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Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct

Nature Protocols volume 1, pages 1133–1139 (2006)Cite this article

Abstract

Small fish are a popular laboratory model for studying gene expression and function by transgenesis. If, however, the transgenes are not readily detectable by visual inspection, a large number of embryos must be injected, raised and screened to identify positive founder fish. Here, we describe a strategy to efficiently generate and preselect transgenic lines harbouring any transgene of interest. Co-injection of a selectable reporter construct (e.g., GFP), together with the transgene of interest on a separate plasmid using the I-SceI meganuclease approach, results in co-distribution of the two plasmids. The quality of GFP expression within the F0 generation therefore reflects the quality of injection and allows efficient and reliable selection of founder fish that are also positive for the second transgene of interest. In our experience, a large fraction (up to 50%) of GFP-positive fish will also be transgenic for the second transgene, thus providing a rapid (within 3–4 months) and efficient way to establish transgenic lines for any gene of interest in medaka and zebrafish.

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Figure 1
Figure 2: Injection setup.
Figure 3: 'The ideal needle': Micropipettes for injection into zebrafish or medaka embryos.
Figure 4: Open versus closed needles for medaka and zebrafish.
Figure 5: Schematic drawing of the agarose mold.
Figure 6: The little metal hook can be used to collect medaka embryos.
Figure 7
Figure 8: Overview of the procedure and crossing scheme.

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Acknowledgements

We thank Aldona Nowicka and Diana Hofmann for expert fish husbandry and Felix Loosli for critical input to the manuscript. This work was supported by a grant from the European Union (FP6 Strep: Plurigenes) to J.W.

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Author notes

  1. Martina Rembold and Kajori Lahiri: These authors contributed equally to this work.

Authors and Affiliations

  1. Developmental Biology Unit, EMBL Heidelberg, Meyerhofstrasse 1, Heidelberg, 69117, Germany

    Martina Rembold & Joachim Wittbrodt

  2. Max-Planck Institut fuer Entwicklungsbiologie, Spemannstrasse 35, Tuebingen, 72076, Germany

    Kajori Lahiri & Nicholas S. Foulkes

Authors
  1. Martina Rembold
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  2. Kajori Lahiri
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  3. Nicholas S. Foulkes
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  4. Joachim Wittbrodt
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Corresponding authors

Correspondence to Nicholas S. Foulkes or Joachim Wittbrodt.

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Rembold, M., Lahiri, K., Foulkes, N. et al. Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct. Nat Protoc 1, 1133–1139 (2006). https://doi.org/10.1038/nprot.2006.165

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