Targeted gene disruption in Cryptococcus neoformans using double-joint PCR with split dominant selectable markers.
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).
Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea.
Min Su
MS
Kim
Seo-Young
SY
Kim
Kwang-Woo
KW
Jung
Yong-Sun
YS
Bahn
D003455:Cryptococcus neoformans / Q000235:genetics*; D017353:Gene Deletion; D005819:Genetic Markers / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods*; D016133:Polymerase Chain Reaction
D005819:Genetic Markers