High-efficiency gene targeting in Schizosaccharomyces pombe using a modular, PCR-based approach with long tracts of flanking homology.
Bähler et al.(1998) recently described a PCR-based system for the deletion, tagging and overexpression of endogenous genes in the fission yeast Schizosaccharomyces pombe. A small set of PCR primers can be used to generate gene-targeting substrates from each of several modules that differ in the selectable marker (ura4(+) or kanMX6), the presence or absence of specific epitope tags (HA, Myc, GST or GFP), the position in which the epitopes will be inserted (C- or N-terminal), and the presence or absence of a regulatable promoter (the nmt1 promoter). This is a straightforward and powerful system: nine different genes were C-terminal tagged at an average efficiency of 73%, using primers producing only 60-81 bp of homology. In contrast, when studying three transcriptionally-silent genes (rec8(+), rec10(+) and rec11(+)) we obtained an average homologous integration efficiency of 4% for 12 targeting constructs when using primers that contained 80 bp of homology. By using a PCR-based increase in the amount of flanking homology to >/=250 bp, we obtained homologous integration efficiencies of up to 100%. Thus, loci of S. pombe that are refractory to gene targeting when using short tracts of homology can be readily modified by increasing the extent of homology flanking the targeting modules. This straightforward and cost-effective approach might therefore be the one of choice for the modification of S. pombe loci in general and of targeting-refractory loci in particular.
Krawchuk
M D
MD
Department of Biochemistry, Vanderbilt University School of Medicine, 621 Light Hall, Nashville, TN 37232-0146, USA.
Wahls
W P
WP
D002874:Chromosome Mapping; D018390:Gene Targeting; D016133:Polymerase Chain Reaction; D012568:Schizosaccharomyces / Q000235:genetics*