Skip to main content

Site-Specific Recombination Using PhiC31 Integrase

  • Chapter
  • First Online:
Site-directed insertion of transgenes

Part of the book series: Topics in Current Genetics ((TCG,volume 23))

Abstract

The integrase from phage φC31 of Streptomyces bacteria is an attractive recombinase for use in generating transgenic organisms and developing gene and cell therapeutic strategies. In nature, φC31 integrase mediates integration by interacting with specific sites in the phage and bacterial genomes. When applied to eukaryotes, φC31 integrase provides efficient unidirectional recombination between its own attB and attP sites or between an attB site on an incoming plasmid and a native genomic pseudo attP site that resembles attP. To date, the φC31 system has been used to generate stable transgenic organisms from multiple species, including plants, insects, and vertebrates. The features of the φC31 system also make it particularly amenable to therapeutic strategies. φC31 integrase has been used in potential therapies for numerous genetic diseases including hemophila, muscular dystrophy, and skin disorders. Additionally, the φC31 system has recently been used to modify human embryonic stem cells and to generate induced pluripotent stem cells. The φC31 system can also be combined with other recombinases to create advanced genome engineering strategies. In the future, the use of φC31 integrase may facilitate the development of new gene and cell therapies, as well as the generation of targeted transgenic organisms.

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

Access this chapter

Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  • Allen BG, Weeks DL (2005) Transgenic Xenopus laevis embryos can be generated using φC31 integrase. Nat Methods 2(12):975–979

    Article  CAS  Google Scholar 

  • Allen BG, Weeks DL (2006) Using φC31 integrase to make transgenic Xenopus laevis Embryos. Nat Protoc 1(3):1248–1257

    Article  CAS  Google Scholar 

  • Allen BG, Weeks DL (2009) Bacteriophage φC31 integrase mediated transgenesis in Xenopus laevis for protein expression at endogenous levels. In: Carrol DJ (ed) Microinjection: methods and applications, vol 518. Humana, New York

    Google Scholar 

  • Andreas S, Schwenk F, Küter-Luks B, Faust N, Kühn 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(11):2299–2306

    Article  CAS  Google Scholar 

  • Aneja MK, Imker R, Rudolph C (2007) Phage phiC31 integrase-mediated genomic integration and long-term gene expression in the lung after nonviral gene delivery. J Gene Med 9:967–975

    Article  CAS  Google Scholar 

  • Aneja MK, Geiger J, Imker R, Üzgün S, Kormann M, Hasenpusch G, Maucksch C, Rudolph C (2009) Optimization of Streptomyces bacteriophage φC31 integrase system to prevent post integrative gene silencing in pulmonary type II cells. Exp Mol Med 41(12):919–934

    Article  CAS  Google Scholar 

  • Bertoni C, Jarrahian S, Wheeler TM, Li Y, Olivares EC, Calos MP, Rando TA (2006) Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration. Proc Natl Acad Sci 103(2):419–424

    Article  CAS  Google Scholar 

  • Brown WRA, Lee NCO, Xu Z, Smith MCM (2011) Serine recombinases as tools for genome engineering. Methods 53(4):372–379

    Article  CAS  Google Scholar 

  • Calos MP (2006) The φC31 integrase system for gene therapy. Curr Gene Ther 6:633–645

    Article  CAS  Google Scholar 

  • Chalberg TW, Genise HL, Vollrath D, Calos MP (2005) φC31 integrase confers genomic integration and long-term transgene expression in rat retina. Invest Opthalmol Vis Sci 46(6):2140–2146

    Article  Google Scholar 

  • Chalberg TW, Portlock JL, Olivares EC, Thyagarajan B, Kirby PJ, Hillman RT, Hoelters J, Calos MP (2006) Integration specificity of phage φC31 integrase in the human genome. J Mol Biol 357:28–48

    Article  CAS  Google Scholar 

  • Chavez CL, Calos MP (2011) Therapeutic applications of the phiC31 integrase system. Curr Gene Ther 11(5):375–381

    Article  CAS  Google Scholar 

  • Chavez CL, Keravala A, Woodard LE, Hillman RT, Stowe TR, Chu JN, Calos MP (2010) Kinetics and longevity of φC31 integrase in mouse liver and cultured cells. Hum Gene Ther 21:1287–1297

