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Recombineering Homologous Recombination Constructs in Drosophila.

Carrerira-Rosario, A., Scoggin, S., Shalaby, N.A., Williams, N.D., Hiesinger, P.R. and Buszczak, M. – 2013

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.

Title
Recombineering Homologous Recombination Constructs in Drosophila.
Author
Carrerira-Rosario, A., Scoggin, S., Shalaby, N.A., Williams, N.D., Hiesinger, P.R. and Buszczak, M.
Publisher
MYJoVE Corporation
Keywords
Genetics, Issue 77, Bioengineering, Molecular Biology, Biomedical Engineering, Physiology, Drosophila melanogaster, genetics (animal and plant), Recombineering, Drosophila, Homologous Recombination, Knock-out, recombination, genetic engineering, gene targeting, gene, genes, DNA, PCR, Primers, sequencing, animal model
Date
2013-07-13
Identifier
doi: 10.3791/50346
Appeared in
J Vis Exp. (77): 50346
Language
eng
Type
Text
Rights
© 2013, Journal of Visualized Experiments