Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
Nat Methods. 2011 Feb;8(2):159-64. doi: 10.1038/nmeth.1550. Epub 2011 Jan 9.
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, in which an important shared function may only be revealed by mutating all family members. Here we describe the 'green monster' technology that enables precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein reporter gene, is subjected to repeated rounds of mating, meiosis and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci in 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 test the technology, we generated a single broadly drug-sensitive strain of Saccharomyces cerevisiae bearing precise deletions of all 16 ATP-binding cassette transporters within clades associated with multidrug resistance.
表型可能会掩盖具有重叠功能的基因的功能。这种现象在基因家族中最为明显，只有通过突变所有家族成员才能揭示出一个重要的共享功能。在这里，我们描述了“绿妖怪”技术，该技术可实现许多基因的精确缺失。在该方法中，具有可诱导绿色荧光蛋白报告基因标记的缺失株群体，经过反复的交配、减数分裂和流式细胞术富集，导致多个缺失基因座在单个细胞中聚集。绿妖怪策略在具有性别或等位基因分离替代方式的任何物种中都可用于组装其他工程修饰。为了测试该技术，我们生成了一个单一的、广泛的药物敏感的酿酒酵母菌株，该菌株具有与多药耐药相关的所有 16 个 ATP 结合盒转运蛋白的精确缺失。
