Gateway compatible transposon vector to genetically modify human embryonic kidney and adipose-derived stromal cells

Autor/innen

  • S. Petrakis
  • T. Rasko
  • L. Mates
  • Z. Ivics
  • Z. Izsvak
  • K. Kouzi-Koliakou
  • G. Koliakos

Journal

  • Biotechnology Journal

Quellenangabe

  • Biotechnol J 7 (7): 891-897

Zusammenfassung

  • The Gateway Technology cloning system and the transposon technology represent state-of-the-art laboratory techniques. Combination of these molecular tools may allow rapid cloning of target genes into expression vectors. Here, we describe a novel Gateway Technology compatible transposon plasmid that combines the advantages of the Gateway recombination cloning with the Sleeping Beauty (SB) transposon-mediated transgene integrations. In our system the transposition is catalyzed by the novel hyperactive SB100x transposase providing the high efficient and precise transgene integrations into the host genome. A Gateway compatible transposon plasmid was generated in which the potential target gene may be fused with a Yellow Fluorescent Protein (YFP) tag at the N'- terminal. The vector utilizes the CAGGS promoter to control the fusion protein expression. The transposon expression vector encoding fusion YFP-IFNB1 protein together with the hyperactive SB100x transposase was used to generate stable cell lines in human embryonic kidney (HEK293) and rat adipose-derived stromal cells (ASC). HEK293 and ASCs stably expressed and secreted human interferon-beta protein up to 4 weeks after transfection. The generated Gateway compatible transposon plasmid may be utilized for numerous experimental approaches, such as gene therapy or high-throughput screening methods in primary cells, representing a valuable molecular tool for laboratory applications.


DOI

doi:10.1002/biot.201100471