Fig. 1 | Nature Communications

Fig. 1

From: Genetic and epigenetic features direct differential efficiency of Xist-mediated silencing at X-chromosomal and autosomal locations

Fig. 1

Generation of a tetracycline-responsive Xist expression system in ES cells. a Targeting strategy to generate Tg-E clones. The Xist TSS was replaced with a bidirectional tetracycline-responsive Ptight promoter, a DsRed reporter gene, and a neomycin resistance cassette. b PCR amplification with primers indicated in a followed by a Tsp509I RFLP digest of PCR product to identify clones with a correctly targeted Cast/Ei allele. Correct targeting results in loss of Cast/Ei-specific band, as shown for clones 7, 41, and 56. Arrows indicate size of PCR product and Tsp509I restriction fragments. F1 F1 2-1 polymorphic 129/Sv-Cast/Ei mother cell line, M marker, H water. c PCR amplification of a fragment of the X-linked gene Atrx and Pf1MI digest of the PCR product to verify the presence of two X chromosomes. Arrows indicate size of PCR product and size of Pf1MI restriction fragments. d, f Schematic representation of the strategy used to generate Tg-X, Tg-X;8 d, and Tg-12 clones f. The Xist transgene was randomly integrated into F1 2-1 40,XX,t(X;8) or 41,XX,dup12 ESC lines and neomycin-resistant clones were screened by DNA FISH. e, g DNA FISH screening of Tg-X, Tg-X;8 e, and Tg-12 g clones. Arrows indicate the chromosome carrying the Xist transgene. Positive Tg-X;8 clones show co-localization of three DNA FISH signals on the translocated X;8 chromosome corresponding to the Xist endogenous locus, the ectopic transgene, and the chromosome 8 portion of the X;8 translocation. Three independent ES clones were generated (267, 339, 203b). Positive Tg-X clones show two DNA FISH signals on the wild-type Cast/Ei X corresponding to the endogenous Xist locus and to the ectopic transgene, respectively. Four independent ES clones were generated (86, 190, 85, 109). Positive Tg-12 clones show co-localization of two DNA FISH signals on one of the three copies of chromosome 12, corresponding to the Xist-inducible transgene and to chromosome 12, respectively. Six independent ES clones have been generated (251, 160, 273, 292, 55, 228). Scale bars represent 5 μm

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