Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Meganuclease-assisted generation of stable transgenics in the sea anemone Nematostella vectensis

Nature Protocols volume 12, pages 1844–1854 (2017)Cite this article

Subjects

Abstract

The sea anemone Nematostella vectensis is a model system used by a rapidly growing research community for comparative genomics, developmental biology and ecology. Here, we describe a microinjection procedure for creating stable transgenic lines in Nematostella based on meganuclease (I-SceI)-assisted integration of a transgenic cassette into the genome. The procedure describes the preparation of the reagents, microinjection of the transgenesis vector and the husbandry of transgenic animals. The microinjection setup differs from those of previously published protocols by the use of a holding capillary mounted on an inverted fluorescence microscope. In one session of injections, a single researcher can microinject up to 1,300 zygotes with a reporter construct digested with the meganuclease I-SceI. Under optimal conditions, fully transgenic heterozygous F1 animals can be obtained within 4–5 months of the injections, with a germ-line transmission efficiency of ∼3%. The method is versatile and, after a short training phase, can be carried out by any researcher with basic training in molecular biology. Flexibility of construct design enables this method to be used for numerous applications, including the functional dissection of cis-regulatory elements, subcellular localization of proteins, detection of protein-binding partners, ectopic expression of genes of interest, lineage tracing and cell-type-specific reporter gene expression.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Microinjection setup and injection series.
Figure 2: Transgenesis vector and transgenic polyps.
Figure 3: Transgenesis efficiency.

Similar content being viewed by others

References

  1. Genikhovich, G. & Technau, U. The starlet sea anemone Nematostella vectensis: an anthozoan model organism for studies in comparative genomics and functional evolutionary developmental biology. Cold Spring Harb. Protoc. http://dx.doi.org/10.1101/pdb.emo129 (2009).

  2. Steele, R.E., David, C.N. & Technau, U. A genomic view of 500 million years of cnidarian evolution. Trends Genet. 27, 7–13 (2011).

    Article  CAS  Google Scholar 

  3. Technau, U. & Steele, R.E. Evolutionary crossroads in developmental biology: Cnidaria. Development 138, 1447–1458 (2011).

    Article  CAS  Google Scholar 

  4. Technau, U. et al. Maintenance of ancestral complexity and non-metazoan genes in two basal cnidarians. Trends Genet. 21, 633–639 (2005).

    Article  CAS  Google Scholar 

  5. Putnam, N.H. et al. Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Science 317, 86–94 (2007).

    Article  CAS  Google Scholar 

  6. Tulin, S., Aguiar, D., Istrail, S. & Smith, J. A quantitative reference transcriptome for Nematostella vectensis early embryonic development: a pipeline for de novo assembly in emerging model systems. Evodevo 4, 16 (2013).

    Article  CAS  Google Scholar 

  7. Kusserow, A. et al. Unexpected complexity of the Wnt gene family in a sea anemone. Nature 433, 156–160 (2005).

    Article  CAS  Google Scholar 

  8. Lee, P.N., Pang, K., Matus, D.Q. & Martindale, M.Q. A WNT of things to come: evolution of Wnt signaling and polarity in cnidarians. Semin. Cell Dev. Biol. 17, 157–167 (2006).

    Article  Google Scholar 

  9. Rentzsch, F., Fritzenwanker, J.H., Scholz, C.B. & Technau, U. FGF signalling controls formation of the apical sensory organ in the cnidarian Nematostella vectensis. Development 135, 1761–1769 (2008).

    Article  CAS  Google Scholar 

  10. Layden, M.J., Boekhout, M. & Martindale, M.Q. Nematostella vectensis achaete-scute homolog NvashA regulates embryonic ectodermal neurogenesis and represents an ancient component of the metazoan neural specification pathway. Development 139, 1013–1022 (2012).

    Article  CAS  Google Scholar 

  11. Saina, M., Genikhovich, G., Renfer, E. & Technau, U. BMPs and chordin regulate patterning of the directive axis in a sea anemone. Proc. Natl. Acad. Sci. USA 106, 18592–18597 (2009).

    Article  CAS  Google Scholar 

  12. Wikramanayake, A.H. et al. An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation. Nature 426, 446–450 (2003).

    Article  CAS  Google Scholar 

  13. Ikmi, A., McKinney, S.A., Delventhal, K.M. & Gibson, M.C. TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis. Nat. Commun. 5, 5486 (2014).

    Article  CAS  Google Scholar 

  14. Kraus, Y., Aman, A., Technau, U. & Genikhovich, G. Pre-bilaterian origin of the blastoporal axial organizer. Nat. Commun. 7, 11694 (2016).

