Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe
Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe
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作者：
Hentges, P;Van Driessche, B;Carr, AM
在裂殖酵母中构建多个突变菌株的简便性受到可用遗传标记数量的限制。在这里，我们描述了三种新的用于聚合酶链式反应介导的基因破坏的盒，它们可以与常用的分裂酵母标记结合使用来进行多个基因缺失。其中，NatMX6、hphMX6和bleMX6标记分别对纳豆菌素(NAT)、潮霉素B和平阳霉素产生耐药性。这些盒由外源序列组成，以提高靶点整合的频率，并具有类似于常用的pFA6a-kanMX6模块化质粒系统的结构。这使得现有菌株中的kanMX6标记可以与三个新的盒中的任何一个进行简单的交换。或者，可以使用为模块化kanMX6盒设计的寡核苷酸引物来制造用于基因破坏的转化PCR片段。我们说明了使用新型抗生素盒与现有遗传标记相结合，构建具有六个独立基因中断的突变株。版权所有(C)2005 John Wiley&Sons，Ltd.
The ease of construction of multiple mutant strains in Schizosaccharomyces pombe is limited by the number of available genetic markers. We describe here three new cassettes for PCR-mediated gene disruption that can be used in combination with commonly used fission yeast markers to make multiple gene deletions. The natMX6, hphMX6 and bleMX6 markers give rise to resistance towards the antibiotics nourseothricin (NAT), hygromycin B and phleomycin, respectively. The cassettes are composed of exogenous sequences to increase the frequency of integration at targeted loci, and have a structure similar to the commonly used pFA6a-kanMX6 modular plasmid system. This allows a simple exchange of the kanMX6 marker in existing strains with any of the three new cassettes. Alternatively, oligonucleotide primers designed for the modular kanMX6 cassettes can be used to make the transforming PCR fragments for gene disruption. We illustrate the construction of a mutant strain with six independent gene disruptions, using the novel antibiotic cassettes in combination with existing genetic markers. Copyright (c) 2005 John Wiley & Sons, Ltd.