High-throughput knockout screen in fission yeast.
We have designed the most efficient strategy to knock out genes in fission yeast Schizosaccharomyces pombe on a large scale. Our technique is based on knockout constructs that contain regions homologous to the target gene cloned into vectors carrying dominant drug-resistance markers. Most of the steps are carried out in a 96-well format, allowing simultaneous deletion of 96 genes in one batch. Based on our knockout technique, we designed a strategy for cloning knockout constructs for all predicted fission yeast genes, which is available in a form of a searchable database http://mendel.imp.ac.at/Pombe_deletion/. We validated this technique in a screen where we identified novel genes required for chromosome segregation during meiosis. Here, we present our protocol with detailed instructions. Using this protocol, one person can knock out 96 S. pombe genes in 8 days.
Gregan
Juraj
J
Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, 1030 Vienna, Austria. juraj.gregan@univie.ac.at
Rabitsch
Peter K
PK
Rumpf
Cornelia
C
Novatchkova
Maria
M
Schleiffer
Alexander
A
Nasmyth
Kim
K
D025141:Drug Resistance, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers; D005822:Genetic Vectors; D010957:Plasmids; D012440:Saccharomyces / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D005819:Genetic Markers