    Article  CAS  Google Scholar 

  15. Park, F. Lentiviral vectors: are they the future of animal transgenesis? Physiol. Genomics 31, 159–173 (2007).

    Article  CAS  Google Scholar 

  16. Wittlieb, J., Khalturin, K., Lohmann, J.U., Anton-Erxleben, F. & Bosch, T.C. Transgenic Hydra allow in vivo tracking of individual stem cells during morphogenesis. Proc. Natl. Acad. Sci. USA 103, 6208–6211 (2006).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  18. Pavlopoulos, A., Oehler, S., Kapetanaki, M.G. & Savakis, C. The DNA transposon Minos as a tool for transgenesis and functional genomic analysis in vertebrates and invertebrates. Genome Biol. 8 (Suppl. 1), S2 (2007).

    Article  Google Scholar 

  19. Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  Google Scholar 

  20. Sternberg, S.H. & Doudna, J.A. Expanding the biologist's toolkit with CRISPR-Cas9. Mol. Cell 58, 568–574 (2015).

    Article  CAS  Google Scholar 

  21. Ishibashi, S., Love, N.R. & Amaya, E. A simple method of transgenesis using I-SceI meganuclease in Xenopus. Methods Mol. Biol. 917, 205–218 (2012).

    Article  CAS  Google Scholar 

  22. Khalturin, K. et al. Transgenic stem cells in Hydra reveal an early evolutionary origin for key elements controlling self-renewal and differentiation. Dev. Biol. 309, 32–44 (2007).

    Article  CAS  Google Scholar 

  23. Siebert, S., Anton-Erxleben, F. & Bosch, T.C. Cell type complexity in the basal metazoan Hydra is maintained by both stem cell based mechanisms and transdifferentiation. Dev. Biol. 313, 13–24 (2008).

    Article  CAS  Google Scholar 

  24. Nakamura, Y., Tsiairis, C.D., Ozbek, S. & Holstein, T.W. Autoregulatory and repressive inputs localize Hydra Wnt3 to the head organizer. Proc. Natl. Acad. Sci. USA 108, 9137–9142 (2011).

    Article  CAS  Google Scholar 

  25. Fritzenwanker, J.H. & Technau, U. Induction of gametogenesis in the basal cnidarian Nematostella vectensis(Anthozoa). Dev. Genes Evol. 212, 99–103 (2002).

    Article  Google Scholar 

  26. Thermes, V. et al. I-SceI meganuclease mediates highly efficient transgenesis in fish. Mech. Dev. 118, 91–98 (2002).

    Article  CAS  Google Scholar 

  27. Grabher, C., Joly, J.S. & Wittbrodt, J. Highly efficient zebrafish transgenesis mediated by the meganuclease I-SceI. Methods Cell Biol. 77, 381–401 (2004).

    Article  CAS  Google Scholar 

  28. Ogino, H., McConnell, W.B. & Grainger, R.M. Highly efficient transgenesis in Xenopus tropicalis using I-SceI meganuclease. Mech. Dev. 123, 103–113 (2006).

    Article  CAS  Google Scholar 

  29. Pan, F.C., Chen, Y., Loeber, J., Henningfeld, K. & Pieler, T. I-SceI meganuclease-mediated transgenesis in Xenopus. Dev. Dyn. 235, 247–252 (2006).

    Article  Google Scholar 

  30. Wang, Y. et al. The meganuclease I-SceI containing nuclear localization signal (NLS-I-SceI) efficiently mediated mammalian germline transgenesis via embryo cytoplasmic microinjection. PLoS One 9, e108347 (2014).

    Article  Google Scholar 

  31. Bevacqua, R.J. et al. Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos. Theriogenology 80, 104–113.e1-29 (2013).

    Article  CAS  Google Scholar 

  32. Fernandez-Martinez, L.T. & Bibb, M.J. Use of the meganuclease I-SceI of Saccharomyces cerevisiae to select for gene deletions in actinomycetes. Sci. Rep. 4, 7100 (2014).

    Article  Google Scholar 

  33. Cohen-Tannoudji, M. et al. I-SceI-induced gene replacement at a natural locus in embryonic stem cells. Mol. Cell Biol. 18, 1444–1448 (1998).

    Article  CAS  Google Scholar 

  34. Choulika, A., Perrin, A., Dujon, B. & Nicolas, J.F. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol. Cell Biol. 15, 1968–1973 (1995).

    Article  CAS  Google Scholar 

  35. Choulika, A., Perrin, A., Dujon, B. & Nicolas, J.F. The yeast I-Sce I meganuclease induces site-directed chromosomal recombination in mammalian cells. C R Acad. Sci. III 317, 1013–1019 (1994).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  37. Renfer, E., Amon-Hassenzahl, A., Steinmetz, P.R. & Technau, U. A muscle-specific transgenic reporter line of the sea anemone, Nematostella vectensis. Proc. Natl. Acad. Sci. USA 107, 104–108 (2010).

    Article  CAS  Google Scholar 

  38. Nakanishi, N., Renfer, E., Technau, U. & Rentzsch, F. Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms. Development 139, 347–357 (2012).

