Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe.
We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was > or = 50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe.
Bähler
J
J
Department of Biology, University of North Carolina, Chapel Hill 27599-3280, USA.
Wu
J Q
JQ
Longtine
M S
MS
Shah
N G
NG
McKenzie
A
A
Steever
A B
AB
Wach
A
A
Philippsen
P
P
Pringle
J R
JR
D004352:Drug Resistance, Microbial / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D005819:Genetic Markers; D005839:Gentamicins / Q000494:pharmacology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012568:Schizosaccharomyces / Q000235:genetics*; D014170:Transformation, Genetic
D005819:Genetic Markers; D005839:Gentamicins; C010680:antibiotic G 418