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Transposon-mediated BAC transgenesis in zebrafish

Nature Protocols volume 6, pages 1998–2021 (2011)Cite this article

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Abstract

Bacterial artificial chromosomes (BACs) are widely used in studies of vertebrate gene regulation and function because they often closely recapitulate the expression patterns of endogenous genes. Here we report a step-by-step protocol for efficient BAC transgenesis in zebrafish using the medaka Tol2 transposon. Using recombineering in Escherichia coli, we introduce the iTol2 cassette in the BAC plasmid backbone, which contains the inverted minimal cis-sequences required for Tol2 transposition, and a reporter gene to replace a target locus in the BAC. Microinjection of the Tol2-BAC and a codon-optimized transposase mRNA into fertilized eggs results in clean integrations in the genome and transmission to the germline at a rate of ∼15%. A single person can prepare a dozen constructs within 3 weeks, and obtain transgenic fish within approximately 3–4 months. Our protocol drastically reduces the labor involved in BAC transgenesis and will greatly facilitate biological and biomedical studies in model vertebrates.

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Figure 1: Flowchart outlining the experimental procedures described in this protocol and anticipated timing for each step.
Figure 2: iTol2, kan and galK cassettes for BAC recombineering.
Figure 3: Recombineering the iTol2 cassette and reporter gene into a BAC clone.
Figure 4: Microinjection of the Tol2-BAC plasmid and excision assay.
Figure 5: Restriction analysis of BAC plasmid DNA before and after recombineering the iTol2-amp and Gal4-VP16 cassettes.
Figure 6: Microinjection setup and breeding and raising of transgenic zebrafish.
Figure 7: Generation and identification of Tol2-BAC transgenic fish.
Figure 8: Analysis of Tol2-BAC integrations.

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References

  1. Giraldo, P. & Montoliu, L. Size matters: use of YACs, BACs and PACs in transgenic animals. Transgenic. Res. 10, 83–103 (2001).

    Article  CAS  Google Scholar 

  2. Nagy, A., Gertsenstein, M., Vintersten, K. & Behringer, R. Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edn. (Cold Spring Harbor Press, Cold Spring Harbor, New York, USA, 2001).

  3. Vintersten, K., Testa, G., Naumann, R., Anastassiadis, K. & Stewart, A.F. Bacterial artificial chromosome transgenesis through pronuclear injection of fertilized mouse oocytes. Methods Mol. Biol. 415, 83–100 (2008).

    CAS  PubMed  Google Scholar 

  4. Copeland, N.G., Jenkins, N.A. & Court, D.L. Recombineering: a powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2, 769–779 (2001).

    Article  CAS  Google Scholar 

  5. Heintz, N. BAC to the future: the use of bac transgenic mice for neuroscience research. Nat. Rev. Neurosci. 2, 861–870 (2001).

    Article  CAS  Google Scholar 

  6. Jessen, J.R., Willett, C.E. & Lin, S. Artificial chromosome transgenesis reveals long-distance negative regulation of rag1 in zebrafish. Nat. Genet. 23, 15–16 (1999).

    Article  CAS  Google Scholar 

  7. Yang, Z. et al. Modified bacterial artificial chromosomes for zebrafish transgenesis. Methods 39, 183–188 (2006).

    Article  CAS  Google Scholar 

  8. Higashijima, S.-I. Transgenic zebrafish expressing fluorescent proteins in central nervous system neurons. Dev. Growth Diff. 50, 407–413 (2008).

    Article  CAS  Google Scholar 

  9. Lieschke, G.J. & Currie, P.D. Animal models of human disease: zebrafish swim into view. Nat. Rev. Genet. 8, 353–367 (2007).

    Article  CAS  Google Scholar 

  10. Asakawa, K. et al. Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish. Proc. Natl. Acad. Sci. USA 105, 1255–1260 (2008).

    Article  CAS  Google Scholar 

  11. Scott, E. et al. Targeting neural circuitry in zebrafish using Gal4 enhancer trapping. Nat. Methods 4, 323–326 (2007).

    Article  CAS  Google Scholar 

  12. Emelyanov, A. & Parinov, S. Mifepristone-inducible LexPR system to drive and control gene expression in transgenic zebrafish. Dev. Biol. 320, 113–121 (2008).

    Article  CAS  Google Scholar 

  13. Langenau, D.M. et al. Cre/lox-regulated zebrafish model with conditional myc-induced T-cell acute lymphoblastic leukemia. Proc. Natl. Acad. Sci. USA 102, 6068–6073 (2005).

    Article  CAS  Google Scholar 

  14. Knopf, F. et al. Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish. Proc. Natl. Acad. Sci. USA 107, 19933–19938 (2010).

    Article  CAS  Google Scholar 

  15. Chandler, K.J. et al. Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression. Mamm. Genome 18, 693–708 (2007).

    Article  CAS  Google Scholar 

  16. Garrick, D., Fiering, S., Martin, D.I. & Whitelaw, E. Repeat-induced gene silencing in mammals. Nat. Genet. 18, 56–59 (1998).

