An efficient strategy for TALEN-mediated genome engineering in Drosophila

Nucleic Acids Res. 2013 Sep;41(17):e163. doi: 10.1093/nar/gkt638. Epub 2013 Jul 22.

Abstract

In reverse genetics, a gene's function is elucidated through targeted modifications in the coding region or associated DNA cis-regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila. We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F1 generation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • DNA-Binding Proteins / chemistry
  • Drosophila melanogaster / genetics*
  • Endodeoxyribonucleases / chemistry
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism*
  • Gene Targeting / methods*
  • Genetic Engineering / methods*
  • Genome, Insect
  • Homologous Recombination
  • Mutagenesis*
  • Plasmids / administration & dosage
  • Protein Structure, Tertiary

Substances

  • DNA-Binding Proteins
  • Endodeoxyribonucleases