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Interhomolog Homologous Recombination in Mouse Embryonic Stem Cells

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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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APA

Vanoli, F., Prakash, R., White, T., & Jasin, M. (2021). Interhomolog Homologous Recombination in Mouse Embryonic Stem Cells. In Methods in Molecular Biology (Vol. 2153, pp. 127–143). Humana Press Inc. https://doi.org/10.1007/978-1-0716-0644-5_10

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