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Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector

Nature Biotechnology volume 18, pages 81–84 (2000)Cite this article

An Erratum to this article was published on 01 May 2000

Abstract

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.

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Figure 1: G1 transgenic silkworms positive for GFP.
Figure 2: Organization of the piggyBac-derived constructs.
Figure 3: Southern blot hybridization of DNA from transformed and control animals.
Figure 4: Genomic DNA sequences surrounding piggyBac insertions, with the TTAA duplicated sequence appearing at all 5′ and 3′ insert boundaries.

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Acknowledgements

We thank Marie-Andrée Guérin, Valérie Carraro, Seo Kazuko, and Yamazaki Hiroko for technical assistance, and Hervé Bossin and Yukuhiro Kenji for their help. We also thank Paul Brey and Charlie Roth for editorial help. The group in Tsukuba was supported by the Japanese MAFF. The group in Villeurbanne was supported by the CNRS and the French MENRT and the group in La Mulatière by the INRA; both also received support from the Europe Union (Contract n° CI1*CT94-0092) and the DRET (Contract n° 95/099).

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Authors and Affiliations

  1. National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki, 305-8634, Japan

    Toshiki Tamura, Toshio Kanda, Abraham Eappen, Mari Kamba & Natuo Kômoto

  2. Centre de Génétique Moléculaire et Cellulaire, CNRS, Villeurbanne, 69622, France

    Chantal Thibert, Jean-Claude Prudhomme & Pierre Couble

  3. Unité Nationale Séricicole, INRA, La Mulatière, 69150, France

    Corinne Royer, Jean-Luc Thomas, Bernard Mauchamp & Gérard Chavancy

  4. USDA ARS CMAVE, Gainesville, FL

    Paul Shirk

  5. Department of Biological Science, University of Notre Dame, Notre Dame, IN

    Malcolm Fraser

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  1. Toshiki Tamura
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Correspondence to Pierre Couble.

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Tamura, T., Thibert, C., Royer, C. et al. Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol 18, 81–84 (2000). https://doi.org/10.1038/71978

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