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Research Article

A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors

Hildur V. Colot, Gyungsoon Park, Gloria E. Turner, Carol Ringelberg, Christopher M. Crew, Liubov Litvinkova, Richard L. Weiss, Katherine A. Borkovich, and Jay C. Dunlap
  1. *Department of Genetics, Dartmouth Medical School, HB7400, Hanover, NH 03755;
  2. ‡Department of Plant Pathology, University of California, Riverside, CA 92521; and
  3. §Department of Chemistry and Biochemistry, 405 Hilgard Avenue, University of California, Los Angeles, CA 90095

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PNAS July 5, 2006 103 (27) 10352-10357; first published June 26, 2006; https://doi.org/10.1073/pnas.0601456103
Hildur V. Colot
*Department of Genetics, Dartmouth Medical School, HB7400, Hanover, NH 03755;
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Gyungsoon Park
‡Department of Plant Pathology, University of California, Riverside, CA 92521; and
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Gloria E. Turner
§Department of Chemistry and Biochemistry, 405 Hilgard Avenue, University of California, Los Angeles, CA 90095
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Carol Ringelberg
*Department of Genetics, Dartmouth Medical School, HB7400, Hanover, NH 03755;
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Christopher M. Crew
‡Department of Plant Pathology, University of California, Riverside, CA 92521; and
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Liubov Litvinkova
‡Department of Plant Pathology, University of California, Riverside, CA 92521; and
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Richard L. Weiss
§Department of Chemistry and Biochemistry, 405 Hilgard Avenue, University of California, Los Angeles, CA 90095
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Katherine A. Borkovich
‡Department of Plant Pathology, University of California, Riverside, CA 92521; and
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Jay C. Dunlap
*Department of Genetics, Dartmouth Medical School, HB7400, Hanover, NH 03755;
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  • For correspondence: jay.c.dunlap@dartmouth.edu
  1. Edited by David D. Perkins, Stanford University, Stanford, CA, and approved March 29, 2006

  2. ↵†H.V.C. and G.P. contributed equally to this work. (received for review February 21, 2006)

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Abstract

The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.

  • filamentous fungi
  • functional genomics
  • knockouts
  • recombinational cloning

Footnotes

  • ↵‖To whom correspondence should be addressed. E-mail: jay.c.dunlap{at}dartmouth.edu
  • Author contributions: H.V.C., G.P., G.E.T., K.A.B., and J.C.D. designed research; H.V.C., G.P., G.E.T., C.R., C.M.C., and L.L. performed research; H.V.C., G.P., G.E.T., C.R., C.M.C., R.L.W., K.A.B., and J.C.D. contributed new reagents/analytic tools; H.V.C., G.P., G.E.T., and K.A.B. analyzed data; and H.V.C., G.P., G.E.T., K.A.B., and J.C.D. wrote the paper.

  • Conflict of interest statement: No conflicts declared.

  • This paper was submitted directly (Track II) to the PNAS office.

  • Abbreviation:
    VM,
    Vogel’s minimal medium.
    • Received February 21, 2006.
    • © 2006 by The National Academy of Sciences of the USA

    Freely available online through the PNAS open access option.

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    A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors
    Hildur V. Colot, Gyungsoon Park, Gloria E. Turner, Carol Ringelberg, Christopher M. Crew, Liubov Litvinkova, Richard L. Weiss, Katherine A. Borkovich, Jay C. Dunlap
    Proceedings of the National Academy of Sciences Jul 2006, 103 (27) 10352-10357; DOI: 10.1073/pnas.0601456103

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    A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors
    Hildur V. Colot, Gyungsoon Park, Gloria E. Turner, Carol Ringelberg, Christopher M. Crew, Liubov Litvinkova, Richard L. Weiss, Katherine A. Borkovich, Jay C. Dunlap
    Proceedings of the National Academy of Sciences Jul 2006, 103 (27) 10352-10357; DOI: 10.1073/pnas.0601456103
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