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Strategies for selection marker-free swine transgenesis using the Sleeping Beauty transposon system

Transgenic Research volume 20, pages 1125–1137 (2011)Cite this article

Abstract

Swine transgenesis by pronuclear injection or cloning has traditionally relied on illegitimate recombination of DNA into the pig genome. This often results in animals containing concatemeric arrays of transgenes that complicate characterization and can impair long-term transgene stability and expression. This is inconsistent with regulatory guidance for transgenic livestock, which also discourages the use of selection markers, particularly antibiotic resistance genes. We demonstrate that the Sleeping Beauty (SB) transposon system effectively delivers monomeric, multi-copy transgenes to the pig embryo genome by pronuclear injection without markers, as well as to donor cells for founder generation by cloning. Here we show that our method of transposon-mediated transgenesis yielded 38 cloned founder pigs that altogether harbored 100 integrants for five distinct transposons encoding either human APOBEC3G or YFP-Cre. Two strategies were employed to facilitate elimination of antibiotic genes from transgenic pigs, one based on Cre-recombinase and the other by segregation of independently transposed transgenes upon breeding.

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Acknowledgments

The authors would like to thank Sandra Horn from the Mouse Genetics lab at the University of Minnesota for help with pig pronuclear injection. We thank Steve Terlouw, Bradley Didion, Richard Koppang, Ann Marie Paprocki, Charity Syverson, Greg Mell, Gordy Gunderson and Eric James for pig cloning, founder management and sample collection and Ludwig and Becky Simmet for their generous support. The authors would also like to thank Zsuzsanna Izsvák, Zoltán Ivics and Lajos Mátés for providing SB100X. This project was supported by National Research Initiative Competitive Grant no. 2008-35205-18852 from the USDA National Institute of Food and Agriculture.

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Affiliations

  1. The Center for Genome Engineering, University of Minnesota, Minneapolis, MN, 55455, USA

    Daniel F. Carlson, Wenfang Tan, Perry B. Hackett & Scott C. Fahrenkrug

  2. Department of Animal Science, University of Minnesota, Saint Paul, MN, 55108, USA

    Daniel F. Carlson, John R. Garbe, Wenfang Tan & Scott C. Fahrenkrug

  3. Spring Point Project, Minneapolis, MN, 55402, USA

    Mike J. Martin

  4. Minitube Biotechnology Center, Mt. Horeb, WI, 53572, USA

    John R. Dobrinsky

  5. Department of Genetics, Cell Biology and Development, Minneapolis, MN, 55455, USA

    Perry B. Hackett

  6. Recombinetics, 2751 Hayes Street, Minneapolis, MN, 55418, USA

    Scott C. Fahrenkrug

  7. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, 55902, USA

    Karl J. Clark

Authors
  1. Daniel F. Carlson
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  2. John R. Garbe
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  3. Wenfang Tan
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  4. Mike J. Martin
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  5. John R. Dobrinsky
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  6. Perry B. Hackett
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  7. Karl J. Clark
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  8. Scott C. Fahrenkrug
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Corresponding author

Correspondence to Scott C. Fahrenkrug.

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Carlson, D.F., Garbe, J.R., Tan, W. et al. Strategies for selection marker-free swine transgenesis using the Sleeping Beauty transposon system. Transgenic Res 20, 1125–1137 (2011). https://doi.org/10.1007/s11248-010-9481-7

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  • DOI: https://doi.org/10.1007/s11248-010-9481-7

Keywords

  • Swine transgenesis
  • Sleeping Beauty
  • Transposon
  • Cre recombinase
  • Cloning
  • Pronuclear injection