Recombineering Homologous Recombination Constructs in Drosophila
Recombineering Homologous Recombination Constructs in Drosophila
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DOI：
10.3791/50346
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发表时间：
2013-07-01
影响因子：
1.2
通讯作者：
Buszczak, Michael
中科院分区：
文献类型：
--
作者：
Carreira-Rosario, Arnaldo;Scoggin, Shane;Buszczak, Michael
快速、大规模操作内源基因位点技术的持续发展将扩大果蝇作为人类疾病相关研究的遗传模型生物的用途。近年来，同源重组和重组工程等技术取得了进步。然而，对于大多数基因来说，产生明确的无效突变或标记内源蛋白仍然是一项艰巨的工作。在这里，我们描述并演示了使用基于重组工程的克隆方法来生成可用于体内靶向和操纵内源基因座的载体的技术。具体来说，我们建立了三种技术的组合：（1）BAC转基因/重组工程，（2）末端同源重组和（3）Gateway技术，为操纵内源基因组位点提供稳健、高效和灵活的方法。在此协议中，我们提供了有关如何（1）设计单个载体，（2）如何使用间隙修复将基因组 DNA 的大片段克隆到同源重组载体中，以及（3）如何使用第二轮重组工程替换或标记这些载体中感兴趣的基因的分步详细信息。最后，我们还将提供如何在体内动员这些盒以产生敲除或通过敲入标记基因的方案。这些方法可以轻松地并行应用于多个目标，并提供及时有效地操纵果蝇基因组的手段。
The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/ recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.