    Article  CAS  Google Scholar 

  17. Dorer, D.R. Do transgene arrays form heterochromatin in vertebrates? Transgenic. Res. 6, 3–10 (1997).

    Article  CAS  Google Scholar 

  18. Stuart, G.W., McMurray, J.V. & Westerfield, M. Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos. Development 103, 403–412 (1988).

    CAS  PubMed  Google Scholar 

  19. Amsterdam, A., Lin, S. & Hopkins, N. The Aequorea victoria green fluorescent protein can be used as a reporter in live zebrafish embryos. Dev. Biol. 171, 123–129 (1995).

    Article  CAS  Google Scholar 

  20. Long, Q. et al. GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene. Development 124, 4105–4111 (1997).

    CAS  PubMed  Google Scholar 

  21. Higashijima, S., Okamoto, H., Ueno, N., Hotta, Y. & Eguchi, G. High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin. Dev. Biol. 192, 289–299 (1997).

    Article  CAS  Google Scholar 

  22. Rembold, M., Lahiri, K., Foulkes, N.S. & Wittbrodt, J. Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct. Nat. Protoc. 1, 1133–1139 (2006).

    Article  CAS  Google Scholar 

  23. Lin, S. et al. Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish. Science 265, 666–669 (1994).

    Article  CAS  Google Scholar 

  24. Gaiano, N., Allende, M., Amsterdam, A., Kawakami, K. & Hopkins, N. Highly efficient germ-line transmission of proviral insertions in zebrafish. Proc. Natl. Acad. Sci. USA 93, 7777–7782 (1996).

    Article  CAS  Google Scholar 

  25. Ellingsen, S. et al. Large-scale enhancer detection in the zebrafish genome. Development 132, 3799–811 (2005).

    Article  CAS  Google Scholar 

  26. Wang, D. et al. Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions. Proc. Natl. Acad. Sci. USA 104, 12428–12433 (2007).

    Article  CAS  Google Scholar 

  27. Thomas, C.E., Ehrhardt, A. & Kay, M.A. Progress and problems with the use of viral vectors for gene therapy. Nat. Rev. Genet. 4, 346–358 (2003).

    Article  CAS  Google Scholar 

  28. Fadool, J.M., Hartl, D.L. & Dowling, J.E. Transposition of the mariner element from Drosophila mauritiana in zebrafish. Proc. Natl. Acad. Sci. USA 95, 5182–516 (1998).

    Article  CAS  Google Scholar 

  29. Kawakami, K. et al. A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev. Cell 7, 133–144 (2004).

    Article  CAS  Google Scholar 

  30. Davidson, A.E. et al. Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon. Dev. Biol. 263, 191–202 (2003).

    Article  CAS  Google Scholar 

  31. Kawakami, K. Tol2: a versatile gene transfer vector in vertebrates. Genome Biol. 8 (Suppl 1): S7 (2007).

    Article  Google Scholar 

  32. Korzh, V. Transposons as tools for enhancer trap screens in vertebrates. Genome Biol. 8 (Suppl 1): S8 (2007).

    Article  Google Scholar 

  33. Suster, M.L., Sumiyama, K. & Kawakami, K. Transposon-mediated BAC transgenesis in zebrafish and mice. BMC Genomics 10, 477 (2009).

    Article  Google Scholar 

  34. Kawakami, K. & Shima, A. Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene 240, 239–244 (1999).

    Article  CAS  Google Scholar 

  35. Balciunas, D. et al. Harnessing a high cargo-capacity transposon for genetic applications in vertebrates. PLoS Genet. 2, e169 (2006).

    Article  Google Scholar 

  36. Urasaki, A., Morvan, G. & Kawakami, K. Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics 174, 639–649 (2006).

    Article  CAS  Google Scholar 

  37. Suster, M.L. et al. A novel conserved evx1 enhancer links spinal interneuron morphology and cis-regulation from fish to mammals. Dev. Biol. 325, 422–433 (2009).

    Article  CAS  Google Scholar 

  38. Sharan, K., Thomason, L.C., Kuznetsov, S.G. & Court, D.L. Recombineering: a homologous recombination-based method of genetic engineering. Nat. Protoc. 4, 206–223 (2009).

    Article  CAS  Google Scholar 

  39. Zhang, Z.D. et al. Statistical analysis of the genomic distribution and correlation of regulatory elements in the ENCODE regions. Genome Res. 17, 787–797 (2007).

    Article  CAS  Google Scholar 

  40. Murphy, K.C. Use of bacteriophage l recombination functions to promote gene replacement in Escherichia coli. J. Bacteriol. 180, 2063–2071 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Murphy, K.C., Campellone, K.G. & Poteete, A.R. PCR-mediated gene replacement in Escherichia coli. Gene 246, 321–330 (2000).

    Article  CAS  Google Scholar 

  42. Zhang, Y., Buchholz, F., Muyrers, J.P. & Stewart, A.F. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20, 123–128 (1998).

