Transgenic Research

, Volume 20, Issue 1, pp 29–45

Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition

  • Daniel F. Carlson
  • Aron M. Geurts
  • John R. Garbe
  • Chang-Won Park
  • Artur Rangel-Filho
  • Scott M. O’Grady
  • Howard J. Jacob
  • Clifford J. Steer
  • David A. Largaespada
  • Scott C. Fahrenkrug
Original Paper

DOI: 10.1007/s11248-010-9386-5

Cite this article as:
Carlson, D.F., Geurts, A.M., Garbe, J.R. et al. Transgenic Res (2011) 20: 29. doi:10.1007/s11248-010-9386-5

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.

Keywords

Sleeping BeautyTransposonTransgenesisMouseRatMethylation

Abbreviations

SB

Sleeping Beauty

TnT

Transpositional transgenesis

CpG

Cytosine-phosphodiester-guanine

Supplementary material

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Daniel F. Carlson
    • 1
    • 4
  • Aron M. Geurts
    • 1
    • 2
    • 3
  • John R. Garbe
    • 4
  • Chang-Won Park
    • 5
  • Artur Rangel-Filho
    • 2
  • Scott M. O’Grady
    • 4
  • Howard J. Jacob
    • 2
  • Clifford J. Steer
    • 3
    • 5
  • David A. Largaespada
    • 1
    • 3
  • Scott C. Fahrenkrug
    • 1
    • 4
  1. 1.The Center for Genome EngineeringMinneapolisUSA
  2. 2.Human and Molecular Genetics CenterMedical College of WisconsinMilwaukeeUSA
  3. 3.Department of Genetics, Cell Biology and DevelopmentUniversity of MinnesotaMinneapolisUSA
  4. 4.Department of Animal ScienceUniversity of MinnesotaSaint PaulUSA
  5. 5.Institute of Human Genetics and Department of MedicineUniversity of MinnesotaMinneapolisUSA