Gateway-compatible transposon vector to genetically modify human embryonic kidney and adipose-derived stromal cells.
Τίτλος | Gateway-compatible transposon vector to genetically modify human embryonic kidney and adipose-derived stromal cells. |
Publication Type | Journal Article |
Year of Publication | 2012 |
Authors | Petrakis, S., Raskó T., Mátés L., Ivics Z., Izsvák Z., Kouzi-Koliakou K., & Koliakos G. |
Journal | Biotechnol J |
Volume | 7 |
Issue | 7 |
Pagination | 891-7 |
Date Published | 2012 Jul |
ISSN | 1860-7314 |
Λέξεις κλειδιά | Adipose Tissue, Animals, Bacterial Proteins, Cloning, Molecular, DNA Transposable Elements, Gene Expression, Genetic Engineering, Genetic Vectors, HEK293 Cells, Humans, Interferon-beta, Luminescent Proteins, Rats, Rats, Wistar, Recombinant Fusion Proteins, Stromal Cells |
Abstract | 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. |
DOI | 10.1002/biot.201100471 |
Alternate Journal | Biotechnol J |
PubMed ID | 22323455 |