Department of Chemistry and Biotechnology, School of Science, Computing and Engineering Technologies, Swinburne University of Technology, Hawthorn, VIC, Australia.
Methods Mol Biol. 2022;2495:49-66. doi: 10.1007/978-1-0716-2301-5_3.
The piggyBac transposon system has been adapted to be a highly efficient genome engineering tool for transgenesis of eukaryotic cells and organisms. As with other methods of transgenesis, incorporation of an inducible promoter, such as a tetracycline-responsive element, enables inducible transgene expression. Here, we describe an efficient method of using the piggyBac system to create stably transfected mammalian cell lines, including inducible transgene expression. Gibson assembly is used to construct the required vectors as it enables multiple DNA fragments to be seamlessly assembled in a single isothermal reaction. We demonstrate an application of this approach to generate a stably transfected pluripotent stem cell line that can be induced to express a transcription factor transgene and rapidly differentiate into neurons in a single step.
piggyBac 转座子系统已被改编为一种高效的基因组工程工具，用于真核细胞和生物的转基因。与其他转基因方法一样，整合诱导型启动子，如四环素反应元件，可实现诱导型转基因表达。在这里，我们描述了一种使用 piggyBac 系统创建稳定转染哺乳动物细胞系的有效方法，包括诱导型转基因表达。Gibson 组装用于构建所需的载体，因为它能够在单个等温反应中无缝组装多个 DNA 片段。我们展示了这种方法的一个应用，生成了一种稳定转染的多能干细胞系，该细胞系可以被诱导表达转录因子转基因，并在一步中快速分化为神经元。
