Figure 7.

(A) Schematic diagram describing mammalian vectors used for the expression of targeted Mtases. (B) Upper panel showing western blot confirming expression levels of 4aFH, 4bFH and 4aQC targeted Mtase enzymes from vector described in (A). Lower panel showing the results of RT–PCR of 4aZfFH RNA at 3, 6 and 12 days post-transfection with 4aZfFH targeted Mtase expression vector. β-Actin was used an internal control. PCR was performed for 28, 30, 32 and 34 cycles. (C) Bisulphite sequencing results for genomic target site DNA, 14 days post-transient transfection with targeted Mtase or control vector. The target site sequence is given at the top of the figure. HpaII sites are in boldface and the 12 bp zinc-finger recognition site is boxed. Below this is a schematic diagram showing target site and flanking CpG site distribution (represented by circles in sequencing data) spanning ∼160 bp. Large ticks indicate HpaII sites, small ticks indicate HhaI sites. Zinc-finger recognition sites for the 4aZf protein are represented by filled rectangles. The expression vector transiently transfected in each experiment is indicated next to each sequencing results block. Filled circles indicate observed methylation.