Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection.
Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection.
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通过性别分类和荧光选择的循环淘汰多基因冗余。
DOI：
10.1038/nmeth.1550
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发表时间：
2011-02
期刊：
影响因子：
48
通讯作者：
Roth, Frederick P.
中科院分区：
文献类型：
--
作者：
Suzuki, Yo;St Onge, Robert P.;Mani, Ramamurthy;King, Oliver D.;Heilbut, Adrian;Labunskyy, Vyacheslav M.;Chen, Weidong;Pham, Linda;Zhang, Lan V.;Tong, Amy H. Y.;Nislow, Corey;Giaever, Guri;Gladyshev, Vadim N.;Vidal, Marc;Schow, Peter;Lehar, Joseph;Roth, Frederick P.
可能揭示基因功能的表型可能会被具有重叠功能的基因所掩盖。这种现象在基因家族中最为著名，其中重要的共享功能只有通过使所有家族成员突变才能揭示。在这里，我们描述了“绿色怪物”技术，使许多基因的精确删除。在该方法中，使具有由诱导型绿色荧光蛋白（GFP）报告基因标记的每个缺失的缺失菌株群体经历重复轮的交配、减数分裂和流式细胞术富集。这导致单个细胞内多个缺失基因座的聚集。绿色怪物策略潜在地适用于在具有性别或等位基因分类的替代手段的任何物种中组装其他工程改造。为了证明该技术，我们产生了一个单一的广泛的药物敏感性菌株的酿酒酵母轴承精确删除的所有16个三磷酸腺苷结合盒转运相关的多药耐药性的分支内。
Phenotypes that might otherwise reveal a gene’s function can be obscured by genes with overlapping function. This phenomenon is best-known within gene families, where an important shared function may only be revealed by mutating all family members. Here we describe the ‘Green Monster’ technology enabling the precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein (GFP) reporter gene, is subjected to repeated rounds of mating, meiosis, and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci within single cells. The Green Monster strategy is potentially applicable to assembling other engineered alterations in any species with sex or alternative means of allelic assortment. To demonstrate the technology, we generated a single broadly drug-sensitive strain of Saccharomyces cerevisiae bearing precise deletions of all 16 adenosine triphosphate-binding cassette transporters within clades associated with multi-drug resistance.
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