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High-throughput knockout screen in fission yeast

Nature Protocols volume 1, pages 2457–2464 (2006)Cite this article

Abstract

We have designed the most efficient strategy to knock out genes in fission yeast Schizosaccharomyces pombe on a large scale. Our technique is based on knockout constructs that contain regions homologous to the target gene cloned into vectors carrying dominant drug-resistance markers. Most of the steps are carried out in a 96-well format, allowing simultaneous deletion of 96 genes in one batch. Based on our knockout technique, we designed a strategy for cloning knockout constructs for all predicted fission yeast genes, which is available in a form of a searchable database http://mendel.imp.ac.at/Pombe_deletion/. We validated this technique in a screen where we identified novel genes required for chromosome segregation during meiosis. Here, we present our protocol with detailed instructions. Using this protocol, one person can knock out 96 S. pombe genes in 8 days.

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Figure 1: Flowchart of the knockout strategy.
Figure 2: Maps of cloning vectors pCloneNat1 and pCloneHyg1.
Figure 3: Dilution of primers.
Figure 4: Amplification of homology regions by PCR.
Figure 5: Pooling of PCR-amplified homology regions—preparation for cloning.
Figure 6: Comparison of ligated homology regions with the gel preview at http://mendel.imp.ac.at/Pombe_deletion/.
Figure 7: E. coli transformants ordered in 96-well plates.
Figure 8: PCR checking of yeast transformants.

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Acknowledgements

This work was supported by Boehringer Ingelheim and partly by Austrian Industrial Research Promotion Fund (FFF) and Austrian Science Fund (FWF) grants. We thank Georg Dietzl and Barry Dickson (IMP, Vienna) for help with setting up the cloning protocol and Stephen Kearsey (University of Oxford, UK) for helpful suggestions and reagents.

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Author notes

  1. Juraj Gregan and Peter K Rabitsch: These authors contributed equally to this work.

Authors and Affiliations

  1. Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, Vienna, 1030, Austria

    Juraj Gregan, Peter K Rabitsch, Maria Novatchkova, Alexander Schleiffer & Kim Nasmyth

  2. Department of Chromosome Biology, Laboratories, University of Vienna, Dr Bohr-Gasse 1, Vienna, 1030, Austria

    Juraj Gregan & Cornelia Rumpf

  3. Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK

    Kim Nasmyth

Authors
  1. Juraj Gregan
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  2. Peter K Rabitsch
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  4. Maria Novatchkova
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Correspondence to Juraj Gregan.

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Gregan, J., Rabitsch, P., Rumpf, C. et al. High-throughput knockout screen in fission yeast. Nat Protoc 1, 2457–2464 (2006). https://doi.org/10.1038/nprot.2006.385

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