Aberrant Epigenetic Silencing Is Triggered by a Transient Reduction in Gene Expression
Figure 2
Dox exposure induces PTRE-HPRT inactivation.
(A) Reducing expression of PTRE-HPRT by growing cells for 1 week in medium containing 1 µg/ml Dox increased the frequency of gene inactivation, as measured by TG-resistance. TG-resistance was measured by washing out Dox, selecting cells with 5 µg/ml TG, and counting surviving colonies after 2 weeks of continuous selection. During Dox treatment or the equivalent period without treatment, cells were maintained in medium containing puromycin and G418 to maintain the PTRE-HPRT and tTA constructs respectively, but without azaserine/hypoxanthine (AzHx) selection. Only cells that express HPRT can grow in AzHx selection. (B) TG-resistance frequencies increased as a function of time HP13 cells were exposed to Dox before starting TG selection. HP13 cells were continuously cultured in 1 µg/ml Dox for 3 weeks, and TG-resistance was measured at different points during the Dox treatment (3, 5, 7, 14, and 21 days). A parallel control culture was maintained in medium containing puromycin and G418 without Dox, and TG-resistance was measured after 21 days (Untreated).