Advertisement

Recombineering Linear BACs

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1227)

Abstract

Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.

Key words

Linear BAC Recombineering E. coli Genomic DNA Chromosome Phage N15 Plasmid 

Notes

Acknowledgements

The authors are grateful to Nikolai Ravin for providing N15 reagents and to Sek-Chuen Chow for support and encouragement. Q.C. is grateful to Monash University Malaysia for a HDR Scholarship. This work was partly funded by a Fundamental Research Grant Scheme FRGS/1/2011/ST/MUSM/02/2 from the Ministry of Higher Education Malaysia to K.N.

References

  1. 1.
    Kazuki Y, Hoshiya H, Takiguchi M et al (2011) Refined human artificial chromosome vectors for gene therapy and animal transgenesis. Gene Ther 18:384–393PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Kakeda M, Nagata K, Osawa K et al (2011) A new chromosome 14-based human artificial chromosome (HAC) vector system for efficient transgene expression in human primary cells. Biochem Biophys Res Commun 415:439–444PubMedCrossRefGoogle Scholar
  3. 3.
    Ferdows MS, Barbour AG (1989) Megabase-sized linear DNA in the bacterium Borrelia burgdorferi, the Lyme disease agent. Proc Natl Acad Sci U S A 86:5969–5973PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Allardet-Servent A, Michaux-Charachon S, Jumas-Bilak E et al (1993) Presence of one linear and one circular chromosome in the Agrobacterium tumefaciens C58 genome. J Bacteriol 175:7869–7874PubMedPubMedCentralGoogle Scholar
  5. 5.
    Lezhava A, Mizukami T, Kajitani T et al (1995) Physical map of the linear chromosome of Streptomyces griseus. J Bacteriol 177:6492–6498PubMedPubMedCentralGoogle Scholar
  6. 6.
    Hertwig S (2007) Linear plasmids and prophages in gram-negative bacteria. In: Meinhardt F, Klassen R (eds) Microbial linear plasmids. Springer, BerlinGoogle Scholar
  7. 7.
    Deneke J, Ziegelin G, Lurz R et al (2000) The protelomerase of temperate Escherichia coli phage N15 has cleaving-joining activity. Proc Natl Acad Sci U S A 97:7721–7726PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Ravin NV, Strakhova TS, Kuprianov VV (2001) The protelomerase of the phage-plasmid N15 is responsible for its maintenance in linear form. J Mol Biol 312:899–906PubMedCrossRefGoogle Scholar
  9. 9.
    Ooi YS, Warburton PE, Ravin NV et al (2008) Recombineering linear DNA that replicate stably in E. coli. Plasmid 59:63–71PubMedCrossRefGoogle Scholar
  10. 10.
    Narayanan K, Chen Q (2011) Bacterial artificial chromosome mutagenesis using recombineering. J Biomed Biotechnol 2011:971296PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Narayanan K, Williamson R, Zhang Y et al (1999) Efficient and precise engineering of a 200 kb beta-globin human/bacterial artificial chromosome in E. coli DH10B using an inducible homologous recombination system. Gene Ther 6:442–447PubMedCrossRefGoogle Scholar
  12. 12.
    Narayanan K, Sim EU, Ravin NV et al (2009) Recombination between linear double-stranded DNA substrates in vivo. Anal Biochem 387:139–141PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Kaufman RM, Pham CT, Ley TJ (1999) Transgenic analysis of a 100-kb human beta-globin cluster-containing DNA fragment propagated as a bacterial artificial chromosome. Blood 94:3178–3184PubMedGoogle Scholar
  14. 14.
    Guzman LM, Belin D, Carson MJ et al (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130PubMedPubMedCentralGoogle Scholar
  15. 15.
    Grant SG, Jessee J, Bloom FR et al (1990) Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A 87:4645–4649PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Narayanan K, Warburton PE (2003) DNA modification and functional delivery into human cells using Escherichia coli DH10B. Nucleic Acids Res 31:e51PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Osoegawa K, Woon PY, Zhao B et al (1998) An improved approach for construction of bacterial artificial chromosome libraries. Genomics 52:1–8PubMedCrossRefGoogle Scholar
  18. 18.
    Narayanan K (2008) Intact recombineering of highly repetitive DNA requires reduced induction of recombination enzymes and improved host viability. Anal Biochem 375:394–396PubMedCrossRefGoogle Scholar
  19. 19.
    Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580PubMedCrossRefGoogle Scholar
  20. 20.
    Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.School of ScienceMonash University MalaysiaBandar SunwayMalaysia
  2. 2.Monash University MalaysiaBandar SunwayMalaysia
  3. 3.Icahn School of Medicine at Mount SinaiNew YorkUSA

Personalised recommendations