Skip to main content
SpringerLink
Log in

Site-specific gene integration in cultured silkworm cells mediated by φC31 integrase

  • Original Paper
  • Published:
Molecular Genetics and Genomics Aims and scope Submit manuscript

Abstract

The integrase from the Streptomyces bacteriophage φC31 carries out efficient recombination between an attP site in the phage genome and an attB site in the host chromosome. In the present study, we have used the φC31 integrase system to mediate site-specific recombination in the cultured silkworm cell line BmN4. A plasmid containing a cDNA encoding DsRed flanked by two φC31 attP sites was co-transfected together with a helper plasmid encoding the φC31 integrase into a cell line in which φC31 attB sites inserted between a baculovirus IE2 promoter, and a polyadenylation signal are present in one chromosome. Seven days after transfection, expression of DsRed was observed in transformed cells. Nucleotide sequence analysis demonstrated that the expected recombination between the attB and attP sites had been precisely carried out by the φC31 integrase. These results indicate that the φC31 site-specific recombination system should be widely applicable for efficient site-specific gene integration into silkworm chromosomes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Andreas S, Schwenk F, Kuter-Luks B, Faust N, Kuhn R (2002) Enhanced efficiency through nuclear localization signal fusion on phage φC31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells. Nucleic Acids Res 30:2299–2306

    Article  PubMed  CAS  Google Scholar 

  • Araki K, Araki M, Yamamura K (1997) Targeted integration of DNA using mutant lox sites in embryonic stem cells. Nucleic Acids Res 25:868–872

    Article  PubMed  CAS  Google Scholar 

  • Argos P, Landy A, Abremski K, Egan JB, Haggard-Ljungquist E, Hoess RH, Kahn ML, Kalionis B, Narayana SVL, Pierson III LS, Sternberg N, Leong JM (1986) The integrase family of site-specific recombinases: regional similarities and global diversity. EMBO J 5:433–440

    PubMed  CAS  Google Scholar 

  • Belteki G, Gertsenstein M, Ow DW, Nagy A. (2003) Site-specific cassette exchange and germline transmission with mouse ES cells expressing φC31 integrase. Nat Biotechnol 21:321–324

    Article  PubMed  CAS  Google Scholar 

  • Dymecki SM (1996) Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice. Proc Natl Acad Sci USA 93:6191–6196

    Article  PubMed  CAS  Google Scholar 

  • Groth AC, Olivares EC, Thyagarajan B, Calos MP (2000) A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci USA 97:5995–6000

    Article  PubMed  CAS  Google Scholar 

  • Groth AC, Fish M, Nusse R, Calos MP (2004) Construction of transgenic Drosophila by using the site-specific integrase from phage φC31. Genetics 166:1775–1782

    Article  PubMed  CAS  Google Scholar 

  • Guzman LM, Belin D, Carson MJ, Becwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130

    PubMed  CAS  Google Scholar 

  • Kalderon D, Roberts BL, Richardson WD, Smith AE (1984) A short amino acid sequence able to specify nuclear location. Cell 39:499–509

    Article  PubMed  CAS  Google Scholar 

  • Koch KS, Aoki T, Wang Y, Atkinson AE, Gleiberman AS, Glebov OK, Leffert HL (2000) Site-specific integration of targeted DNA into animal cell genomes. Gene 249:135–144

    Article  PubMed  CAS  Google Scholar 

  • Kolb AF (2001) Selection-marker-free modification of the murine β-casein gene using a lox2722 site. Anal Biochem 290:260–271

    Article  PubMed  CAS  Google Scholar 

  • Kuhstoss S, Rao RN (1991) Analysis of the integration function of the streptomycete bacteriophage φC31. J Mol Biol 222:897–908

    Article  PubMed  CAS  Google Scholar 

  • Lakso M, Sauer B, Mosinger B Jr, Lee EJ, Manning RW, Yu SH, Mulder KL, Westphal H (1992) Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci USA 89:6232–6236

    Article  PubMed  CAS  Google Scholar 

  • Lee G, Saito I (1998) Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene 216:55–65

    Article  PubMed  CAS  Google Scholar 

  • Lee JM (2003) Characterization and construction of constitutive gene expression system in Bombyx mori. Ph.D. Thesis, Kyushu University

  • Lewandoski M (2001) Conditional control of gene expression in the mouse. Nat Rev Genet 2:743–755

    Article  PubMed  CAS  Google Scholar 

  • Mori H, Yamao M, Nakazawa H, Sugahara Y, Shirai N, Matsubara F, Sumida M, Imamura T (1995) Transovarian transmission of a foreign gene in the silkworm, Bombyx mori, by Autographa californica nuclear polyhedrosis virus. Biotechnology (NY) 13:1005–1007

    Article  CAS  Google Scholar 

  • Nagy A (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26:99–109

    Article  PubMed  CAS  Google Scholar 

  • Olivares EC, Hollis RP, Chalberg TW, Meuse L, Kay MA, Calos MP (2002) Site-specific genomic integration produces therapeutic factor IX levels in mice. Nat Biotechnol 20:1124–1128

