Use of site-specific recombination to regenerate selectable markers.
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.
Cregg
J M
JM
Salk Institute Biotechnology/Industrial Associates, Inc., La Jolla, CA 92037.
Madden
K R
KR
D015139:Blotting, Southern; D002872:Chromosome Deletion; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D005819:Genetic Markers; D010957:Plasmids; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
D004271:DNA, Fungal; D005819:Genetic Markers