Site-Specific Recombination Using PhiC31 Integrase

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


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.


Gene therapy Induced pluripotent stem cells Phage integrase Recombinases Site-specific integration Transgenic organisms 


  1. Allen BG, Weeks DL (2005) Transgenic Xenopus laevis embryos can be generated using φC31 integrase. Nat Methods 2(12):975–979CrossRefGoogle Scholar
  2. Allen BG, Weeks DL (2006) Using φC31 integrase to make transgenic Xenopus laevis Embryos. Nat Protoc 1(3):1248–1257CrossRefGoogle Scholar
  3. 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 YorkGoogle Scholar
  4. 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):2299–2306CrossRefGoogle Scholar
  5. 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 9:967–975CrossRefGoogle Scholar
  6. 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):919–934CrossRefGoogle Scholar
  7. 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):419–424CrossRefGoogle Scholar
  8. Brown WRA, Lee NCO, Xu Z, Smith MCM (2011) Serine recombinases as tools for genome engineering. Methods 53(4):372–379CrossRefGoogle Scholar
  9. Calos MP (2006) The φC31 integrase system for gene therapy. Curr Gene Ther 6:633–645CrossRefGoogle Scholar
  10. 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):2140–2146CrossRefGoogle Scholar
  11. 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 357:28–48CrossRefGoogle Scholar
  12. Chavez CL, Calos MP (2011) Therapeutic applications of the phiC31 integrase system. Curr Gene Ther 11(5):375–381CrossRefGoogle Scholar
  13. 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 21:1287–1297CrossRefGoogle Scholar
  14. 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):e189CrossRefGoogle Scholar
  15. 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 17:1077–1094CrossRefGoogle Scholar
  16. 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):2325–2331CrossRefGoogle Scholar
  17. 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):13999–14004CrossRefGoogle Scholar
  18. 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 6:226–235CrossRefGoogle Scholar
  19. Grindley NDF, Whiteson KL, Rice PA (2006) Mechanisms of site-specific recombination. Annu Rev Biochem 75:567–605CrossRefGoogle Scholar
  20. 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):5995–6000CrossRefGoogle Scholar
  21. Groth AC, Fish M, Nusse R, Calos MP (2004) Construction of transgenic Drosophila by using the site-specific integrase from phage ΦC31. Genetics 166:1775–1782CrossRefGoogle Scholar
  22. 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):399–408CrossRefGoogle Scholar
  23. 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 1:79CrossRefGoogle Scholar
  24. 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 16:1383–1394CrossRefGoogle Scholar
  25. 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 8:646–653CrossRefGoogle Scholar
  26. 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):1692–1704CrossRefGoogle Scholar
  27. 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 72:673–687CrossRefGoogle Scholar
  28. 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 276:135–146CrossRefGoogle Scholar
  29. 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 8:1008–1017CrossRefGoogle Scholar
  30. 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 173:299–305CrossRefGoogle Scholar
  31. 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 18:842–848CrossRefGoogle Scholar
  32. 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 138:2625–2630CrossRefGoogle Scholar
  33. Kuhstoss S, Rao RN (1991) Analysis of the integration function of the Streptomycete bacteriophage φC31. J Mol Biol 222:897–908CrossRefGoogle Scholar
  34. 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):e788CrossRefGoogle Scholar
  35. 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 75:1163–1175CrossRefGoogle Scholar
  36. 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 14:1207–1215CrossRefGoogle Scholar
  37. Lister JA (2010) Transgene excision in zebrafish using the phiC31 integrase. Genesis 48:137–143CrossRefGoogle Scholar
  38. Liu J, Jeppesen I, Nielsen K, Jensen TG (2006) PhiC31 integrase induces chromosomal aberrations in primary human fibroblasts. Gene Ther 13:1188–1190CrossRefGoogle Scholar
  39. 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 9:31–38CrossRefGoogle Scholar
  40. 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):1459–1471CrossRefGoogle Scholar
  41. Lu J, Maddison LA, Chen W (2011) PhiC31 integrase induces efficient site-specific excision in zebrafish. Transgenic Res 20(1):183–189CrossRefGoogle Scholar
  42. Lutz KA, Corneille S, Azhagiri AK, Svab Z, Maliga P (2004) A novel approach to plastid transformation utilizes the phiC31 phage integrase. Plant J 37:906–913CrossRefGoogle Scholar
  43. 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 345:984–988CrossRefGoogle Scholar
  44. 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):5462–5471CrossRefGoogle Scholar
  45. 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):e14587CrossRefGoogle Scholar
  46. Monetti C, Nishino K, Biechele S, Zhang P, Baba T, Woltjen K, Nagy A (2011) PhiC31 integrase facilitates genetic approaches combining multiple recombinases. Methods 53:380–385CrossRefGoogle Scholar
  47. Nakayama G, Kawaguchi Y, Koga K, Kusakabe T (2006) Site-specific gene integration in cultured silkworm cells mediated by φC31 integrase. Mol Genet Genomics 275:1–8CrossRefGoogle Scholar
  48. 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):49–51CrossRefGoogle Scholar
  49. 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 182:1089–1100CrossRefGoogle Scholar
  50. Nimmo DD, Alphey L, Meredith JM, Eggleston P (2006) High efficiency site-specific genetic engineering of the mosquito genome. Insect Mol Biol 15(2):129–136CrossRefGoogle Scholar
  51. 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 34:47–59CrossRefGoogle Scholar
  52. Olivares EC, Hollis RP, Calos MP (2001) Phage R4 integrase mediates site-specific integration in human cells. Gene 278:167–176CrossRefGoogle Scholar
  53. 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 20:1124–1128CrossRefGoogle Scholar
  54. 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):1166–1170CrossRefGoogle Scholar
  55. 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 14:923–928CrossRefGoogle Scholar
  56. 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 276:155–163CrossRefGoogle Scholar
  57. 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):9715–9720CrossRefGoogle Scholar
  58. 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 17:871–876CrossRefGoogle Scholar
  59. 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):679–687CrossRefGoogle Scholar
  60. 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 14:514–522CrossRefGoogle Scholar
  61. Rausch H, Lehmann M (1991) Structural analysis of the actinophage φC31 attachment site. Nucleic Acids Res 19(19):5187–5189CrossRefGoogle Scholar
  62. Raymond CS, Soriano P (2007) High-efficiency FLP and φC31 site-specific recombination in mammalian cells. PLoS One 2(1):e162CrossRefGoogle Scholar
  63. 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 27:1821–1831CrossRefGoogle Scholar
  64. 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):18171–18176CrossRefGoogle Scholar
  65. Sharma N, Moldt B, Dalsgaard T, Jensen TG, Mikkelsen JG (2008) Regulated gene insertion by steroid-induced φC31 integrase. Nucleic Acids Res 36(11):e67CrossRefGoogle Scholar
  66. 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):1346–1356CrossRefGoogle Scholar
  67. 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):7902–7907CrossRefGoogle Scholar
  68. 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 10:17CrossRefGoogle Scholar
  69. 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 95:5505–5510CrossRefGoogle Scholar
  70. 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, TotowaGoogle Scholar
  71. 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):3926–3934CrossRefGoogle Scholar
  72. 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 26:119–126CrossRefGoogle Scholar
  73. 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):312–319CrossRefGoogle Scholar
  74. 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 17:217–226CrossRefGoogle Scholar
  75. 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):e11367CrossRefGoogle Scholar
  76. 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):19467–19472CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2013

Authors and Affiliations

  1. 1.Department of GeneticsStanford University School of MedicineStanfordUSA

Personalised recommendations