    Article  PubMed  CAS  Google Scholar 

  • Orban PC, Chui D, Marth JD (1992) Tissue- and site-specific DNA recombination in transgenic mice. Proc Natl Acad Sci USA 89:6861–6865

    Article  PubMed  CAS  Google Scholar 

  • Pena CEA, Kahlenberg JM, Hatfull GF (1999) Protein-DNA complexes in mycobacteriophage L5 integrative recombination. J Bacteriol 181:454–461

    PubMed  CAS  Google Scholar 

  • Rausch H, Lehmann M (1991) Structural analysis of the actinophage φC31 attachment site. Nucleic Acids Res 19:5187–5189

    Article  PubMed  CAS  Google Scholar 

  • Rodriguez CI, Buchholz F, Galloway J, Sequerra R, Kasper J, Ayala R, Stewart AF, Dymecki SM (2000) High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat Genet 25:139–140

    Article  PubMed  CAS  Google Scholar 

  • Schalke T, Bode J (1994) Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry 33:12746–12751

    Article  PubMed  Google Scholar 

  • Seibler J, Schubeler D, Fiering S, Groudine M, Bode J (1998) DNA cassette exchange in ES cells mediated by Flp recombinase: an efficient strategy for repeated modification of tagged loci by marker-free constructs. Biochemistry 37:6229–6234

    Article  PubMed  CAS  Google Scholar 

  • Seidman CE, Struhl K, Sheen J, Jessen T (1997) Introduction of plasmid DNA into cells. In: Ausubel FA, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current protocols in molecular biology, Wiley, New York, pp 1.8.1–1.8.10

    Google Scholar 

  • Smith-Mungo L, Chan IT, Landy A (1994) Structure of the P22 att site—conservation and divergence in the λmotif of recombinogenic complexes. J Biol Chem 269:20798–20805

    PubMed  CAS  Google Scholar 

  • Stark WM, Boocock MR, Sherratt DJ (1992) Catalysis by site-specific recombinases. Trends Genet 8:432–439

    Article  PubMed  CAS  Google Scholar 

  • Strauss WM (1998) Preparation of genomic DNA from mammalian tissue. In: Ausubel FA, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Curr protocols in molecular biology. Wiley, New York, pp 2.2.1––2.2.3

    Google Scholar 

  • Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC, Couble P (2000) Germline transformation of silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol 18:81–84

    Article  PubMed  CAS  Google Scholar 

  • Thomason LC, Calendar R, Ow DW (2001) Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage φC31 site-specific recombination system. Mol Genet Genomics 265:1031–1038

    Article  PubMed  CAS  Google Scholar 

  • Thorpe HM, Smith MCM (1998) In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci USA 95:5505–5510

    Article  PubMed  CAS  Google Scholar 

  • Thorpe HM, Wilson SE, Smith MCM (2000) Control of directionality in the site-specific recombination system of the Streptomyces phage φC31. Mol Microbiol 38:232–241

    Article  PubMed  CAS  Google Scholar 

  • Thyagarajan B, Olivares EC, Hollis RP, Ginsburg DS, Calos MP (2001) Site-specific genomic integration in mammalian cells mediated by phage φC31 integrase. Mol Cell Biol 21:3926–3934

    Article  PubMed  CAS  Google Scholar 

  • Tomita S, Kanda T, Imanishi S, Tamura T (1999) Yeast FLP recombinase-mediated excision in cultured cells and embryos of the silkworm, Bombyx mori (Lepidoptera: Bombycidae). Appl Entomol Zool (Jpn) 34:371–377

    Google Scholar 

  • Voziyanov Y, Pathania S, Jayaram M (1999) A general model for site-specific recombination by the integrase family recombinases. Nucleic Acids Res 27:930–941

    Article  PubMed  CAS  Google Scholar 

  • Yoshida Y, Hamada H (1997) Adenovirus-mediated inducible gene expression through tetracycline-controllable transactivator with nuclear localization signal. Biochem Biophys Res Commun 230:426–430

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported in part by grants-in-aids (Nos. 17380037 and 17658028) and National Bioresource Project from the Ministry of Education, Science, and Culture of Japan

Author information

Authors and Affiliations

  1. Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki, 812-8581, Fukuoka, Japan

    Gaku Nakayama, Yutaka Kawaguchi, Katsumi Koga & Takahiro Kusakabe

Authors
  1. Gaku Nakayama
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yutaka Kawaguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Katsumi Koga
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Takahiro Kusakabe
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Takahiro Kusakabe.

Additional information

Communicated by G. Georgiev

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nakayama, G., Kawaguchi, Y., Koga, K. et al. Site-specific gene integration in cultured silkworm cells mediated by φC31 integrase. Mol Genet Genomics 275, 1–8 (2006). https://doi.org/10.1007/s00438-005-0026-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-005-0026-3

Keywords

Search

Navigation