Gateway-compatible transposon vector to genetically modify human embryonic kidney and adipose-derived stromal cells.
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.
Biohellenika Biotechnology company, Thessaloniki, Greece. spetrakis@biohellenika.gr
Spyros
S
Petrakis
Tamas
T
Raskó
Lajos
L
Mátés
Zoltan
Z
Ivics
Zsuzsanna
Z
Izsvák
Kokkona
K
Kouzi-Koliakou
George
G
Koliakos
D000273:Adipose Tissue / Q000166:cytology / Q000502:physiology*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D016899:Interferon-beta / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology / Q000502:physiology
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D016899:Interferon-beta