TY - JOUR
T1 - An efficient gene-disruption method in Cryptococcus neoformans by double-joint PCR with NAT-split markers
AU - Kim, Min Su
AU - Kim, Seo Young
AU - Yoon, Ja Kyung
AU - Lee, Yin Won
AU - Bahn, Yong Sun
PY - 2009/12/18
Y1 - 2009/12/18
N2 - Targeted gene disruption via biolistic transformation and homologous recombination is a method widely used to identify and investigate the function of genes in Cryptococcus neoformans that causes fatal fungal meningitis if not timely treated. Currently, most laboratories employ the overlap-PCR method to generate a gene-disruption cassette with dominant selectable markers, such as nourseothricin acetyltransferase (NAT). However, the conventional overlap-PCR method is often found to be inefficient because of the presence of multiple templates and of the long length of the final overlap-PCR products. In this report, we suggested an efficient gene-disruption method for C. neoformans, termed a double-joint PCR with NAT-split markers. Here we demonstrated that the gene-disruption cassette generated using double-joint PCR with NAT-split markers can be used successfully for targeted C. neoformans gene disruption with the advantages of providing a more convenient construction of gene-disruption cassettes and high targeted-integration frequency.
AB - Targeted gene disruption via biolistic transformation and homologous recombination is a method widely used to identify and investigate the function of genes in Cryptococcus neoformans that causes fatal fungal meningitis if not timely treated. Currently, most laboratories employ the overlap-PCR method to generate a gene-disruption cassette with dominant selectable markers, such as nourseothricin acetyltransferase (NAT). However, the conventional overlap-PCR method is often found to be inefficient because of the presence of multiple templates and of the long length of the final overlap-PCR products. In this report, we suggested an efficient gene-disruption method for C. neoformans, termed a double-joint PCR with NAT-split markers. Here we demonstrated that the gene-disruption cassette generated using double-joint PCR with NAT-split markers can be used successfully for targeted C. neoformans gene disruption with the advantages of providing a more convenient construction of gene-disruption cassettes and high targeted-integration frequency.
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U2 - 10.1016/j.bbrc.2009.10.089
DO - 10.1016/j.bbrc.2009.10.089
M3 - Article
C2 - 19852932
AN - SCOPUS:70449703215
SN - 0006-291X
VL - 390
SP - 983
EP - 988
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 3
ER -