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

• Published in: Biotechnology journal Vol. 7; no. 7; p. 891
• Main Authors: Petrakis, Spyros, Raskó, Tamas, Mátés, Lajos, Ivics, Zoltan, Izsvák, Zsuzsanna, Kouzi-Koliakou, Kokkona, Koliakos, George
• Format: Journal Article
• Language: English
• Published: Germany 01.07.2012
• Subjects: Adipose Tissue - cytology; Adipose Tissue - physiology; Animals; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cloning, Molecular - methods; DNA Transposable Elements; Gene Expression; Genetic Engineering - methods; Genetic Vectors - genetics; HEK293 Cells; Humans; Interferon-beta - genetics; Interferon-beta - metabolism; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Rats; Rats, Wistar; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Stromal Cells - cytology; Stromal Cells - physiology
• ISSN: 1860-7314
• DOI: 10.1002/biot.201100471

The Gateway technology cloning system and transposon technology represent state-of-the-art laboratory techniques. Combination of these molecular tools allows rapid cloning of target genes into expression vectors. Here, we describe a novel Gateway technology-compatible transposon plasmid that combines the advantages of 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, and provides highly efficient and precise transgene integrations into the host genome. A Gateway-compatible transposon plasmid was generated in which the potential target gene can be fused with a yellow fluorescent protein (YFP) tag at the N-terminal. The vector utilizes the CAGGS promoter to control fusion protein expression. The transposon expression vector encoding the YFP-interferon-β protein (IFNB1) fusion 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). ASCs and HEK293 cells stably expressed and secreted the human IFNB1 for up to 4 weeks after transfection. The generated Gateway-compatible transposon plasmid can 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.