Gateway‐compatible transposon vector to genetically modify human embryonic kidney and adipose‐derived 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.

Autologous administration of adipose stromal cells improves skin flap survival through neovascularization: An experimental study

“…After the addition of an equal volume of phosphate‐buffer saline (PBS), the mixture was isolated for 15 minutes at room temperature, until three distinct layers were formed. The middle layer, containing the stromal cells, was aspirated with a syringe and then centrifuged for 10 minutes at 600 × g. The sample was, thereby, divided into two layers, the lower one including the stromal cells …”

Sleeping Beautytransposition: from biology to applications

“…The characterized expression cassette can be exchanged relatively simply when the SB cassette is combined with other molecular engineering tools (Grabundzija et al, 2013;Petrakis et al, 2012). A powerful strategy could be used to express a series of expression cassettes from the same genomic locus by cassette exchange using Cre or FLP recombinases (Garrels et al, 2016a;Grabundzija et al, 2013).…”

Stem cell models of polyglutamine diseases and their use in cell-based therapies

“…Would it be possible to produce a robust pathogenic polyQ phenotype in MSCs? To this end, we generated an inducible mesenchymal stem cell model for the polyQ disease SCA1 using the Sleeping Beauty transposon system (Petrakis et al, 2012a ). These cells stably overexpress the mutant gene ataxin-1 (ATXN1Q82) in fusion with Yellow Fluorescent Protein (YFP) under the Tet-On promoter.…”

“…The Sleeping Beauty transposon system is one of them; it has major advantages over viral vectors including high transgene expression, ease of handling and more importantly biosafety (Galla et al, 2011 ). In addition, it can be used for the genetic modification of MSCs and the generation of cell lines stably expressing a gene of interest (Petrakis et al, 2012a ).…”