烟曲霉高通量基因置换研究

Can Zhao, Marcin G. Fraczek, Lauren Dineen, Ressa Lebedinec, Juliane Macheleidt, Thorsten Heinekamp, Daniela Delneri, Paul Bowyer, Axel A. Brakhage, Michael Bromley
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引用次数: 31

摘要

烟曲霉是一种人类病原体,是侵袭性和慢性曲霉病的主要病原,每年导致数十万人死亡。很少有抗真菌药物可用于治疗烟曲霉引起的感染,并且对我们所拥有的抗真菌药物正在产生耐药性。我们对驱动致病性和耐药性的分子机制的理解受到缺乏大型突变体收集的阻碍,这限制了我们进行功能基因组学分析的能力。在这里,我们提出了一种高通量基因敲除方法,该方法结合了高度可重复的融合PCR方法,可以通过多孔格式转换程序生成基因替换盒。这个过程可以在5天内由一个人产生96个无效突变体,每个突变体的成本不到18英镑(24美元),我们的实验室正在使用这个过程来生成烟熏螨的条形码全基因组敲除文库。©2019作者。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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High-Throughput Gene Replacement in Aspergillus fumigatus

Aspergillus fumigatus is a human pathogen and the principal etiologic agent of invasive and chronic aspergillosis leading to several hundreds of thousands of deaths every year. Very few antifungals are available to treat infections caused by A. fumigatus, and resistance is developing to those we have. Our understanding of the molecular mechanisms that drive pathogenicity and drug resistance have been hampered by the lack of large mutant collections, which limits our ability to perform functional genomics analysis. Here we present a high-throughput gene knockout method that combines a highly reproducible fusion PCR method to enable generation of gene replacement cassettes with a multiwell format transformation procedure. This process can be used to generate 96 null mutants within 5 days by a single person at a cost of less than £18 ($24) per mutant and is being employed in our laboratory to generate a barcoded genome-wide knockout library in A. fumigatus. © 2019 The Authors.

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Current Protocols in Microbiology
Current Protocols in Microbiology Immunology and Microbiology-Parasitology
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期刊介绍: Current Protocols in Microbiology provides detailed, step-by-step instructions for analyzing bacteria, animal and plant viruses, fungi, protozoans and other microbes. It offers updated coverage of emerging technologies and concepts, such as biofilms, quorum sensing and quantitative PCR, as well as proteomic and genomic methods. It is the first comprehensive source of high-quality microbiology protocols that reflects and incorporates the new mandates and capabilities of this robust and rapidly evolving discipline.
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