Targeted gene disruption in cryptococcus neoformans using double-joint PCR with split dominant selectable markers

Min Su Kim, Seo Young Kim, Kwang Woo Jung, Yong Sun Bahn

Research output: Chapter in Book/Report/Conference proceedingChapter

14 Citations (Scopus)

Abstract

Cryptococcus neoformans causes fatal meningoencephalitis if not timely treated. Targeted gene disruption for functional analysis of a gene involves overlap PCR for the production of gene disruption cassettes carrying dominant selectable markers, followed by biolistic transformation. However, the conventional overlap PCR method between two flanking regions of the target gene and selectable marker is often inefficient due to the long length of the PCR product and the presence of multiple templates. Here we describe double-joint PCR with split dominant selectable markers for the more convenient generation of a gene-disruption cassette in C. neoformans with high targeted integration frequency (Kim et al., Biochem. Biophys. Res. Commun 390(3):983-988, 2009).

Original languageEnglish
Title of host publicationHost-Fungus Interactions
Subtitle of host publicationMethods and Protocols
EditorsAlexandra Brand, Donna MacCallum
Pages67-84
Number of pages18
DOIs
Publication statusPublished - 2012 Mar 16

Publication series

NameMethods in Molecular Biology
Volume845
ISSN (Print)1064-3745

Fingerprint

Cryptococcus neoformans
Joints
Polymerase Chain Reaction
Genes
Biolistics
Meningoencephalitis

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

Cite this

Kim, M. S., Kim, S. Y., Jung, K. W., & Bahn, Y. S. (2012). Targeted gene disruption in cryptococcus neoformans using double-joint PCR with split dominant selectable markers. In A. Brand, & D. MacCallum (Eds.), Host-Fungus Interactions: Methods and Protocols (pp. 67-84). (Methods in Molecular Biology; Vol. 845). https://doi.org/10.1007/978-1-61779-539-8_5
Kim, Min Su ; Kim, Seo Young ; Jung, Kwang Woo ; Bahn, Yong Sun. / Targeted gene disruption in cryptococcus neoformans using double-joint PCR with split dominant selectable markers. Host-Fungus Interactions: Methods and Protocols. editor / Alexandra Brand ; Donna MacCallum. 2012. pp. 67-84 (Methods in Molecular Biology).
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Kim, MS, Kim, SY, Jung, KW & Bahn, YS 2012, Targeted gene disruption in cryptococcus neoformans using double-joint PCR with split dominant selectable markers. in A Brand & D MacCallum (eds), Host-Fungus Interactions: Methods and Protocols. Methods in Molecular Biology, vol. 845, pp. 67-84. https://doi.org/10.1007/978-1-61779-539-8_5

Targeted gene disruption in cryptococcus neoformans using double-joint PCR with split dominant selectable markers. / Kim, Min Su; Kim, Seo Young; Jung, Kwang Woo; Bahn, Yong Sun.

Host-Fungus Interactions: Methods and Protocols. ed. / Alexandra Brand; Donna MacCallum. 2012. p. 67-84 (Methods in Molecular Biology; Vol. 845).

Research output: Chapter in Book/Report/Conference proceedingChapter

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AB - Cryptococcus neoformans causes fatal meningoencephalitis if not timely treated. Targeted gene disruption for functional analysis of a gene involves overlap PCR for the production of gene disruption cassettes carrying dominant selectable markers, followed by biolistic transformation. However, the conventional overlap PCR method between two flanking regions of the target gene and selectable marker is often inefficient due to the long length of the PCR product and the presence of multiple templates. Here we describe double-joint PCR with split dominant selectable markers for the more convenient generation of a gene-disruption cassette in C. neoformans with high targeted integration frequency (Kim et al., Biochem. Biophys. Res. Commun 390(3):983-988, 2009).

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Kim MS, Kim SY, Jung KW, Bahn YS. Targeted gene disruption in cryptococcus neoformans using double-joint PCR with split dominant selectable markers. In Brand A, MacCallum D, editors, Host-Fungus Interactions: Methods and Protocols. 2012. p. 67-84. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-61779-539-8_5