Efficient stable gene transfer into human cells by the Sleeping Beauty transposon vectors |
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Authors: | Zsuzsanna Izsvk Marinee KL Chuah Thierry VandenDriessche Zoltn Ivics |
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Institution: | aMax Delbrück Center for Molecular Medicine, Berlin, Germany;bFlanders Institute for Biotechnology (VIB), Vesalius Research Center, University of Leuven, Leuven, Belgium;cInstitute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary |
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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. |
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Keywords: | Sleeping Beauty Transposon Non-viral Gene delivery CD34+ HeLa Gene expression Transfection Nucleofection |
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