Faculty Collaboration Database

Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition. Transgenic Res 2011 Feb;20(1):29-45

Date

03/31/2010

Pubmed ID

20352328

Pubmed Central ID

PMC3516389

DOI

10.1007/s11248-010-9386-5

Scopus ID

2-s2.0-78651398529 (requires institutional sign-in at Scopus site)   25 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.

Author List

Carlson DF, Geurts AM, Garbe JR, Park CW, Rangel-Filho A, O'Grady SM, Jacob HJ, Steer CJ, Largaespada DA, Fahrenkrug SC

Author

Aron Geurts PhD Professor in the Physiology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Animals, Genetically Modified
DNA Methylation
DNA Transposable Elements
Embryo, Mammalian
Enhancer Elements, Genetic
Gene Transfer Techniques
Humans
Mice
Rats
Transgenes
Transposases