Figure 6. The impact of a promoter driving Neo^R expression within AAV-based targeting vectors on gene targeting efficiency.

(A) PIGA gene targeting efficiency achieved with AAV-based targeting vectors carrying various promoters. The cell lines denoted in the graphs were infected with the targeting vectors shown in Figure 5A, selected with G418, processed for fluorescence-labeling of GPI-anchors with FLAER, and analyzed by fluorescence FCM. The ratios of FLAER-negative cells which represent PIGA gene-targeted cells are shown (mean ± s.e.m.; n = 3). (B) Schematic representation of an experimental system determining gene targeting efficiency using a Hyg^R–5′ EGFP fusion reporter gene. The Hyg^R–5′ EGFP reporter vector (top) was introduced into DLD-1, and a cell clone stably expressing the Hyg^R–5′ EGFP gene was established. This reporter clone was then infected with AAV-based targeting vectors harboring various promoters (bottom), selected with G418, and FCM-analyzed. The diagram is not drawn to scale. (C) The gene targeting efficiencies determined based on the Hyg^R–EGFP reporter system. Shown are the ratios of GFP positive cells which represent the frequency of homologous recombination events occurring between the reporter and the targeting vectors (mean ± s.e.m.; n = 3). An unrelated AAV vector harboring the Neo^R gene was used for V.C. For abbreviations, refer to legend for Figure 1.