    Article  CAS  Google Scholar 

  • Dafhnis-Calas F, Xu Z, Haines S, Malla SK, Smith MCM, Brown WRA (2005) Iterative in vivo assembly of large and complex transgenes by combining the activities of φC31 integrase and Cre recombinase. Nucleic Acids Res 33(22):e189

    Article  Google Scholar 

  • Ehrhart A, Engler JA, Xu H, Cherry AM, Kay MA (2006) Molecular analysis of chromosomal rearragements in mammalian cells after phiC31-mediated integration. Hum Gene Ther 17:1077–1094

    Article  Google Scholar 

  • Fish MP, Groth AC, Calos MP, Nusse R (2007) Creating transgenic Drosophila by microinjecting the site-specific φC31 integrase mRNA and a transgene-containing donor plasmid. Nat Protoc 2(10):2325–2331

    Article  CAS  Google Scholar 

  • Gao G, McMahon C, Chen J, Rong YS (2008) A powerful method combining homologous recombination and site-specific recombination for targeted mutagenesis in Drosophila. Proc Natl Acad Sci 105(37):13999–14004

    Article  CAS  Google Scholar 

  • Gils M, Marillonnet S, Werner S, Grützner R, Giritch A, Engler C, Schachschneider R, Klimyuk V, Gleba Y (2008) A novel hybrid seed system for plants. Plant Biotechnol J 6:226–235

    Article  CAS  Google Scholar 

  • Grindley NDF, Whiteson KL, Rice PA (2006) Mechanisms of site-specific recombination. Annu Rev Biochem 75:567–605

    Article  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 97(11):5995–6000

    Article  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  CAS  Google Scholar 

  • Held PK, Olivares EC, Aguilar CP, Finegold M, Calos MP, Grompe M (2005) In vivo correction of murine hereditary tyrosinemia type I by φC31 integrase-mediated gene delivery. Mol Ther 11(3):399–408

    Article  CAS  Google Scholar 

  • Hollis RP, Stoll SM, Sclimenti CR, Lin J, Chen-Tasi Y, Calos MP (2003) Phage integrases for the construction and manipulation of transgenic mammals. Reprod Biol Endocrinol 1:79

    Article  Google Scholar 

  • Inoue K, Sone T, Oneyama C, Nishiumi F, Kishine H, Sasaki Y, Andoh T, Okada M, Chesnut JD, Imamoto F (2009) A versatile nonviral vector system for tetracycline-dependent one-step conditional induction of transgene expression. Gene Ther 16:1383–1394

    Article  CAS  Google Scholar 

  • Ishikawa Y, Tanaka N, Uchiyama T, Kumaki S, Tsuchiya S, Kugoh H, Oshimura M, Calos MP, Sugamura K (2006) Phage φC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines. J Gene Med 8:646–653

    Article  CAS  Google Scholar 

  • Karow M, Chavez CL, Farruggio AP, Geisinger JM, Keravala A, Jung WE, Lan F, Wu JC, Chen-Tsai Y, Calos MP (2011) Site-specific recombinase strategy to create iPS cells efficiently with plasmid DNA. Stem Cells 29(11):1692–1704

    Article  Google Scholar 

  • Kempe K, Rubtsova M, Berger C, Kumlehn J, Schollmeier C, Gils M (2010) Transgene excision from wheat chromosomes by phage φC31 integrase. Plant Mol Biol 72:673–687

    Article  CAS  Google Scholar 

  • Keravala A, Groth AC, Jarrahian S, Thyagarajan B, Hoyt JJ, Kirby P, Calos MP (2006a) ‘A diversity of serine phage integrases mediate site-specific recombination in mammalian cells. Mol Genet Genomics 276:135–146

    Article  CAS  Google Scholar 

  • Keravala A, Portlock JL, Nash JA, Vitrant DG, Robbins PD, Calos MP (2006b) PhiC31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints. J Gene Med 8:1008–1017

    Article  CAS  Google Scholar 

  • Keravala A, Ormerod BK, Palmer TD, Calos MP (2008) Long-term transgene expression in mouse neural progenitor cells modified with φC31 integrase. J Neurosci Methods 173:299–305

