Recombination-Mediated Genetic Engineering of Large Genomic DNA Transgenes

  • Radoslaw Kamil Ejsmont
  • Peter Ahlfeld
  • Andrei Pozniakovsky
  • A. Francis Stewart
  • Pavel Tomancak
  • Mihail SarovEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 772)


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 


  1. 1.
    Zhang Y, Buchholz F, Muyrers JP et al (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20:123–128PubMedCrossRefGoogle Scholar
  2. 2.
    Murphy KC (1998) Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180:2063–2071PubMedGoogle Scholar
  3. 3.
    Muyrers JP, Zhang Y, Testa G et al (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res 27:1555–1557PubMedCrossRefGoogle Scholar
  4. 4.
    Testa G, Zhang Y, Vintersten K et al (2003) Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles. Nat Biotechnol 21:443–447PubMedCrossRefGoogle Scholar
  5. 5.
    Wang J, Sarov M, Rientjes J et al (2006) An improved recombineering approach by adding RecA to lambda Red recombination. Mol Biotechnol 32:43–53PubMedCrossRefGoogle Scholar
  6. 6.
    Sarov M, Schneider S, Pozniakovski A et al (2006) A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nat Methods 3:839–844PubMedCrossRefGoogle Scholar
  7. 7.
    Poser I, Sarov M, Hutchins JRA et al (2008) BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat Methods 5:409–415PubMedCrossRefGoogle Scholar
  8. 8.
    Ejsmont RK, Sarov M, Winkler S et al (2009) A toolkit for high-throughput, cross-species gene engineering in Drosophila. Nat Methods 6:435–437PubMedCrossRefGoogle Scholar
  9. 9.
    Groth AC, Fish M, Nusse R et al (2004) Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166:1775–1782PubMedCrossRefGoogle Scholar
  10. 10.
    Sharma RC, Schimke RT (1996) Preparation of electrocompetent E coli using salt-free growth medium. Biotechniques 20:42–44PubMedGoogle Scholar
  11. 11.
    Maresca M, Erler A, Fu J et al (2010) Single-stranded heteroduplex intermediates in lambda Red homologous recombination. BMC Mol Biol 11:54PubMedCrossRefGoogle Scholar
  12. 12.
    Bischof J, Maeda RK, Hediger M et al (2007) An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci USA 104:3312–3317PubMedCrossRefGoogle Scholar

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
    Email author
  1. 1.Max Planck Institute of Molecular Cell Biology and GeneticsDresdenGermany

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