Figure 5.

Morphology and Southern analysis of A9 hybrids harboring demethylated human fragile X expansions that were retransferred from EC cells. Microcells were generated from MLEC30.1 and fused to A9 cells. A, Morphology of a hybrid clone (16*). Bar = 100 μm. B, Isolated clones harboring chromosomes of both murine parents and a human FMR1 gene, analyzed on EcoRI-plus-EagI blots hybridized to Ox1.9. The results for nine clones, indicated by numbers, are shown. Notably sharp and intense signals of full-mutation fragments with unmethylated EagI sites were obtained in many clones; for example, 16* (315 CGGs), 22 (265 CGGs), and 24 (280 CGGs). C, One of the A9 hybrids (16*), subcloned to further prove mitotic stability of the expansion. The unmethylated full-mutation allele could be amplified by cloning (lanes 1–4) and did not experience any significant change of repeat size upon clonal expansion.