    Article  CAS  Google Scholar 

  • Keravala A, Chavez CL, Hu G, Woodard LE, Monahan PE, Calos MP (2011) Long-term phenotypic correction in factor IX knockout mice by using phiC31 integrase-mediated gene therapy. Gene Ther 18:842–848

    Article  CAS  Google Scholar 

  • Kontarakis Z, Pavlopoulos A, Kiupakis A, Konstantinides N, Douris V, Averof M (2011) A versatile strategy for gene trapping and trap conversion in emerging model organisms. Development 138:2625–2630

    Article  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  CAS  Google Scholar 

  • Labbé GMC, Nimmo DD, Alphey L (2010) piggybac- and phiC31-mediated genetic transformation of the Asian tiger mosquito, Aedes albopictus (Skuse). PLoS Negl Trop Dis 4(8):e788

    Article  Google Scholar 

  • Leighton PA, Van de Lavior M-C, Diamond JH, Xia C, Etches RJ (2008) Genetic modification of primordial germ cells by gene trapping, gene targeting, and φC31 integrase. Mol Reprod Dev 75:1163–1175

    Article  CAS  Google Scholar 

  • Lieu PT, Machleidt T, Thyagarajan B, Fontes A, Frey E, Fuerstenau-Sharp M, Thompson DV, Swamilingiah GM, Derebail SS, Piper D, Chesnut JD (2009) Generation of site-specific retargeting platform cell lines for drug discovery using phiC31 and R4 integrases. J Biomol Screen 14:1207–1215

    Article  CAS  Google Scholar 

  • Lister JA (2010) Transgene excision in zebrafish using the phiC31 integrase. Genesis 48:137–143

    Article  CAS  Google Scholar 

  • Liu J, Jeppesen I, Nielsen K, Jensen TG (2006) PhiC31 integrase induces chromosomal aberrations in primary human fibroblasts. Gene Ther 13:1188–1190

    Article  CAS  Google Scholar 

  • Liu J, Skjørringe T, Gjetting T, Jensen TG (2009a) PhiC31 integrase induces a DNA damage response and chromosomal rearrangements in human adult fibroblasts. BMC Biotechnol 9:31–38

    Article  Google Scholar 

  • Liu Y, Thyagarajan B, Lakshmipathy U, Xue H, Lieu P, Fontes A, MacArthur CC, Scheyhing K, Rao MS, Chesnut JD (2009b) Generation of platform human embryonic stem cell lines that allow efficient targeting at a predetermined genomic location. Stem Cells Dev 18(10):1459–1471

    Article  CAS  Google Scholar 

  • Lu J, Maddison LA, Chen W (2011) PhiC31 integrase induces efficient site-specific excision in zebrafish. Transgenic Res 20(1):183–189

    Article  CAS  Google Scholar 

  • Lutz KA, Corneille S, Azhagiri AK, Svab Z, Maliga P (2004) A novel approach to plastid transformation utilizes the phiC31 phage integrase. Plant J 37:906–913

    Article  CAS  Google Scholar 

  • Ma Q-W, Sheng H-Q, Yan J-B, Cheng S, Huang Y, Chen-Tsai Y, Ren Z-R, Huang S-Z, Zeng Y-T (2006) Identification of pseudo attP sites for phage φC31 integrase in bovine genome. Biochem Biophys Res Commun 345:984–988

    Article  CAS  Google Scholar 

  • Maucksch C, Aneja MK, Hennen E, Bohla A, Hoffmann F, Elfinger M, Rosenecker J, Rudolph C (2008) Cell type differences in activity of the Streptomyces bacteriophage φC31 integrase. Nucleic Acids Res 36(17):5462–5471

    Article  CAS  Google Scholar 

  • Meredith JM, Basu S, Nimmo DD, Larget-Thiery I, Warr EL, Underhill A, McArthur CC, Carter V, Hurd H, Bourgouin C, Eggleston P (2011) Site-specific integration and expression of an anti-malarial gene in transgenic Anopheles gambiae significantly reduces Plasmodium infections. PLoS One 6(1):e14587

    Article  CAS  Google Scholar 

  • Monetti C, Nishino K, Biechele S, Zhang P, Baba T, Woltjen K, Nagy A (2011) PhiC31 integrase facilitates genetic approaches combining multiple recombinases. Methods 53:380–385

