Figure 2.
Stability of XIST reactivation in active X hybrid clones treated with 5aCdr and DNA methylation analysis. (A) XIST-positive clones derived from the 5aCdr-treated GM06318 culture were expanded and maintained for several generations to determine the stability of XIST reactivation. XIST expression was analyzed in these clones and X8–6T2S1 by semiquantitative RT-PCR as described in Materials and Methods; shown is a Southern blot analysis of XIST:MIC2 RT-PCR products. All samples were run at two concentrations in the RT reaction (0.5 or 1.0 μg RNA/reaction). (B) Methylation analysis of the 5′ region of XIST. Genomic DNA was digested with either PstI alone or with PstI and SacII together. SacII sites in the 5′ region of XIST are unmethylated on the expressed allele and methylated on the repressed allele in human cells or in cell hybrids. 4F5, 7H10, and 9B7 are 5aCdr-treated GM06318 clones that exhibited strong XIST reactivation at a very early stage of growth. Clones derived from 5aCdr-treated normal male fibroblasts were analyzed after growing to ≈3 × 106 cells.