework Program of the European Union, Integrated Project 'MitoCheck' (LSHG-CT-2004-503464), and by NGFN2 grant SMP-RNAi (01GR0402). Work in the laboratories of J.-M.P. and K.M. is supported by Boehringer Ingelheim, the GenAu Program, the Austrian Research Promotion Agency (FFG), the European Science Foundation and the Austrian Science Fund (FWF) via the EuroDynaProgram. A.F.S. received funding from the 6th Framework Program of the European Union, Integrated Project 'Heroic' (LSHG-CT-2005-018883). K.P.W. is supported by grant 1R01HG004428-01 from the National Human Genome Research Institute of the US National Institutes of Health. R.K. is supported by a long-term fellowship of the Human Frontier Science Program Organization. Y.T. was supported by the Uehara Memorial Foundation.

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Correspondence to A Francis Stewart or Jan-Michael Peters or Frank Buchholz or Anthony A Hyman.

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Competing interests

Y.Z. and A.F.S. are shareholders, and Y.Z. is an employee, of GeneBridges GmbH, which holds the exclusive commercial rights to the Red/ET recombineering methodologies.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1 and 2, Supplementary Methods (PDF 17838 kb)

Supplementary Table 3

Binding Sites for XBP1-S in MCF-7 Cells Identified by Chromatin Immunoprecipitation (for Chromosome 4, 15, 18, and 20). (XLS 315 kb)

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