Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

An efficient method for gene disruption in Neurospora crassa

B D Aronson1, K M Lindgren, J C Dunlap

  • 1Dartmouth Medical School, Department of Biochemistry, Hanover, NH 03755-3844.

Molecular & General Genetics : MGG
|February 1, 1994
PubMed
Summary

This study presents a new protocol for efficient gene disruption in fungi, even with low homologous recombination rates. The method uses flanking selectable markers and PCR to rapidly identify targeted gene replacements in Neurospora crassa.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Fungal Light Sensing at the Bench and Beyond.

Advances in genetics·2016
Same author

The circadian Per1 and Per2 genes influence alcohol intake, reinforcement, and blood alcohol levels.

Behavioural brain research·2013
Same author

Functional characterization of MAT1-1-specific mating-type genes in the homothallic ascomycete Sordaria macrospora provides new insights into essential and nonessential sexual regulators.

Eukaryotic cell·2010
Same author

A circadian clock in Neurospora: how genes and proteins cooperate to produce a sustained, entrainable, and compensated biological oscillator with a period of about a day.

Cold Spring Harbor symposia on quantitative biology·2008
Same author

Circadian output, input, and intracellular oscillators: insights into the circadian systems of single cells.

Cold Spring Harbor symposia on quantitative biology·2008
Same author

Microarray and real-time PCR analyses reveal mating type-dependent gene expression in a homothallic fungus.

Molecular genetics and genomics : MGG·2006

Area of Science:

  • Molecular Biology
  • Genetics
  • Mycology

Background:

  • Homologous recombination is crucial for gene targeting but can be inefficient in some fungal systems.
  • Low recombination rates necessitate strategies to enrich for or easily screen homologous integration events over ectopic ones.
  • Traditional methods like Southern analysis are insufficient for identifying homologous recombinants in low-efficiency systems.

Purpose of the Study:

  • To develop an efficient protocol for isolating Neurospora crassa strains with null alleles in target genes.
  • To overcome challenges posed by low homologous recombination frequencies in fungal gene manipulation.
  • To provide a method applicable to various fungal systems for gene disruption and reporter gene fusion studies.

Main Methods:

  • Utilized a selectable marker flanking a disrupted plasmid-borne gene copy to enrich for homologous recombination events.

Related Experiment Videos

  • Employed a polymerase chain reaction (PCR) assay for rapid identification of homologous recombinants.
  • Applied the protocol to disrupt the Neurospora ccg-1 gene.

    • Main Results:

    • Achieved a seven-fold enrichment for putative homologous site replacement events.
    • Successfully identified 3 Neurospora ccg-1 gene disruptions out of 129 primary transformants using the PCR assay.
    • Demonstrated the protocol's effectiveness in a system with low homologous recombination rates.

    Conclusions:

    • The developed protocol efficiently isolates fungal strains with targeted gene disruptions, even in low homologous recombination scenarios.
    • This method enhances the identification of homologous recombinants compared to traditional molecular techniques.
    • The protocol is versatile and applicable for generating null mutations and targeting reporter gene fusions in various fungal species.