Max Delbrück Center for Molecular Medicine, Berlin, Germany.
Methods. 2009 Nov;49(3):287-97. doi: 10.1016/j.ymeth.2009.07.001. Epub 2009 Jul 15.
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.
转座元件可以被视为天然的、非病毒基因传递载体，能够高效地进行基因组插入。Sleeping Beauty（SB）的基于质粒的转座子系统结合了病毒和裸露 DNA 分子的优势。与质粒载体相比，转座子通过精确的、重组酶介导的机制整合到染色体中，为细胞中感兴趣的基因提供长期表达。与病毒系统相比，转座子的优势包括其简单性和改善的安全性/毒性特征。此外，超活性 SB100X 是第一个克服非病毒传递功效的基于质粒的递送系统。转座子递送系统由转座酶和整合盒组成，由转座酶识别。基于质粒的转座子递送系统可以与任何非病毒递送方法结合使用。在这里，我们提供了两个详细的方案，用于在培养细胞中应用 SB 介导的非病毒基因转移。在我们的第一个例子中，我们使用基于脂质的递送方法与基于转座子的整合系统结合，应用于易于转染的（HeLa）细胞系。其次，我们展示了如何通过核转染在临床上相关的、难以转染的细胞（造血干细胞，HSCs）中实现 40-50%的转基因稳定表达。所提供的方案适用于培养中的任何脊椎动物细胞。
