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Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition

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Abstract

Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline.

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Abbreviations

SB:

Sleeping Beauty

TnT:

Transpositional transgenesis

CpG:

Cytosine-phosphodiester-guanine

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Acknowledgments

The authors would like to thank Dr. Randy Daughters of the University of Minnesota for providing the HSA-CCTG300 transgene, Sandra Wagner of the University of Minnesota Mouse Genetics lab for mouse PNI, Drs. Wanda Filipiak and Thom Saunders at the University of Michigan Transgenic Animal Model Core for training in rat PNI and Dr. Yang Da at the University of Minnesota for statistical support.

Funding

This work was in part supported by a U of MN Academic Health Center Faculty Development Grant and National Institutes of Health grant 5R56DK074010-02 to Drs. O’Grady, Steer and Fahrenkrug.

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Authors and Affiliations

  1. The Center for Genome Engineering, Minneapolis, MN, USA

    Daniel F. Carlson, Aron M. Geurts, David A. Largaespada & Scott C. Fahrenkrug

  2. Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

    Aron M. Geurts, Artur Rangel-Filho & Howard J. Jacob

  3. Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA

    Aron M. Geurts, Clifford J. Steer & David A. Largaespada

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

    Daniel F. Carlson, John R. Garbe, Scott M. O’Grady & Scott C. Fahrenkrug

  5. Institute of Human Genetics and Department of Medicine, University of Minnesota, Minneapolis, MN, 55455, USA

    Chang-Won Park & Clifford J. Steer

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  1. Daniel F. Carlson
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  2. Aron M. Geurts
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  3. John R. Garbe
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  4. Chang-Won Park
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  5. Artur Rangel-Filho
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  6. Scott M. O’Grady
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  7. Howard J. Jacob
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  8. Clifford J. Steer
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  9. David A. Largaespada
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  10. Scott C. Fahrenkrug
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Correspondence to Scott C. Fahrenkrug.

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Carlson, D.F., Geurts, A.M., Garbe, J.R. et al. Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition. Transgenic Res 20, 29–45 (2011). https://doi.org/10.1007/s11248-010-9386-5

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