García-Pedrajas María D, Nadal Marina, Denny Timothy, Baeza-Montañez Lourdes, Paz Zahi, Gold Scott E
Estación Experimental "La Mayora", CSIC, Algarrobo-Costa, Málaga, Spain.
Methods Mol Biol. 2010;638:55-76. doi: 10.1007/978-1-60761-611-5_5.
Gene deletion is one of the most powerful tools to study gene function. In the genomics era there is great demand for fast, simple high-throughput methods for gene deletion to study the roles of the large numbers of genes that are being identified. Here we present an approach that speeds up the process of generation of deletion mutants by greatly simplifying the production of gene deletion constructs. With this purpose we have developed a method, which we named DelsGate (Deletion via Gateway), that combines PCR and Gateway cloning technology together with the use of the I-SceI homing endonuclease to generate precise deletion constructs in a very simple, universal and robust manner in just 2 days. DelsGate consists of standard PCR of only the 5' and 3' 1 kb gene flanks directly followed by in vitro Gateway cloning and final generation of the circular deletion construct by in vivo recombination in Escherichia coli. For use in DelsGate we have modified a Gateway cloning vector to include selectable markers for the transformation of Ascomycetes and the Basidiomycete fungus Ustilago maydis. The PCR and transformation steps of DelsGate should be well suited for high-throughput approaches to gene deletion construction in fungal species. We describe here the entire process, from the generation of the deletion construct with DelsGate to the analysis of the fungal transformants to test for gene replacement, with the Basidiomycete fungus Ustilago maydis. Application of DelsGate to other fungal species is also underway. Additionally, we describe how this basic approach can be adapted to other genetic manipulations with minor changes. We specifically describe its application to create unmarked deletions in Ralstonia solanacearum, a Gram-negative phytopathogenic bacterium.
基因缺失是研究基因功能最强大的工具之一。在基因组学时代，对快速、简单的高通量基因缺失方法有巨大需求，以研究大量已鉴定基因的作用。在此，我们提出一种方法，通过极大简化基因缺失构建体的产生过程来加速缺失突变体的生成。为此，我们开发了一种方法，命名为DelsGate（通过Gateway进行缺失），该方法将PCR和Gateway克隆技术与I-SceI归巢内切酶的使用相结合，以非常简单、通用且可靠的方式在短短2天内生成精确的缺失构建体。DelsGate包括仅对5'和3'端1 kb基因侧翼进行标准PCR，随后直接进行体外Gateway克隆，并通过大肠杆菌体内重组最终生成环状缺失构建体。为用于DelsGate，我们对一个Gateway克隆载体进行了修饰，使其包含用于子囊菌和担子菌真菌玉米黑粉菌转化的选择标记。DelsGate的PCR和转化步骤应非常适合真菌物种中基因缺失构建的高通量方法。我们在此描述整个过程，从用DelsGate生成缺失构建体到分析真菌转化体以测试基因替换，以担子菌真菌玉米黑粉菌为例。DelsGate在其他真菌物种中的应用也在进行中。此外，我们描述了如何对这一基本方法进行微小改变以适用于其他基因操作。我们特别描述了其在革兰氏阴性植物病原菌青枯雷尔氏菌中创建无标记缺失的应用。
