Fig. 4.

Unmethylated FM alleles do not become silenced on differentiation into neurons. The repeat size, methylation status, and FMR1 mRNA levels of the indicated cultures were monitored as described in the “Methods” section. The “+” and “−” signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. A late passage culture of 37D ESCs containing little, if any methylated alleles, was differentiated into neurons as described in the “Methods” section. Methylation levels were measured by qMS-PCR on the indicated number of days after the initiation of neuronal differentiation. See Additional file 3: Figure S3 for representative images during neuronal differentiation of 37D cells