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 μm 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.
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Communicated by C.P. Hollenberg
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Cregg, J.M., Madden, K.R. Use of site-specific recombination to regenerate selectable markers. Molec. Gen. Genet. 219, 320–323 (1989). https://doi.org/10.1007/BF00261194
- 2 μm plasmid
- FLP gene
- Pichia pastoris
- Methylotrophic yeast