Premium
Gateway‐compatible transposon vector to genetically modify human embryonic kidney and adipose‐derived stromal cells.
Author(s) -
Petrakis Spyros,
Raskó Tamas,
Mátés Lajos,
Ivics Zoltan,
Izsvák Zsuzsanna,
KouziKoliakou Kokkona,
Koliakos George
Publication year - 2012
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201100471
Subject(s) - transposase , sleeping beauty transposon system , biology , transposable element , hek 293 cells , plasmid , transposon mutagenesis , fusion protein , transgene , cloning (programming) , gene , genetics , computational biology , microbiology and biotechnology , genome , recombinant dna , computer science , programming language
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.