Figure 3

Comparison between SDFs and ssODNs for correction of 1567G>A mutations in β-galactosidase genes. CHO-K1 cells were co-transfected with the pCH110 1567G>A plasmid and correcting ssODNs (0.25 μM) or SDFs (7.5 nM) using 15 μg Lipofectamine (Invitrogen) [51]. Two days after transfection β-galactosidase enzyme activity was measured using a β-Galactosidase Enzyme Assay system (Promega) according to the manufacturer's protocol. ssODNs were designed to target the antisense strand (AS) of the β-galactosidase sequence in the region of the 1567G>A mutation. Two different lengths were employed: 25nt (AS-ssODN, 25nt) and 35nt (AS-ssODN, 35nt), both containing a centrally located cytosine in order to induce a mismatch with the targeted DNA. A Cy3-conjugated ssODN (AS-ssODN, 35nt, Cy3-conjugated) was included to test the effect of additional 5'-end protection. SDFs were synthesized using the pCH110 659G>A plasmid as template as previously described. The 480 bp SDF-molecule contained the mismatched base 270 bp from the 5'-end. As negative controls pCH110 1567G>A plasmid alone, a non-correcting SDF (constructed using the pCH110 1567G>A plasmid as template) and SDF without plasmid transfection were used.