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Related Experiment Video

Updated: Jun 26, 2025

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans
07:32

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Published on: March 19, 2015

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Biolistic Transformation of Cryptococcus neoformans.

Dena L Toffaletti1, Jennifer L Tenor1, John R Perfect2

  • 1Duke University School of Medicine, Department of Medicine, Division of Infectious Diseases, Durham, NC, USA.

Methods in Molecular Biology (Clifton, N.J.)
|May 17, 2024
PubMed
Summary

Biolistic transformation is a key method for genetically modifying Cryptococcus neoformans yeast. This protocol details gene deletion and restoration using biolistic particle delivery and homologous recombination.

Keywords:
Biolistic transformationCre-recombinaseCryptococcus neoformansDrug selection cassetteGene deletionGene reconstitutionGeneticinHomologous recombinationNourseothricinloxP

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Yeast Genetics

Background:

  • Biolistic transformation is a widely used technique for genetic manipulation in various organisms, including the pathogenic yeast Cryptococcus neoformans.
  • Genetic modification of C. neoformans is essential for understanding its biology and developing antifungal strategies.
  • Traditional methods often require selectable markers, which can be limiting for sequential genetic alterations.

Purpose of the Study:

  • To provide a detailed working protocol for gene deletion and restoration in Cryptococcus neoformans using biolistic transformation.
  • To highlight the utility of biolistic transformation as a robust tool for constructing genetically modified yeast strains.
  • To discuss the application of selectable markers and recyclable systems for efficient genetic engineering.

Main Methods:

  • Utilizing a biolistic particle system to deliver DNA-coated gold beads into Cryptococcus neoformans cells via a helium shock wave.
  • Employing homologous recombination with large overlapping DNA fragments to facilitate targeted gene insertion or deletion.
  • Implementing dominant selectable markers such as nourseothricin or Geneticin for strain selection.
  • Discussing the use of recyclable marker systems like Cre-loxP or CRISPR for generating multiple gene deletions.

Main Results:

  • Successful gene deletion and restoration in Cryptococcus neoformans strains using the described biolistic transformation protocol.
  • Demonstration of homologous recombination efficiency enhanced by using large DNA fragments.
  • Validation of dominant selectable markers for identifying transformed yeast cells.
  • Confirmation that biolistic transformation remains a viable and effective method for yeast genetic engineering.

Conclusions:

  • Biolistic transformation is a reliable method for genetic manipulation in Cryptococcus neoformans, enabling targeted gene alterations.
  • The protocol facilitates the construction of custom genetically modified yeast strains for research purposes.
  • Advancements in marker systems offer enhanced capabilities for complex genetic modifications in C. neoformans.