期刊
CELL REPORTS
卷 35, 期 3, 页码 -出版社
CELL PRESS
DOI: 10.1016/j.celrep.2021.109014
关键词
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类别
资金
- Ministry of Health, Labour and Welfare of Japan
- Japan Agency for Medical Research and Development (AMED) [JP20wm0225002, JP20he0822006, JP20fk0108264, JP20he0822008, JP20wm0225003, JP20fk0108267, JP19fk0108113, JP20wm0125010]
- Japan Society for the Promotion of Science (KAKENHI) [JP19K24679]
- Japan Science and Technology Agency (JST) (Moonshot RD) [JPMJMS2025]
- JSPS research fellowship for young scientists [19J12641]
- Grants-in-Aid for Scientific Research [19J12641] Funding Source: KAKEN
A PCR-based, bacterium-free method has been developed to generate SARS-CoV-2 infectious clones, allowing for a better understanding of the virus through the construction of reporter and mutant viruses.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). Although multiple mutations have been observed in SARS-CoV-2, functional analysis of each mutation of SARS-CoV-2 has been limited by the lack of convenient mutagenesis methods. In this study, we establish a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. The construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (similar to 5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. This reverse genetics system may potentially advance further understanding of SARS-CoV-2.
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