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Related Experiment Video

Updated: Jun 19, 2026

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

An efficient gene-disruption method in Cryptococcus neoformans by double-joint PCR with NAT-split markers.

Min Su Kim1, Seo-Young Kim, Ja Kyung Yoon

  • 1Department of Biotechnology, Center for Fungal Pathogenesis, Yonsei University, Seoul 120-749, Republic of Korea.

Biochemical and Biophysical Research Communications
|October 27, 2009
PubMed
Summary

We developed an efficient double-joint PCR method using NAT-split markers for targeted gene disruption in Cryptococcus neoformans. This approach simplifies cassette construction and achieves high integration frequency, improving fungal gene function studies.

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Last Updated: Jun 19, 2026

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Area of Science:

  • Mycology
  • Molecular Biology
  • Genetics

Background:

  • Targeted gene disruption is crucial for understanding gene function in Cryptococcus neoformans, a pathogen causing fatal fungal meningitis.
  • Current methods using overlap-PCR for generating gene-disruption cassettes with markers like nourseothricin acetyltransferase (NAT) are often inefficient due to multiple templates and long PCR products.

Purpose of the Study:

  • To develop and demonstrate an efficient gene-disruption method for Cryptococcus neoformans.
  • To improve the construction of gene-disruption cassettes and increase targeted-integration frequency.

Main Methods:

  • Development of a novel double-joint PCR technique utilizing NAT-split markers.
  • Application of the generated gene-disruption cassette for targeted gene disruption in Cryptococcus neoformans via biolistic transformation and homologous recombination.

Main Results:

  • The double-joint PCR method with NAT-split markers provides a more convenient way to construct gene-disruption cassettes.
  • This new method achieves a high targeted-integration frequency in Cryptococcus neoformans.

Conclusions:

  • The double-joint PCR with NAT-split markers is an efficient and advantageous method for targeted gene disruption in Cryptococcus neoformans.
  • This technique enhances the study of fungal gene function, potentially leading to better therapeutic strategies for fungal meningitis.