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A Simple Method of Transgenesis Using I-Sce I Meganuclease in Xenopus

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Xenopus Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 917))

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Abstract

Here we present a protocol for generating transgenic embryos in Xenopus using I-SceI meganuclease. This method relies on integration of DNA constructs, containing one or two I-SceI meganuclease sites. It is a simpler method than the REMI method of transgenesis, and it is ideally suited for generating transgenic lines in Xenopus laevis and Xenopus tropicalis. In addition to it being simpler than the REMI method, this protocol also results in single copy integration events rather than tandem concatemers. Although the protocol we describe is for X. tropicalis, the method can also be used to generate transgenic lines in X. laevis. We also describe a convenient method for designing and generating complex constructs for transgenesis, named pTransgenesis, based on the Multisite Gateway® cloning, which include I-SceI sites and Tol2 elements to facilitate genome integration.

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References

  1. Kroll KL, Amaya E (1996) Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122(10):3173–3183

    PubMed  CAS  Google Scholar 

  2. Shemesh M, Gurevich M, Harel-Markowitz E, Benvenisti L, Shore LS, Stram Y (2000) Gene integration into bovine sperm genome and its expression in transgenic offspring. Mol Reprod Dev 56(2 Suppl):306–308

    Article  PubMed  CAS  Google Scholar 

  3. Harel-Markowitz E, Gurevich M, Shore LS, Katz A, Stram Y, Shemesh M (2009) Use of sperm plasmid DNA lipofection combined with REMI (restriction enzyme-mediated insertion) for production of transgenic chickens expressing eGFP (enhanced green fluorescent protein) or human follicle-stimulating hormone. Biol Reprod 80(5):1046–1052

    Article  PubMed  CAS  Google Scholar 

  4. Noh W, Kim SW, Dong-Won B, Kim JY, Ro HS (2010) Genetic introduction of foreign genes to Pleurotus eryngii by restriction enzyme-mediated integration. J Microbiol 48(2):253–256

    Article  PubMed  CAS  Google Scholar 

  5. Yan W, Liu X, Shi T, Hao L, Tomley FM, Suo X (2009) Stable transfection of Eimeria tenella: constitutive expression of the YFP-YFP molecule throughout the life cycle. Int J Parasitol 39(1):109–117

    Article  PubMed  CAS  Google Scholar 

  6. Kuspa A, Loomis WF (1992) Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA. Proc Natl Acad Sci U S A 89(18):8803–8807

    Article  PubMed  CAS  Google Scholar 

  7. Dujon B (1980) Sequence of the intron and flanking exons of the mitochondrial 21S rRNA gene of yeast strains having different alleles at the omega and rib-1 loci. Cell 20(1):185–197

    Article  PubMed  CAS  Google Scholar 

  8. Colleaux L, D’Auriol L, Galibert F, Dujon B (1988) Recognition and cleavage site of the intron-encoded omega transposase. Proc Natl Acad Sci U S A 85(16):6022–6026

    Article  PubMed  CAS  Google Scholar 

  9. Thermes V, Grabher C, Ristoratore F, Bourrat F, Choulika A, Wittbrodt J, Joly JS (2002) I-SceI meganuclease mediates highly efficient transgenesis in fish. Mech Dev 118(1–2):91–98

    Article  PubMed  CAS  Google Scholar 

  10. Deschet K, Nakatani Y, Smith WC (2003) Generation of Ci-Brachyury-GFP stable transgenic lines in the ascidian Ciona savignyi. Genesis 35(4):248–259

    Article  PubMed  CAS  Google Scholar 

  11. Pan FC, Chen Y, Loeber J, Henningfeld K, Pieler T (2006) I-SceI meganuclease-mediated transgenesis in Xenopus. Dev Dyn 235(1):247–252

    Article  PubMed  Google Scholar 

  12. Ogino H, McConnell WB, Grainger RM (2006) Highly efficient transgenesis in Xenopus tropicalis using I-SceI meganuclease. Mech Dev 123(2):103–113

    Article  PubMed  CAS  Google Scholar 

  13. Ochiai H, Sakamoto N, Suzuki K, Akasaka K, Yamamoto T (2008) The Ars insulator facilitates I-SceI meganuclease-mediated transgenesis in the sea urchin embryo. Dev Dyn 237(9):2475–2482

    Article  PubMed  CAS  Google Scholar 

  14. Perrin A, Buckle M, Dujon B (1993) Asymmetrical recognition and activity of the I-SceI endonuclease on its site and on intron-exon junctions. EMBO J 12(7):2939–2947

    PubMed  CAS  Google Scholar 

  15. Love NR, Thuret R, Chen Y, Ishibashi S, Sabherwal N, Paredes R, Dorey K, Noble AM, Guille MJ, Sasai Y, Papalopulu N, Amaya E (2011) pTransgenesis: A cross-species, modular transgenesis resource. Development 138(24):5451–5458

    Article  PubMed  CAS  Google Scholar 

  16. Hartley JL, Temple GF, Brasch MA (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10(11):1788–1795

    Article  PubMed  CAS  Google Scholar 

  17. Kawakami K, Takeda H, Kawakami N, Kobayashi M, Matsuda N, Mishina M (2004) A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev Cell 7(1):133–144

    Article  PubMed  CAS  Google Scholar 

  18. Khokha MK, Chung C, Bustamante EL, Gaw LW, Trott KA, Yeh J, Lim N, Lin JC, Taverner N, Amaya E, Papalopulu N, Smith JC, Zorn AM, Harland RM, Grammer TC (2002) Techniques and probes for the study of Xenopus tropicalis development. Dev Dyn 225(4):499–510