    Article  CAS  Google Scholar 

  • Nakayama G, Kawaguchi Y, Koga K, Kusakabe T (2006) Site-specific gene integration in cultured silkworm cells mediated by φC31 integrase. Mol Genet Genomics 275:1–8

    Article  CAS  Google Scholar 

  • Ni J-Q, Markstein M, Binari R, Pfeiffer B, Liu L-P, Villalta C, Booker M, Perkins E, Perrimon N (2008) Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster. Nat Methods 5(1):49–51

    Article  CAS  Google Scholar 

  • Ni J-Q, Liu L-P, Binari R, Hardy R, Shim H-S, Cavallaro A, Booker M, Pfeiffer BD, Markstein M, Wang H, Villalta C, Laverty TR, Perkins LA, Perrimon N (2009) A Drosophila resource of transgenic RNAi lines for neurogenetics. Genetics 182:1089–1100

    Article  CAS  Google Scholar 

  • Nimmo DD, Alphey L, Meredith JM, Eggleston P (2006) High efficiency site-specific genetic engineering of the mosquito genome. Insect Mol Biol 15(2):129–136

    Article  CAS  Google Scholar 

  • Nishiumi F, Sone T, Kishine H, Thyagarajan B, Kogure T, Miyawaki A, Chesnut JD, Imamoto F (2009) Simultaneous single cell stable expression of 2–4 cDNAs in HeLaS3 using φC31 integrase system. Cell Struct Funct 34:47–59

    Article  CAS  Google Scholar 

  • Olivares EC, Hollis RP, Calos MP (2001) Phage R4 integrase mediates site-specific integration in human cells. Gene 278:167–176

    Article  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  CAS  Google Scholar 

  • Ortiz-Urda S, Thyagarajan B, Keene DR, Lin Q, Fang M, Calos MP, Khavari PA (2002) Stable nonviral genetic correction of inherited human skin disease. Nat Med 8(10):1166–1170

    Article  CAS  Google Scholar 

  • Ortiz-Urda S, Thyagarajan B, Keene D, Lin Q, Calos MP, Khavari PA (2003) φC31 integrase-mediated nonviral genetic correction of junctional epidermolysis bullosa. Hum Gene Ther 14:923–928

    Article  CAS  Google Scholar 

  • Ou H-L, Huang Y, Qu L-J, Xu M, Yan J-B, Ren Z-R, Huang S-Z, Zeng Y-T (2009) A φC31 integrase-mediated integration hotspot in favor of transgene expression exists in the bovine genome. FEBS J 276:155–163

    Article  CAS  Google Scholar 

  • Pfeiffer BD, Jenett A, Hammonds AS, Ngo T-TB, Misra S, Murphy C, Scully A, Carlson JW, Wan KH, Laverty TR, Mungall C, Svirskas R, Kadonga JT, Doe CQ, Eisen MB, Celniker SE, Rubin GM (2008) Tools for neuroanatomy and neurogenetics in Drosophila. Proc Natl Acad Sci 105(28):9715–9720

    Article  CAS  Google Scholar 

  • Portlock JL, Keravala A, Bertoni C, Lee S, Rando TA, Calos MP (2006) Long-term increase in mVEGF164 in mouse hindlimb muscle mediated by phage φC31 integrase after nonviral DNA delivery. Hum Gene Ther 17:871–876

    Article  CAS  Google Scholar 

  • Quenneville SP, Chapdelaine P, Rousseau J, Beaulieu J, Caron NJ, Skuk D, Mills P, Olivares EC, Calos MP, Tremblay JP (2004) Nucleofection of muscle-derived stem cells and myoblasts with φC31 integrase: stable expression of a full-length-dystrophin fusion gene by human myoblasts. Mol Ther 10(4):679–687

    Article  CAS  Google Scholar 

  • Quenneville SP, Chapdelaine RJ, Tremblay JP (2007) Dystrophin expression in host muscle following transplantation of muscle precursor cells modified with the phiC31 integrase. Gene Ther 14:514–522

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Raymond CS, Soriano P (2007) High-efficiency FLP and φC31 site-specific recombination in mammalian cells. PLoS One 2(1):e162

