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High-throughput knockout screen in fission yeast.

ABSTRACT: 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.

SUBMITTER: Gregan J

PROVIDER: S-EPMC2957175 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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High-throughput knockout screen in fission yeast.

Gregan Juraj J Rabitsch Peter K PK Rumpf Cornelia C Novatchkova Maria M Schleiffer Alexander A Nasmyth Kim K

Nature protocols 20060101 5

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 predict ...[more]

PMID: 17406492

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