Cregg J M, Madden K R
Salk Institute Biotechnology/Industrial Associates, Inc., La Jolla, CA 92037.
Mol Gen Genet. 1989 Oct;219(1-2):320-3. doi: 10.1007/BF00261194.
A method which allows the repeated use of a single selectable marker in DNA transformations was demonstrated. This marker regeneration method employed portions of the Saccharomyces cerevisiae 2 microns circle plasmid: the inverted repeat sequences (FRTs), and the FLP gene whose product, a site-specific recombinase, catalyzes recombination events between FRTs. When FRTs were oriented as direct repeats and integrated into the genome of the yeast Pichia pastoris, FLP-mediated recombination resulted in the efficient and precise deletion of DNA located between the repeats. In the example described, the S. cerevisiae ARG4 gene, placed between a set of FRTs and integrated into Pichia in a prior transformation, was deleted by FLP, thereby regenerating an arginine-requiring phenotype in the P. pastoris strain.
展示了一种在DNA转化中允许重复使用单个选择标记的方法。这种标记再生方法利用了酿酒酵母2微米环状质粒的部分序列：反向重复序列（FRTs），以及FLP基因，其产物是一种位点特异性重组酶，催化FRTs之间的重组事件。当FRTs以正向重复的方式定向并整合到巴斯德毕赤酵母的基因组中时，FLP介导的重组导致重复序列之间的DNA高效且精确地缺失。在所描述的例子中，置于一组FRTs之间并在先前转化中整合到毕赤酵母中的酿酒酵母ARG4基因被FLP删除，从而在巴斯德毕赤酵母菌株中再生出需要精氨酸的表型。
