绿色怪物承载多基因缺失的酵母菌株的产生过程
Summary
绿色妖怪方法能够快速组装的多个标记的报道基因编码绿色荧光蛋白的缺失。这种方法是基于对驾驶酵母菌株通过各种各样的缺失性和携带更多的缺失的细胞的荧光为基础的浓缩的重复循环。
Abstract
只有当测试是在一个特定的遗传背景或环境条件1,2的突变表型基因缺失往往发现。有这样的例子，许多基因需要被删除，揭露隐藏的功能基因3,4。尽管有潜在的重要发现，主要涉及三个或更多的基因的遗传相互作用的探索。多突变体相互作用的穷举搜索将是不切实际的因缺失可能的组合数量之多。然而，研究选定的几套基因，如套旁系同源具有更大的先验机会共享一个共同的功能，将信息。
酵母中的酿酒酵母（Saccharomyces cerevisiae）中 ，是通过更换通过同源重组的可选择标记基因与基因敲除。由于标记的数目是有限的，方法已被开发用于除去和重用的sam Ë标记5,6,7,8,9,10。然而，依次工程多个突变，使用这些方法是耗时的，因为所需要的时间以产生缺失的数量呈线性变化关系。
在这里，我们描述了常规工程酵母11多个缺失的绿色怪物的方法。在该方法中，绿色荧光蛋白（GFP）报告用于定量标签菌株缺失纳入根据每种菌株中所含的缺失（ 图1）的数量。各种各样的GFP-标记的缺失通过酵母交配和减数分裂反复多轮耦合与流式细胞富集携带更多的这些缺失导致的积累缺失株（ 图2）的菌株。并行执行多个进程，与每个每轮将一个或多个缺失的方法，可以减少应变施工所需要的时间。
内容“>的第一个步骤是，以制备单倍体单突变体被称为'ProMonsters，其中每个执行在一个已删除的位点和”工具箱“位点之一的GFP记者要么绿色妖怪GMToolkit-a或GMToolkit-α在can1Δ位点（ 图3），使用从酵母缺失集合12株，GFP标记的缺失可以方便地产生取代共同KanMX4盒中存在的一个普遍GFP – URA3片段与这些菌株。包含每个GMToolkit：无论是一个 -或α-交配型特定单倍体选择标记1，准确的两个标记，当两个GMToolkits是本，统称允许二倍体选择之一。第二个步骤是进行性循环通过的随机分类并/或减数分裂RECOMB的的缺失位点，通过它可以结合在一个单一的细胞ination，伴随着每一个周期的交配和产孢。
Protocol
1。生成ProMonsters 准备通用GFP替换盒扩增TETO 2-GFP标记和URA3标记从质粒pYOGM012的（氨苄青霉素抗性）使用引物序列： ：GGATCCCCGGGTTAATTAAGGCGCGCCAGATCTGTTTAGCTTGCC CAAGCTCCTCGAGTAATTCG和GGCGTTAGTATCGAATCGACAGCAGTATAGCGACCAGCATTCAC GTACCGGGTAATAACTGATATAAT（粗体地区提供磁带允许的KanMX4有针对性的更换侧翼区的同源性）。应进行的PCR反应Phusion聚合酶，HF缓冲液，?…
Representative Results
当携带4 GFP标记的缺失（ycl033cΔyer042wΔykl069wΔyol118cΔ） 的 a-单倍体菌株杂交的α-单倍体菌株携带四个缺失（ycl033cΔydl242wΔydl227cΔyer042wΔ），有两个缺失（ycl033cΔyer042wΔ）共享由两个菌株，代表结果得到的。培养的YPDA培养基中G418和NAT选择二倍体交配混合物。将得到的二倍体在孢子形成的介质中培养。将孢子分散治疗和超声ZYMOLYASE，，发芽在单倍体选择媒体。然后将…
Discussion
我们开发的绿色怪物的方法，我们关注的不同的GFP更换磁带的可能性之间的重组，从而导致基因组重排。成功地进行了多轮交配和减数分裂的细胞，减轻对这种可能性是我们的选择。细胞重新排列的基因组预计将不太适合交配后的单元格不相同的重排。事实上，我们并没有观察到任何导致基因组不稳定之间的重组GFP箱11。但是，我们不能完全排除这种可能性，所以建议用户测试生成的株重…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国防部高级研究计划局的合同N66001-12-C-4039支持YS，RSL，Alfred P. Sloan基金会的资助和美国国立健康补助R01 HG003224和R21 CA130266 FPRFPR也从加拿大高级研究所的奖学金，由加拿大卓越研究教席计划的支持。
Materials
| Name of the reagent or instrument | Company | Catalogue number | Comments (optional) |
| G418 | Sigma-Aldrich | A1720 | Dissolve in water and filter-sterilize (0.2-μm filter). Stock concentration: 200 mg/ml. Store at 4 °C. |
| ClonNAT (nourseothricin) | WERNER BioAgents | 5001000 | Dissolve in water and filter-sterilize (0.2-μm filter). Stock concentration: 100 mg/ml. Store at 4 °C. |
| Doxycycline | Sigma-Aldrich | D9891 | Dissolve in 50% ethanol and filter-sterilize (0.2-μm filter). Stock concentration: 10 mg/ml. Make fresh every four weeks. Shield from light using aluminum foil and store at 4 °C. |
| Zymolyase | ZymoResearch | E1005 | |
| Difco yeast nitrogen base w/o amino acids | BD | 291940 | |
| Revolver (rotator for tubes) | Labnet | H5600 | |
| Enduro Gel XL electrophoresis unit | Labnet | E0160 | |
| Sonifier 450 | Branson | 101-063-198 | |
| Microtip for Sonifier 450 | Branson | 101-148-062 | |
| FACSAria cell sorter | BD | ||
| MoFlo cell sorter | Beckman-Coulter | ||
| Biomek FX or equivalent robot | Beckman Coulter | Optional. For setting up genotyping PCRs. |
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Tags
Green Monster ProcessYeast StrainsGene DeletionsPhenotypesGenetic BackgroundEnvironmental ConditionMutation TestingHidden Gene FunctionsGenetic InteractionsMulti-mutant InteractionsParalogsSaccharomyces CerevisiaeGene KnockoutSelectable MarkerHomologous RecombinationMarker RemovalGreen Monster MethodGreen Fluorescent Protein (GFP) Reporter
Cite This Article
Suzuki, Y., Stam, J., Novotny, M., Yachie, N., Lasken, R. S., Roth, F. P. The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions. J. Vis. Exp. (70), e4072, doi:10.3791/4072 (2012).