    Article  CAS  Google Scholar 

  39. Richards, G.S. & Rentzsch, F. Transgenic analysis of a SoxB gene reveals neural progenitor cells in the cnidarian Nematostella vectensis. Development 141, 4681–4689 (2014).

    Article  CAS  Google Scholar 

  40. Layden, M.J., Rentzsch, F. & Rottinger, E. The rise of the starlet sea anemone Nematostella vectensis as a model system to investigate development and regeneration. Wiley Interdiscip. Rev. Dev. Biol. 5, 408–428 (2016).

    Article  Google Scholar 

  41. Belfort, M. & Roberts, R.J. Homing endonucleases: keeping the house in order. Nucleic Acids Res. 25, 3379–3388 (1997).

    Article  CAS  Google Scholar 

  42. Rouet, P., Smih, F. & Jasin, M. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol. Cell Biol. 14, 8096–8106 (1994).

    Article  CAS  Google Scholar 

  43. Doyon, J.B., Pattanayak, V., Meyer, C.B. & Liu, D.R. Directed evolution and substrate specificity profile of homing endonuclease I-SceI. J. Am. Chem. Soc. 128, 2477–2484 (2006).

    Article  CAS  Google Scholar 

  44. Moure, C.M., Gimble, F.S. & Quiocho, F.A. The crystal structure of the gene targeting homing endonuclease I-SceI reveals the origins of its target site specificity. J. Mol. Biol. 334, 685–695 (2003).

    Article  CAS  Google Scholar 

  45. Chiarle, R. et al. Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell 147, 107–119 (2011).

    Article  CAS  Google Scholar 

  46. Layden, M.J., Rottinger, E., Wolenski, F.S., Gilmore, T.D. & Martindale, M.Q. Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis. Nat. Protoc. 8, 924–934 (2013).

    Article  Google Scholar 

  47. Genikhovich, G. & Technau, U. Induction of spawning in the starlet sea anemone Nematostella vectensis, in vitro fertilization of gametes, and dejellying of zygotes. Cold Spring Harb. Protoc. 2009, pdb prot5281 (2009).

    PubMed  Google Scholar 

  48. Ogino, H., McConnell, W.B. & Grainger, R.M. High-throughput transgenesis in Xenopus using I-SceI meganuclease. Nat. Protoc. 1, 1703–1710 (2006).

    Article  CAS  Google Scholar 

  49. Schwaiger, M. et al. Evolutionary conservation of the eumetazoan gene regulatory landscape. Genome Res. 24, 639–650 (2014).

    Article  CAS  Google Scholar 

  50. Layden, M.J. et al. MAPK signaling is necessary for neurogenesis in Nematostella vectensis. BMC Biol. 14, 61 (2016).

    Article  Google Scholar 

  51. DuBuc, T.Q. et al. In vivo imaging of Nematostella vectensis embryogenesis and late development using fluorescent probes. BMC Cell Biol. 15, 44 (2014).

    Article  Google Scholar 

  52. Green, M.R. & Sambrook, J. Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2012).

  53. Gibson, D.G. Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides. Nucleic Acids Res. 37, 6984–6990 (2009).

    Article  CAS  Google Scholar 

  54. Technau, U., Kraus, J.E.M. & Genikhovich, G. Cnidaria. in Evolutionary Developmental Biology of Invertebrates, Vol. 1 (ed. Wanninger, A.) (Springer, 2015).

  55. Ikmi, A. & Gibson, M.C. Identification and in vivo characterization of NvFP-7R, a developmentally regulated red fluorescent protein of Nematostella vectensis. PLoS One 5, e11807 (2010).

    Article  Google Scholar 

Download references

Acknowledgements

We thank the members of the Technau lab, in particular M. Martinussen, W. Prunner, G. Sageder and W. Göschl, for animal husbandry. We are grateful to B. Zimmermann for help with the statistics in Figure 3, A. Demilly for providing photos for Figure 2h,i and A. Cole for critically reading the manuscript. This work was supported by grants from the Austrian Science Fund FWF to U.T. (P 27353, P 25993 and P 24858).

Author information

Authors and Affiliations

  1. Department of Molecular Evolution and Development, Faculty of Life Sciences, University of Vienna, Vienna, Austria

    Eduard Renfer & Ulrich Technau

Authors
  1. Eduard Renfer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ulrich Technau
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

U.T. conceived the paper. E.R. generated the data and took photos. E.R. and U.T. wrote the paper.

Corresponding author

Correspondence to Ulrich Technau.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Renfer, E., Technau, U. Meganuclease-assisted generation of stable transgenics in the sea anemone Nematostella vectensis. Nat Protoc 12, 1844–1854 (2017). https://doi.org/10.1038/nprot.2017.075

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2017.075

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research