Section 66
Chapter 65,416

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Leko, V.; Sripathy, S.; Adrianse, R.L.; Loe, T.; Park, A.; Lao, U.; Foss, E.J.; Bartolomei, M.S.; Bedalov, A.

Journal of Visualized Experiments: Jove 2018(133)


ISSN/ISBN: 1940-087X
PMID: 29553562
DOI: 10.3791/56398
Accession: 065415269

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Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

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