FIG. 7.

Effect of ETnII and MusD internal regions on stably integrated transgenes. (A) Scheme of an integrated transgene. Internal and common sequences indicated in Fig. 5A were cloned in the reverse orientation upstream of the CMV promoter. Common region fragments amplified after ChIP or bisulfite treatment are shown. (B) EGFP expression of ETnII and MusD internal region-containing transgenes during single-cell sorting 13 days after transfection. (C) EGFP expression of ETnII and MusD internal region-containing transgenes monitored as separate clones until day 110 posttransfection, after which they were pooled and subjected to ChIP. A total of 10,000 to 20,000 propidium iodine-negative cells were analyzed. (D) ChIP results for cells with either an ETnII or a MusD internal region transgene. The results are presented as the relative amount of acetylated or methylated histones associated with target samples with respect to input material. Quantitative PCRs on DNA from three separate immunoprecipitations were performed in triplicate; error bars are standard deviations between biological replicates. (E) Bisulfite analysis of the ETnII/MusD common region of the transgene. Each horizontal line represents a single clone. Unmethylated CpGs are shown as open circles and methylated CpGs are shown as solid circles.