Targeted In Situ Gene Correction of Dysfunctional APOE Alleles to Produce Atheroprotective Plasma ApoE3 Protein

Figure 3

Effects of nuclease-protected ssODNs on targeted gene repair. Recombinant cells expressing mutated (nonfluorescent) green fluorescent protein (GFP) were targeted with two ssODNs, one with three phosphorothioate (PTO) residues at each end (a) and the other with four internal PTOs at the central correcting segment (b). Posttargeting, the green cells were counted by flow cytometry (left panels), stained for cell cycle analysis (central panels) and cultured to observe cell growth (right panels). The PTO end-protected ssODN gave a much higher rate of gene correction, as measured by the number of green cells (boxes in left panels). However, this resulted in DNA damage and accrual of cells in the G2 phase, whereas the GFP+ve cells obtained after internally protected ssODN targeting had a markedly greater proportion in G1 (central panel). Significantly, only the occasional divided pair of green cells was noted in cultures treated with end-protected ssODN, whereas actively replicating green cells were evident when the internally protected ssODN was used (right panels).

(a) End-protected 5′3^′PTO ssODN: good efficiency and minimal proliferation

(b) Internal-protected PTO ssODN: medium efficiency and good proliferation