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Fungal Genomics pp 179–189Cite as

High-Throughput Production of Gene Replacement Mutants in Neurospora crassa

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 722))

Abstract

The model filamentous fungus Neurospora crassa has been the focus of functional genomics studies for the past several years. A high-throughput gene knockout procedure has been developed and used to generate mutants for more than two-thirds of the ∼10,000 annotated N. crassa genes. Yeast recombinational cloning was incorporated as an efficient procedure to produce all knockout cassettes. N. crassa strains with the Δmus-51 or Δmus-52 deletion mutations were used as transformation recipients in order to reduce the incidence of ectopic integration and increase homologous recombination of knockout cassettes into the genome. A 96-well format was used for many steps of the procedure, including fungal transformation, isolation of homokaryons, and verification of mutants. In addition, development of software programs for primer design and restriction enzyme selection facilitated the high-throughput aspects of the overall protocol.

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Acknowledgments

This work was supported by National Institute of General Medical Sciences grant P01 GM068087. We thank John Jones for software design and implementation of the LIMS and Gloria Turner for helpful comments on the manuscript.

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Authors and Affiliations

  1. Department of Plant Pathology and Microbiology, University of California, Riverside, CA, USA

    Katherine A. Borkovich

Authors
  1. Gyungsoon Park
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  2. Hildur V. Colot
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  3. Patrick D. Collopy
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  4. Svetlana Krystofova
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  5. Christopher Crew
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  6. Carol Ringelberg
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  7. Liubov Litvinkova
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  8. Lorena Altamirano
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  9. Liande Li
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  10. Susan Curilla
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  11. Wei Wang
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  12. Norma Gorrochotegui-Escalante
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  13. Jay C. Dunlap
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  14. Katherine A. Borkovich
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Corresponding author

Correspondence to Katherine A. Borkovich .

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Editors and Affiliations

  1. , Dept. of Botany and Plant Pathology, Purdue University, West State Street 915, West Lafayette, 47907, Indiana, USA

    Jin-Rong Xu

  2. Division of Agriculture, Department of Plant Pathology, University of Arkansas, North Campus Drive 495, Fayetteville, 72701, Arkansas, USA

    Burton H. Bluhm

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Park, G. et al. (2011). High-Throughput Production of Gene Replacement Mutants in Neurospora crassa . In: Xu, JR., Bluhm, B. (eds) Fungal Genomics. Methods in Molecular Biology, vol 722. Humana Press. https://doi.org/10.1007/978-1-61779-040-9_13

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  • DOI: https://doi.org/10.1007/978-1-61779-040-9_13

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-039-3

  • Online ISBN: 978-1-61779-040-9

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