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LINE-1 Retrotransposition Assays in Embryonic Stem Cells

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Transposable Elements

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2607))

Abstract

The ongoing mobilization of active non-long terminal repeat (LTR) retrotransposons continues to impact the genomes of most mammals, including humans and rodents. Non-LTR retrotransposons mobilize using an intermediary RNA and a copy-and-paste mechanism termed retrotransposition. Non-LTR retrotransposons are subdivided into long and short interspersed elements (LINEs and SINEs, respectively), depending on their size and autonomy; while active class 1 LINEs (LINE-1s or L1s) encode the enzymatic machinery required to mobilize in cis, active SINEs use the enzymatic machinery of active LINE-1s to mobilize in trans. The mobilization mechanism used by LINE-1s/SINEs was exploited to develop ingenious plasmid-based retrotransposition assays in cultured cells, which typically exploit a reporter gene that can only be activated after a round of retrotransposition. Retrotransposition assays, in cis or in trans, are instrumental tools to study the biology of mammalian LINE-1s and SINEs. In fact, these and other biochemical/genetic assays were used to uncover that endogenous mammalian LINE-1s/SINEs naturally retrotranspose during early embryonic development. However, embryonic stem cells (ESCs) are typically used as a cellular model in these and other studies interrogating LINE-1/SINE expression/regulation during early embryogenesis. Thus, human and mouse ESCs represent an excellent model to understand how active retrotransposons are regulated and how their activity impacts the germline. Here, we describe robust and quantitative protocols to study human/mouse LINE-1 (in cis) and SINE (in trans) retrotransposition using (human and mice) ESCs. These protocols are designed to study the mobilization of active non-LTR retrotransposons in a cellular physiologically relevant context.

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Acknowledgments

M.G.-C. acknowledges the support of Fundación Pública Andaluza “Progreso y Salud,” Seville, Spain. F.J.S.-L. acknowledges the support of an EMERGIA20_00225 Grant from the Council of Economy, Knowledge, Enterprises, and University of the Andalusian Government (Spain). J.L.G.P.’s lab is supported by MINECO-FEDER [SAF2017-89745-R], CTEICU-PAIDI_2020-FEDER [P18-RT-5067] and by a private donation from Ms. Francisca Serrano (Trading y Bolsa para Torpes, Granada, Spain).

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Garcia-Cañadas, M., Sanchez-Luque, F.J., Sanchez, L., Rojas, J., Garcia Perez, J.L. (2023). LINE-1 Retrotransposition Assays in Embryonic Stem Cells. In: Branco, M.R., de Mendoza Soler, A. (eds) Transposable Elements. Methods in Molecular Biology, vol 2607. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2883-6_13

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