Strategies for selection marker-free swine transgenesis using the Sleeping Beauty transposon system

Daniel F. Carlson, John R. Garbe, Wenfang Tan, Mike J. Martin, John R. Dobrinsky, Perry B. Hackett, Karl J. Clark, Scott C. Fahrenkrug

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Swine transgenesis by pronuclear injection or cloning has traditionally relied on illegitimate recombination of DNA into the pig genome. This often results in animals containing concatemeric arrays of transgenes that complicate characterization and can impair long-term transgene stability and expression. This is inconsistent with regulatory guidance for transgenic livestock, which also discourages the use of selection markers, particularly antibiotic resistance genes. We demonstrate that the Sleeping Beauty (SB) transposon system effectively delivers monomeric, multi-copy transgenes to the pig embryo genome by pronuclear injection without markers, as well as to donor cells for founder generation by cloning. Here we show that our method of transposon-mediated transgenesis yielded 38 cloned founder pigs that altogether harbored 100 integrants for five distinct transposons encoding either human APOBEC3G or YFP-Cre. Two strategies were employed to facilitate elimination of antibiotic genes from transgenic pigs, one based on Cre-recombinase and the other by segregation of independently transposed transgenes upon breeding.

Original languageEnglish (US)
Pages (from-to)1125-1137
Number of pages13
JournalTransgenic Research
Volume20
Issue number5
DOIs
StatePublished - Oct 1 2011

Fingerprint

Beauty
Gene Transfer Techniques
transposons
Transgenes
Swine
transgenes
swine
Organism Cloning
molecular cloning
genetically modified organisms
Genome
injection
Injections
genome
Livestock
Microbial Drug Resistance
antibiotic resistance
Genetic Recombination
Genes
Breeding

Keywords

  • Cloning
  • Cre recombinase
  • Pronuclear injection
  • Sleeping Beauty
  • Swine transgenesis
  • Transposon

Cite this

Strategies for selection marker-free swine transgenesis using the Sleeping Beauty transposon system. / Carlson, Daniel F.; Garbe, John R.; Tan, Wenfang; Martin, Mike J.; Dobrinsky, John R.; Hackett, Perry B.; Clark, Karl J.; Fahrenkrug, Scott C.

In: Transgenic Research, Vol. 20, No. 5, 01.10.2011, p. 1125-1137.

Research output: Contribution to journalArticle

Carlson, Daniel F. ; Garbe, John R. ; Tan, Wenfang ; Martin, Mike J. ; Dobrinsky, John R. ; Hackett, Perry B. ; Clark, Karl J. ; Fahrenkrug, Scott C. / Strategies for selection marker-free swine transgenesis using the Sleeping Beauty transposon system. In: Transgenic Research. 2011 ; Vol. 20, No. 5. pp. 1125-1137.
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