    Article  CAS  Google Scholar 

  43. Zhang, Y., Muyrers, J.P., Testa, G. & Stewart, A.F. DNA cloning by homologous recombination in Escherichia coli. Nat. Biotechnol. 18, 1314–1317 (2000).

    Article  CAS  Google Scholar 

  44. Yu, D. et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA 97, 5978–5983 (2000).

    Article  CAS  Google Scholar 

  45. Warming, S., Costantino, N., Court, D.L., Jenkins, N.A. & Copeland, N.G. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 33, e36 (2005).

    Article  Google Scholar 

  46. Cassuto, E. & Radding, C.M. Mechanism for the action of l exonuclease in genetic recombination. Nat. New Biol. 229, 13–16 (1971).

    Article  CAS  Google Scholar 

  47. Little, J.W. An exonuclease induced by bacteriophage l. II. Nature of the enzymatic reaction. J. Biol. Chem. 242, 679–686 (1967).

    CAS  PubMed  Google Scholar 

  48. Carter, D.M. & Radding, C.M. The role of exonuclease and b protein of phage l in genetic recombination. II. Substrate specificity and the mode of action of lambda exonuclease. J. Biol. Chem. 246, 2502–2512 (1971).

    CAS  PubMed  Google Scholar 

  49. Cassuto, E., Lash, T., Sriprakash, K.S. & Radding, C.M. Role of exonuclease and b protein of phage l in genetic recombination. V. Recombination of l DNA in vitro. Proc. Natl. Acad. Sci. USA 68, 1639–1643 (1971).

    Article  CAS  Google Scholar 

  50. Sadowski, I., Ma, J., Triezenberg, S. & Ptashne, M. GAL4-VP16 is an unusually potent transcriptional activator. Nature 335, 563–564 (1988).

    Article  CAS  Google Scholar 

  51. Köster, R.W. & Fraser, S.E. Tracing transgene expression in living zebrafish embryos. Dev. Biol. 233, 329–346 (2001).

    Article  Google Scholar 

  52. Sternberg, N. & Hamilton, D. Bacteriophage P1 site-specific recombination: I. Recombination between loxP sites. J. Mol. Biol. 150, 467–486 (1981).

    Article  CAS  Google Scholar 

  53. Suster, M.L., Kikuta, H., Urasaki, A., Asakawa, K. & Kawakami, K. Transgenesis in zebrafish with the tol2 transposon system. Methods Mol. Biol. 561, 41–63 (2009).

    Article  CAS  Google Scholar 

  54. Baier, H. & Scott, E.K. Genetic and optical targeting of neural circuits and behavior—zebrafish in the spotlight. Curr. Opin. Neurobiol. 19, 553–560.

  55. Friedenreich, H. & Schartl, M. Transient expression directed by homologous and heterologous promoter and enhancer sequences in fish cells. Nucleic Acids Res. 18, 3299–3305 (1990).

    Article  CAS  Google Scholar 

  56. Datta, S., Costantino, N. & Court, D.L. A set of recombineering plasmids for Gram-negative bacteria. Gene 379, 109–115 (2006).

    Article  CAS  Google Scholar 

  57. Lister, J.A., Robertson, C.P., Lepage, T., Johnson, S.L. & Raible, D.W. nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. Development 126, 3757–3767 (1999).

    CAS  PubMed  Google Scholar 

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Acknowledgements

For excellent fish care, we thank T. Kronstad, F. Silva and J. Xu at the Sars International Centre, and A. Ito, M. Suzuki, M. Mizushina, N. Mouri and T. Uematsu at the National Institute of Genetics. We thank A. Urasaki for initial characterization of zT2TP and K. Sumiyama for the pBSK-GFP-pA-FRT-kan-FRT plasmid. This work was supported by core funding from the Sars International Centre, a fellowship from the Japan Society for the Promotion of Science and the Takeda Science Foundation to M.L.S., and grants from the National BioResource Project of Japan and the Ministry of Education, Culture, Sports, Science and Technnology of Japan to K.K.

AUTHOR CONTRIBUTIONS

M.L.S. wrote the manuscript, directed the work at the Sars Centre and generated data and reagents. A.S. contributed reagents and optimized extensively the protocol. G.A. contributed data. K.K directed the work and wrote the manuscript.

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Authors and Affiliations

  1. Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka, Japan

    Maximiliano L Suster, Gembu Abe & Koichi Kawakami

  2. Sars International Center for Marine Molecular Biology, University of Bergen, Bergen, Norway

    Maximiliano L Suster & Anders Schouw

  3. Department of Genetics, Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan

    Koichi Kawakami

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  1. Maximiliano L Suster
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Correspondence to Maximiliano L Suster or Koichi Kawakami.

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Full sequence and graphic map of plasmids used. (PDF 550 kb)

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Suster, M., Abe, G., Schouw, A. et al. Transposon-mediated BAC transgenesis in zebrafish. Nat Protoc 6, 1998–2021 (2011). https://doi.org/10.1038/nprot.2011.416

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