Molecular Methods for Evolutionary Genetics pp 445-458

Part of the Methods in Molecular Biology book series (MIMB, volume 772) | Cite as

Recombination-Mediated Genetic Engineering of Large Genomic DNA Transgenes

  • Radoslaw Kamil Ejsmont
  • Peter Ahlfeld
  • Andrei Pozniakovsky
  • A. Francis Stewart
  • Pavel Tomancak
  • Mihail Sarov
Protocol

Abstract

Faithful gene activity reporters are a useful tool for evo-devo studies enabling selective introduction of specific loci between species and assaying the activity of large gene regulatory sequences. The use of large genomic constructs such as BACs and fosmids provides an efficient platform for exploration of gene function under endogenous regulatory control. Despite their large size they can be easily engineered using in vivo homologous recombination in Escherichia coli (recombineering). We have previously demonstrated that the efficiency and fidelity of recombineering are sufficient to allow high-throughput transgene engineering in liquid culture, and have successfully applied this approach in several model systems. Here, we present a detailed protocol for recombineering of BAC/fosmid transgenes for expression of fluorescent or affinity tagged proteins in Drosophila under endogenous in vivo regulatory control. The tag coding sequence is seamlessly recombineered into the genomic region contained in the BAC/fosmid clone, which is then integrated into the fly genome using ϕC31 recombination. This protocol can be easily adapted to other recombineering projects.

Key words

Red/ET Recombineering FlyFos 

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Radoslaw Kamil Ejsmont
    • 1
  • Peter Ahlfeld
    • 1
  • Andrei Pozniakovsky
    • 1
  • A. Francis Stewart
    • 1
  • Pavel Tomancak
    • 1
  • Mihail Sarov
    • 1
  1. 1.Max Planck Institute of Molecular Cell Biology and GeneticsDresdenGermany

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