Kim Min Su, Kim Seo-Young, Yoon Ja Kyung, Lee Yin-Won, Bahn Yong-Sun
Department of Biotechnology, Center for Fungal Pathogenesis, Yonsei University, Seoul 120-749, Republic of Korea.
Biochem Biophys Res Commun. 2009 Dec 18;390(3):983-8. doi: 10.1016/j.bbrc.2009.10.089. Epub 2009 Oct 21.
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.
通过生物弹道转化和同源重组进行靶向基因破坏是一种广泛用于鉴定和研究新型隐球菌基因功能的方法，若不及时治疗，新型隐球菌会引发致命的真菌性脑膜炎。目前，大多数实验室采用重叠PCR方法来生成带有显性选择标记（如制霉菌素乙酰转移酶（NAT））的基因破坏盒。然而，由于存在多个模板以及最终重叠PCR产物长度较长，传统的重叠PCR方法往往效率低下。在本报告中，我们提出了一种针对新型隐球菌的高效基因破坏方法，称为带有NAT分裂标记的双连接PCR。在此我们证明，使用带有NAT分裂标记的双连接PCR生成的基因破坏盒可成功用于新型隐球菌的靶向基因破坏，其优点是提供了更便捷的基因破坏盒构建方式以及高靶向整合频率。
