Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition.
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.
Carlson
Daniel F
DF
The Center for Genome Engineering, Minneapolis, MN, USA.
Geurts
Aron M
AM
Garbe
John R
JR
Park
Chang-Won
CW
Rangel-Filho
Artur
A
O'Grady
Scott M
SM
Jacob
Howard J
HJ
Steer
Clifford J
CJ
Largaespada
David A
DA
Fahrenkrug
Scott C
SC
D000818:Animals; D030801:Animals, Genetically Modified; D019175:DNA Methylation; D004251:DNA Transposable Elements / Q000235:genetics*; D004622:Embryo, Mammalian; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D018014:Gene Transfer Techniques; D006801:Humans; D051379:Mice; D051381:Rats; D019076:Transgenes; D019895:Transposases / Q000235:genetics
D004251:DNA Transposable Elements; D019895:Transposases