Gregan Juraj, Rabitsch Peter K, Rumpf Cornelia, Novatchkova Maria, Schleiffer Alexander, Nasmyth Kim
Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, 1030 Vienna, Austria.
Nat Protoc. 2006;1(5):2457-64. doi: 10.1038/nprot.2006.385.
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.
我们设计了一种最高效的策略，用于在裂殖酵母粟酒裂殖酵母中大规模敲除基因。我们的技术基于敲除构建体，该构建体包含与克隆到携带显性耐药标记的载体中的靶基因同源的区域。大多数步骤以96孔板形式进行，允许一批同时缺失96个基因。基于我们的敲除技术，我们设计了一种策略，用于克隆所有预测的裂殖酵母基因的敲除构建体，该策略以可搜索数据库http://mendel.imp.ac.at/Pombe_deletion/的形式提供。我们在一个筛选中验证了该技术，在该筛选中我们鉴定出了减数分裂期间染色体分离所需的新基因。在这里，我们提供带有详细说明的方案。使用该方案，一个人可以在8天内敲除96个粟酒裂殖酵母基因。
