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. Author manuscript; available in PMC: 2024 Dec 11.
Published in final edited form as: Nat Genet. 2019 Jan 14;51(2):285–295. doi: 10.1038/s41588-018-0305-7

Figure 6. Xist expression is not necessary for homologous Xic pairing.

Figure 6

A) Schematic representation of the derivation of a homozygous Xist double knock-out (DKO) cell line in PGK12.1 XXTetO cells by CRISPR/Cas9-mediated genetic engineering. B) RNA FISH for nascent Tsix (red) and Huwe1 (white) transcripts as well as processed and accumulating Xist RNA (green) performed in PGK12.1 XXTetO (control) and PGK12.1 XXTetO ΔXist DKO in ESCs and at day 4 of differentiation after LIF withdrawal (depicted are representative cells for each condition). C) Quantification of monoallelic and biallelic Xist RNA accumulation as well as monoallelic and biallelic Tsix expression determined by presence of Xist RNA domains/clusters as well as focal signals for Tsix in RNA FISH. D) DNA FISH for the Xist/Tsix region (BAC 8, red) performed in PGK12.1 XXTetO (control) and PGK12.1 XXTetO ΔXist DKO in ESCs and at day 4 of differentiation after LIF withdrawal. Depicted are representative cells for each condition. E) Fraction of cells with Xic at pairing distance in differentiating PGK12.1 XXTetO (control) and PGK12.1 XXTetO ΔXist DKO cells. 3D distance was determined after DNA FISH for the Xist/Tsix region (BAC 8) as depicted in D). (n = number of cells, scale bar = 2 µm)