Original Paper

Transgenic Research

, Volume 20, Issue 1, pp 29-45

First online:

Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition

  • Daniel F. CarlsonAffiliated withThe Center for Genome EngineeringDepartment of Animal Science, University of Minnesota
  • , Aron M. GeurtsAffiliated withThe Center for Genome EngineeringHuman and Molecular Genetics Center, Medical College of WisconsinDepartment of Genetics, Cell Biology and Development, University of Minnesota
  • , John R. GarbeAffiliated withDepartment of Animal Science, University of Minnesota
  • , Chang-Won ParkAffiliated withInstitute of Human Genetics and Department of Medicine, University of Minnesota
  • , Artur Rangel-FilhoAffiliated withHuman and Molecular Genetics Center, Medical College of Wisconsin
  • , Scott M. O’GradyAffiliated withDepartment of Animal Science, University of Minnesota
  • , Howard J. JacobAffiliated withHuman and Molecular Genetics Center, Medical College of Wisconsin
  • , Clifford J. SteerAffiliated withDepartment of Genetics, Cell Biology and Development, University of MinnesotaInstitute of Human Genetics and Department of Medicine, University of Minnesota
  • , David A. LargaespadaAffiliated withThe Center for Genome EngineeringDepartment of Genetics, Cell Biology and Development, University of Minnesota
    • , Scott C. FahrenkrugAffiliated withThe Center for Genome EngineeringDepartment of Animal Science, University of Minnesota Email author 

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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.

Keywords

Sleeping Beauty Transposon Transgenesis Mouse Rat Methylation