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A recombineering pipeline for functional genomics applied to Caenorhabditis elegans

Nature Methods volume 3, pages 839–844 (2006)Cite this article

Abstract

We present a new concept in DNA engineering based on a pipeline of serial recombineering steps in liquid culture. This approach is fast, straightforward and facilitates simultaneous processing of multiple samples in parallel. We validated the approach by generating green fluorescent protein (GFP)-tagged transgenes from Caenorhabditis briggsae genomic clones in a multistep pipeline that takes only 4 d. The transgenes were engineered with minimal disturbance to the natural genomic context so that the correct level and pattern of expression will be secured after transgenesis. An example transgene for the C. briggsae ortholog of lin-59 was used for ballistic transformation in Caenorhabditis elegans. We show that the cross-species transgene is correctly expressed and rescues RNA interference (RNAi)-mediated knockdown of the endogenous C. elegans gene. The strategy that we describe adapts the power of recombineering in Escherichia coli for fluent DNA engineering to a format that can be directly scaled up for genomic projects.

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Figure 1: Recombineering pipeline.
Figure 2: Recombineering toolkit.
Figure 3: Parallel processing in liquid culture.
Figure 4: Expression pattern of the cblin-59::egfp transgene.
Figure 5: Rescue of an RNAi-induced phenotype by a cross-species third allele.

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Acknowledgements

We thank M. Valvano for the pSCRhaB2 plasmid; the Caenorhabditis Genetics Center for worm strains; and E. Entchev, L. Pelletier, J. Fu and members of the Genomics Department of TU Dresden for discussions. This work was supported by the European Union and Freistaat Sachsen, by Europäischer Fonds für regionale Entwicklung (EFRE) funding (project code 181) through the Sächsisches Staatsministerium für Wissenschaft und Kunst (SMWK) and Technische Universität Dresden (TUD), and by the Bundesminiterium fuer Bildung und Forschung (BMBF) Proteomics Program.

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Authors and Affiliations

  1. Genomics Department, Dresden University of Technology, BioInnovations Centre, Tatzberg 47, Dresden, 01307, Germany

    Mihail Sarov, Susan Schneider, Assen Roguev & A Francis Stewart

  2. Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, 01307, Germany

    Andrei Pozniakovski, Susanne Ernst & A Anthony Hyman

  3. Gene Bridges GmbH, Tatzberg 47, Dresden, 01307, Germany

    Youming Zhang

Authors
  1. Mihail Sarov
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  2. Susan Schneider
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  8. A Francis Stewart
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Contributions

M.S. performed the experiments in Figures 1,3–5; M.S., S.S. and A.P. generated the reagents used in Figure 2; A.R. generated the BAC clone map; S.E. performed the bombardment experiments; Y.Z. provided reagents and essential expertise in Red/ET recombineering; M.S, A.A.H. and A.F.S. conceived the project and prepared the manuscript.

Corresponding author

Correspondence to A Francis Stewart.

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Competing interests

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

Supplementary information

Supplementary Fig. 1

BAC mapping. (PDF 53 kb)

Supplementary Fig. 2

Controlled expression of recombinases from pRedFlp. (PDF 65 kb)

Supplementary Fig. 3

Restriction digest analysis of the 10 tagged transgenes. (PDF 293 kb)

Supplementary Fig. 4

Recombineering outcome with individual clones. (PDF 167 kb)

Supplementary Table 1

Bombardment experiments. (PDF 15 kb)

Supplementary Data (DOC 1105 kb)

Supplementary Methods (PDF 134 kb)

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Sarov, M., Schneider, S., Pozniakovski, A. et al. A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nat Methods 3, 839–844 (2006). https://doi.org/10.1038/nmeth933

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