Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Biochemical Research Methods, Biochemistry & Molecular Biology, Sleeping Beauty, Transposon, Non-viral, Gene delivery, CD34(+), HeLa, Gene expression, Transfection, Nucleofection, MESENCHYMAL STEM-CELLS, EXPRESSION IN-VIVO, TRANSGENE EXPRESSION, SITE SELECTION, SOMATIC-CELLS, ACTIVE GENES, HUMAN GENOME, INTEGRATION, DELIVERY, THERAPY, DNA Transposable Elements, Gene Transfer Techniques, Genetic Vectors, HeLa Cells, Hematopoietic Stem Cells, Humans, Hela Cells, 1103 Clinical Sciences, 3101 Biochemistry and cell biology

Abstract:

Transposable elements can be considered as natural, non-viral gene delivery vehicles capable of efficient genomic insertion. The plasmid-based transposon system of Sleeping Beauty (SB) combines the advantages of viruses and naked DNA molecules. In contrast to plasmid vectors, transposons integrate through a precise, recombinase-mediated mechanism into chromosomes, providing long-term expression of the gene of interest in cells. The advantages of transposons in comparison to viral systems include their simplicity and improved safety/toxicity profiles. In addition, the hyperactive SB100X is the first plasmid-based delivery system that overcomes the efficacy of non-viral delivery. The transposon delivery system consists of the transposase and the integration cassette, recognized by the transposase. The plasmid-based transposon delivery system can be combined with any non-viral delivery method. Here we provide two detailed protocols to apply SB-mediated, non-viral gene transfer in cultured cells. In our first example, we use a lipid-based delivery method in combination with the transposon-based integration system in an easy-to-transfect (HeLa) cell line. Second, we show how to achieve 40-50% stable expression of a transgene in clinically relevant, hard-to-transfect cells (hematopoetic stem cells, HSCs) by nucleofection. The given protocols are adaptable to any vertebrate cells in culture.