High-throughput production of gene replacement mutants in Neurospora crassa

Methods Mol Biol. 2011;722:179-89. doi: 10.1007/978-1-61779-040-9_13.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Blotting, Southern
  • Cloning, Molecular / methods*
  • Electroporation
  • Fungal Proteins / genetics*
  • Fungal Proteins / metabolism
  • Gene Deletion*
  • Genes, Fungal
  • Genome, Fungal
  • Genomics / methods
  • Neurospora crassa / genetics*
  • Recombination, Genetic*
  • Transformation, Genetic

Substances

  • Fungal Proteins