NDLI: The green monster process for the generation of yeast strains carrying multiple gene deletions.

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Year: 2012 Issue: 70

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The green monster process for the generation of yeast strains carrying multiple gene deletions.

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The green monster process for the generation of yeast strains carrying multiple gene deletions.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Suzuki, Yo Stam, Jason Novotny, Mark Yachie, Nozomu Lasken, Roger S. Roth, Frederick P.
Description	Author Affiliation: Suzuki Y ( Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute. ysuzuki@jcvi.org)
Abstract	Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition(1,2). There are examples where many genes need to be deleted to unmask hidden gene functions(3,4). Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative. In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker(5,6,7,8,9,10). However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated. Here we describe the Green Monster method for routinely engineering multiple deletions in yeast(11). In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction. The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci-either Green Monster GMToolkit-a or GMToolkit- at the can1Δ locus (Figure 3). Using strains from the yeast deletion collection(12), GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or -mating-type-specific haploid selection marker(1) and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids. The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/4072
Journal	Journal of Visualized Experiments
Issue Number	70
Language	English
Publisher	MyJove Corp.
Publisher Date	2012-12-15
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Gene Deletion Genes, Fungal Genetic Engineering Saccharomyces Cerevisiae Genetics Gene Knockout Techniques Green Fluorescent Proteins Research Support, N.i.h., Extramural Research Support, Non-u.s. Gov't Research Support, U.s. Gov't, Non-p.h.s. Video-audio Media
Content Type	Text
Resource Type	Article

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