Brown Simon D, Lorenz Alexander
Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.
Bio Protoc. 2016 Dec 20;6(24). doi: 10.21769/BioProtoc.2075.
The ability to utilize different selectable markers for tagging or mutating multiple genes in is hampered by the historical use of only two selectable markers, and ; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells often requires strain construction from scratch. To overcome this problem we and other groups have created transformation cassettes with flanking homologies to and which enable an efficient and time-saving way to exchange markers in existing mutated or tagged fission yeast strains. Here, we present a protocol for single-step marker switching by lithium acetate transformation in fission yeast, . In the following we describe how to swap the marker to a or marker, which provide resistance against the antibiotics G418, nourseothricin (clonNAT) or hygromycin B, respectively. We also detail how to exchange any of the markers for nutritional markers, such as and .
由于历史上仅使用两种选择标记（潮霉素B和新霉素磷酸转移酶基因；后者赋予对抗生素G418（遗传霉素）的抗性），在裂殖酵母中利用不同选择标记对多个基因进行标记或诱变的能力受到了阻碍。最近已经描述了更多的标记，但将这些标记引入酵母细胞通常需要从头构建菌株。为了克服这个问题，我们和其他研究小组创建了与潮霉素B和新霉素磷酸转移酶基因具有侧翼同源性的转化盒，这使得在现有的突变或标记的裂殖酵母菌株中交换标记成为一种高效且省时的方法。在这里，我们介绍了一种通过乙酸锂转化在裂殖酵母中进行单步标记切换的方案。在接下来的内容中，我们将描述如何将潮霉素B标记换成新霉素磷酸转移酶基因或营养缺陷型标记，它们分别提供对抗生素G418、制霉菌素（clonNAT）或潮霉素B的抗性。我们还详细说明了如何将任何一个营养缺陷型标记换成营养标记，如尿嘧啶缺陷型和色氨酸缺陷型。
