Recombineering homologous recombination constructs in Drosophila

Arnaldo Carreira-Rosario, Shane Scoggin, Nevine A. Shalaby, Nathan D.avid Williams, P. Robin Hiesinger, Michael Buszczak

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

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.

Original languageEnglish (US)
JournalJournal of Visualized Experiments
Issue number77
DOIs
StatePublished - Jul 13 2013

Keywords

  • Animal model
  • Bioengineering
  • Biomedical engineering
  • DNA
  • Drosophila
  • Drosophila melanogaster
  • Gene
  • Gene targeting
  • Genes
  • Genetic engineering
  • Genetics
  • Genetics (animal and plant)
  • Homologous recombination
  • Issue 77
  • Knock-out
  • Molecular biology
  • PCR
  • Physiology
  • Primers
  • Recombination
  • Recombineering
  • Sequencing

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Fingerprint Dive into the research topics of 'Recombineering homologous recombination constructs in Drosophila'. Together they form a unique fingerprint.

  • Cite this

    Carreira-Rosario, A., Scoggin, S., Shalaby, N. A., Williams, N. D. A., Robin Hiesinger, P., & Buszczak, M. (2013). Recombineering homologous recombination constructs in Drosophila. Journal of Visualized Experiments, (77). https://doi.org/10.3791/50346