Abstract
Transgenics is a mainstay of functional genomics. Conditionally overexpressing genes of interest (GOIs) helps to reveal their roles in the control of complex biological processes. Complemented by findings in classic animal model systems, recent advances in human embryonic stem cell (hESC) and patient-specific induced pluripotent stem cell (hiPSC) differentiation have led to sophisticated in vitro models of human development and disease. Yet, as transgenic elements encoding inducible systems must be introduced de novo into each genetically unique human stem cell line, robust and straightforward solutions to gene delivery are required. Transposons are a family of mobile DNA elements that have been adapted as experimental tools for stable genomic integration of transgenes. The piggyBac (PB) transposon from Trichoplusia ni presents a number of benefits over classic viral or BAC transgenesis: ease of application, simple integration-site mapping, and the unique capacity for traceless excision. Moreover, their large capacity permits the consolidation of multiple transgene components in a single vector system. In this chapter, we outline the features of a panel of “All-in-One” PB transposons designed for drug-inducible gene expression and provide guidelines to establish and validate populations or clones of transgenic hiPSCs.
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Acknowledgments
We like to recognize Ryoko Hirohata and Tomoko Matsumoto for their valuable technical support in developing these materials and protocols. Our appreciation to all our colleagues who continue to make use of these transposon resources, in various research applications. This work was supported by the Research Center Network for Realization of Regenerative Medicine, Program for Intractable Diseases Research Utilizing Disease-Specific iPS Cells, of the Japan Science and Technology Agency (JST), and the Strategic International Collaborative Research Program of the JST. K.W. is a Hakubi Center Special Project Researcher. S.Y. is a non-salaried scientific advisor of iPS Academia Japan. S-I.K. is a JSPS Fellowship recipient (2011–2013) and JST Researcher.
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Kim, SI., Oceguera-Yanez, F., Sakurai, C., Nakagawa, M., Yamanaka, S., Woltjen, K. (2015). Inducible Transgene Expression in Human iPS Cells Using Versatile All-in-One piggyBac Transposons. In: Turksen, K., Nagy, A. (eds) Induced Pluripotent Stem (iPS) Cells. Methods in Molecular Biology, vol 1357. Humana Press, New York, NY. https://doi.org/10.1007/7651_2015_251
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DOI: https://doi.org/10.1007/7651_2015_251
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