    Article  Google Scholar 

  • Rubtsova M, Kempe K, Gils A, Ismagul A, Weyen J, Gils M (2008) Expression of active Streptomyces phage φC31 integrase in transgenic wheat plants. Plant Cell Rep 27:1821–1831

    Article  CAS  Google Scholar 

  • Schetelig MF, Scolari F, Handler AM, Kittelmann S, Gasperi G, Wimmer EA (2009) Site-specific recombination for the modification of transgenic strains of the Mediterranean fruit fly Ceratitis capitata. Proc Natl Acad Sci 106(43):18171–18176

    Article  CAS  Google Scholar 

  • Sharma N, Moldt B, Dalsgaard T, Jensen TG, Mikkelsen JG (2008) Regulated gene insertion by steroid-induced φC31 integrase. Nucleic Acids Res 36(11):e67

    Article  Google Scholar 

  • Sivalingam J, Krishnan S, Ng WH, Lee SS, Phan TT, Kon OL (2010) Biosafety assessment of site-directed transgene integration in human umbilical cord-lining cells. Mol Ther 18(7):1346–1356

    Article  CAS  Google Scholar 

  • Tasic B, Hippenmeyer S, Wang C, Gamboa M, Zong H, Chen-Tsai Y, Luo L (2011) Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc Natl Acad Sci 108(19):7902–7907

    Article  CAS  Google Scholar 

  • Thomson JG, Chan R, Thilmony R, Yau Y-Y, Ow DW (2010) PhiC31 recombination system demonstrates heritable germinal transmission of site-specific excision from the Arabidopsis genome. BMC Biotechnol 10:17

    Article  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 95:5505–5510

    Article  CAS  Google Scholar 

  • Thyagarajan B, Calos MP (2005) Site-specific integration for high-level protein production in mammalian cells. In: Smales CM, James DC (eds) Methods in molecular biology, vol 308, Therapeutic proteins: methods and protocols. Humana, Totowa

    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(12):3926–3934

    Article  CAS  Google Scholar 

  • Thyagarajan B, Liu Y, Shin S, Lakshmipathy U, Scheyhing K, Xue H, Ellerström C, Strehl HJ, Rao MS, Chesnut JD (2008) Creation of engineered human embryonic stem cell lines using phiC31 integrase. Stem Cells 26:119–126

    Article  CAS  Google Scholar 

  • Watanabe S, Nakamura S, Sakurai T, Akasaka K, Sato M (2010) Improvement of a phiC31 integrase-based gene delivery system that confers high and continuous transgene expression. New Biotechnol 28(4):312–319

    Article  Google Scholar 

  • Woodard LE, Hillman RT, Keravala A, Lee S, Calos MP (2010a) Effect of nuclear localization and hydrodynamic delivery-induced cell division on φC31 integrase activity. Gene Ther 17:217–226

    Article  CAS  Google Scholar 

  • Woodard LE, Keravala A, Jung WE, Wapinsky OL, Yang Q, Felsher DW, Calos MP (2010b) Impact of hydrodynamic injection and phiC31 integrase on tumor latency in mouse model of MYC-induced hepatocellular carcinoma. PLoS One 5(6):e11367

    Article  Google Scholar 

  • Ye L, Chang JC, Lin C, Qi Z, Yu J, Kan YW (2010) Generation of induced pluripotent stem cells using site-specific integration with phage integrase. Proc Natl Acad Sci 107(45):19467–19472

    Article  CAS  Google Scholar 

Download references

Acknowledgements

JMG was supported by a graduate fellowship from the National Science Foundation. Work in the Calos lab is supported by grants to MPC from the National Institutes of Health, the California Institute for Regenerative Medicine, the Muscular Dystrophy Association, and the Jain Foundation. MPC is an inventor on Stanford-owned patents covering phage integrases.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Michele P. Calos .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media B.V.

About this chapter

Cite this chapter

Geisinger, J.M., Calos, M.P. (2013). Site-Specific Recombination Using PhiC31 Integrase. In: Renault, S., Duchateau, P. (eds) Site-directed insertion of transgenes. Topics in Current Genetics, vol 23. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-4531-5_8

Download citation

Publish with us

Policies and ethics