Colot Hildur V, Park Gyungsoon, Turner Gloria E, Ringelberg Carol, Crew Christopher M, Litvinkova Liubov, Weiss Richard L, Borkovich Katherine A, Dunlap Jay C
*Department of Genetics, Dartmouth Medical School, HB7400, Hanover, NH 03755.
Department of Plant Pathology, University of California, Riverside, CA 92521; and.
Proc Natl Acad Sci U S A. 2006 Jul 5;103(27):10352-10357. doi: 10.1073/pnas.0601456103. Epub 2006 Jun 26.
The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.
丝状真菌野生型菌株同源重组率低，阻碍了针对这类生物体的高通量基因敲除程序的开发。在本研究中，我们描述了一种快速创建敲除突变体的方法，该方法利用酵母重组克隆、缺乏非同源末端连接DNA修复的粗糙脉孢菌突变菌株、定制编写的软件工具和机器人技术。为了说明我们的方法，我们创建了携带103个编码转录因子的粗糙脉孢菌基因缺失的菌株。对这些菌株在生长以及无性和有性发育过程中的表征揭示了43%的缺失突变体的表型，其中一半以上的菌株存在多种缺陷。总体而言，该方法在这种丝状真菌中实现了>90%的高通量基因破坏效率，有望应用于同源重组频率低的其他真核生物。