    Article  PubMed  CAS  Google Scholar 

  19. Bronchain OJ, Hartley KO, Amaya E (1999) A gene trap approach in Xenopus. Curr Biol 9(20):1195–1198

    Article  PubMed  CAS  Google Scholar 

  20. Beck CW, Slack JM (1999) Gut specific expression using mammalian promoters in transgenic Xenopus laevis. Mech Dev 88(2):221–227

    Article  PubMed  CAS  Google Scholar 

  21. Yoshida T, Ohkumo T, Ishibashi S, Yasuda K (2005) The 5′-AT-rich half-site of Maf recognition element: a functional target for bZIP transcription factor Maf. Nucleic Acids Res 33(11):3465–3478

    Article  PubMed  CAS  Google Scholar 

  22. Turner DL, Weintraub H (1994) Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev 8(12):1434–1447

    Article  PubMed  CAS  Google Scholar 

  23. Niwa H, Yamamura K, Miyazaki J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108(2):193–199

    Article  PubMed  CAS  Google Scholar 

  24. Schorpp M, Jager R, Schellander K, Schenkel J, Wagner EF, Weiher H, Angel P (1996) The human ubiquitin C promoter directs high ubiquitous expression of transgenes in mice. Nucleic Acids Res 24(9):1787–1788

    Article  PubMed  CAS  Google Scholar 

  25. Friedrich G, Soriano P (1991) Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev 5(9):1513–1523

    Article  PubMed  CAS  Google Scholar 

  26. Hartley KO, Hardcastle Z, Friday RV, Amaya E, Papalopulu N (2001) Transgenic Xenopus embryos reveal that anterior neural development requires continued suppression of BMP signaling after gastrulation. Dev Biol 238(1):168–184

    Article  PubMed  CAS  Google Scholar 

  27. Meadows SM, Salanga MC, Krieg PA (2009) Kruppel-like factor 2 cooperates with the ETS family protein ERG to activate Flk1 expression during vascular development. Development 136(7):1115–1125

    Article  PubMed  CAS  Google Scholar 

  28. Smith SJ, Kotecha S, Towers N, Latinkic BV, Mohun TJ (2002) XPOX2-peroxidase expression and the XLURP-1 promoter reveal the site of embryonic myeloid cell development in Xenopus. Mech Dev 117(1–2):173–186

    Article  PubMed  CAS  Google Scholar 

  29. Koster RW, Fraser SE (2001) Tracing transgene expression in living zebrafish embryos. Dev Biol 233(2):329–346

    Article  PubMed  CAS  Google Scholar 

  30. Rizzo MA, Springer GH, Granada B, Piston DW (2004) An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol 22(4):445–449

    Article  PubMed  CAS  Google Scholar 

  31. Heim R, Tsien RY (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6(2):178–182

    Article  PubMed  CAS  Google Scholar 

  32. Ai HW, Henderson JN, Remington SJ, Campbell RE (2006) Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochem J 400(3):531–540

    Article  PubMed  CAS  Google Scholar 

  33. Karasawa S, Araki T, Nagai T, Mizuno H, Miyawaki A (2004) Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem J 381(Pt 1):307–312

    PubMed  CAS  Google Scholar 

  34. Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV, Lukyanov S (2006) Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3(4):281–286

    Article  PubMed  CAS  Google Scholar 

  35. Tsutsui H, Karasawa S, Shimizu H, Nukina N, Miyawaki A (2005) Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep 6(3):233–238

    Article  PubMed  CAS  Google Scholar 

  36. Ando R, Hama H, Yamamoto-Hino M, Mizuno H, Miyawaki A (2002) An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc Natl Acad Sci U S A 99(20):12651–12656

    Article  PubMed  CAS  Google Scholar 

  37. Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273(5280):1392–1395

    Article  PubMed  CAS  Google Scholar 

  38. Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20(1):87–90

    Article  PubMed  CAS  Google Scholar 

  39. Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22(12):1567–1572

    Article  PubMed  CAS  Google Scholar 

  40. Shcherbo D, Merzlyak EM, Chepurnykh TV, Fradkov AF, Ermakova GV, Solovieva EA, Lukyanov KA, Bogdanova EA, Zaraisky AG, Lukyanov S, Chudakov DM (2007) Bright far-red fluorescent protein for whole-body imaging. Nat Methods 4(9):741–746

    Article  PubMed  CAS  Google Scholar 

  41. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118(2):401–415

    PubMed  CAS  Google Scholar 

  42. Verrou C, Zhang Y, Zurn C, Schamel WW, Reth M (1999) Comparison of the tamoxifen regulated chimeric Cre recombinases MerCreMer and CreMer. Biol Chem 380(12):1435–1438

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. The Healing Foundation Centre, The Faculty of Life Sciences, University of Manchester, Manchester, England, UK

    Shoko Ishibashi & Nick R. Love

  2. Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, Manchester, England, UK

    Enrique Amaya

Authors
  1. Shoko Ishibashi
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  2. Nick R. Love
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  3. Enrique Amaya
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Correspondence to Enrique Amaya .

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Editors and Affiliations

  1. , Institute of Medical Sciences, University of Aberdeen, FORESTERHILL, ABERDEEN, AB25 2ZD, United Kingdom

    STEFAN HOPPLER

  2. , Department of Biological Science, University of Calgary, University Drive NW 2500, Calgary, T2N 1N4, Canada

    Peter D Vize

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Ishibashi, S., Love, N.R., Amaya, E. (2012). A Simple Method of Transgenesis Using I-Sce I Meganuclease in Xenopus . In: HOPPLER, S., Vize, P. (eds) Xenopus Protocols. Methods in Molecular Biology, vol 917. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-992-1_12

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  • DOI: https://doi.org/10.1007/978-1-61779-992-1_12

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-991-4

  • Online ISBN: 978-1-61779-992-1

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