Abstract
Homologous recombination is a critical mechanism for the repair of DNA double-strand breaks (DSBs). It occurs predominantly between identical sister chromatids and at lower frequency can also occur between homologs. Interhomolog homologous recombination (IH-HR) has the potential lead to substantial loss of genetic information, i.e., loss of heterozygosity (LOH), when it is accompanied by crossing over. In this chapter, we describe a system to study IH-HR induced by a defined DSB in mouse embryonic stem cells derived from F1 hybrid mice. This system is based on the placement of mutant selectable marker genes, one of which contains an I-SceI endonuclease cleavage site, on the two homologs such that repair of the I-SceI-generated DSB from the homolog leads to drug resistance. Loss of heterozygosity arising during IH-HR is analyzed using a PCR-based approach. Finally, we present a strategy to analyze the role of BLM helicase in this system.
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Acknowledgments
We would like to thank Jeremy Stark for helpful input. This work was supported by MSK Cancer Center Support Grant/Core Grant (NIH P30CA008748), NIH F32GM110978 (R.P.), American Cancer Society-New York Cancer Research Fund PF-17-136-01-DMC (T.W.), and grants to M.J. from the MSK Functional Genomics Initiative and NIH (R35GM118175, R01CA185660).
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Vanoli, F., Prakash, R., White, T., Jasin, M. (2021). Interhomolog Homologous Recombination in Mouse Embryonic Stem Cells. In: Aguilera, A., Carreira, A. (eds) Homologous Recombination. Methods in Molecular Biology, vol 2153. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0644-5_10
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DOI: https://doi.org/10.1007/978-1-0716-0644-5_10
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