Carreira-Rosario Arnaldo, Scoggin Shane, Shalaby Nevine A, Williams Nathan David, Hiesinger P Robin, Buszczak Michael
Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, USA.
J Vis Exp. 2013 Jul 13(77):e50346. doi: 10.3791/50346.
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.
用于快速、大规模操纵内源性基因座的技术的持续发展，将拓宽黑腹果蝇作为人类疾病相关研究的遗传模式生物的应用范围。近年来出现了同源重组和重组工程等技术进步。然而，对于大多数基因而言，产生明确的无效突变或标记内源性蛋白质仍然是一项艰巨的工作。在这里，我们描述并展示了使用基于重组工程的克隆方法来生成可用于在体内靶向和操纵内源性基因座的载体的技术。具体而言，我们建立了三种技术的组合：（1）BAC转基因/重组工程，（2）末端外同源重组和（3）Gateway技术，以提供一种强大、高效且灵活的操纵内源性基因组基因座的方法。在本方案中，我们提供了关于如何（1）设计单个载体，（2）如何使用缺口修复将基因组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在这些载体内替换或标记感兴趣的基因的详细步骤。最后，我们还将提供一个关于如何在体内移动这些盒式结构以通过敲入产生基因敲除或标记基因的方案。这些方法可以很容易地并行应用于多个靶点，并提供了一种及时、高效地操纵果蝇基因组的手段。
