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

Research output: Contribution to journalArticle

23 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)29-45
Number of pages17
JournalTransgenic Research
Volume20
Issue number1
DOIs
StatePublished - 2011

Keywords

  • Methylation
  • Mouse
  • Rat
  • Sleeping Beauty
  • Transgenesis
  • Transposon

ASJC Scopus subject areas

  • Biotechnology
  • Animal Science and Zoology
  • Agronomy and Crop Science
  • Genetics

Fingerprint Dive into the research topics of 'Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition'. Together they form a unique fingerprint.

Cite this