Figure 1.
Transient GFP expression driven by various constitutive promoters in AAV vectors.
(A) Schematic representation of AAV vectors used for GFP assays. “Promoters” indicates the position where one of the six promoters was incorporated. ITR, inverted terminal repeat; SV40, simian virus 40; NeoR, neomycin phosphotransferase gene; pA, polyadenylation site; EGFP, enhanced GFP. The diagram is not drawn to scale. (B, C) Transient GFP expression in human cell lines. Cell lines denoted within bar graphs were infected with the AAV vectors shown in (A), and GFP signals were quantified by fluorescence FCM after 2-days incubation. Shown are mean GFP intensity (acquired as mean FL1-A) for each infectant relative to data obtained with the CMV promoter (B) and percentage of GFP positive cells within each infectant (C) (mean ± s.e.m.; n = 3). hACTB, human β-actin; hEF-1α, human elongation factor-1α; CAG, cytomegalovirus early enhancer/chicken β-actin; CMV, cytomegalovirus; HSV-TK, herpes simplex virus thymidine kinase; hACTB-R, hACTB reverse direction; N.P., no promoter; V.C., vector control. For V.C., an unrelated AAV vector harboring no EGFP gene was used. *Significantly higher than any of the other promoters and controls (p<0.001 for all pairs). **Significantly higher than CAG, SV40, HSV-TK, hACTB-R, N.P. and V.C. (p = 0.001 versus CAG; p<0.001 versus the others). ***Significantly higher than hACTB, hEF-1α, CAG, HSV-TK, hACTB-R, N.P. and V.C. (p = 0.004 versus hACTB; p<0.001 versus the others). ****The data for SV40 is the highest (p = 0.002 versus CMV; p<0.001 versus the others), and those for CMV is the second highest (p = 0.001 versus hACTB and hEF-1α; p = 0.003 versus HSV-TK; p<0.001 versus CAG, hACTB-R, N.P. and V.C) among the investigated promoters and controls. p-values were determined based on Scheffe’s post hoc test.
Figure 2.
Time-course study of GFP signals expressed by various constitutive promoters in AAV vectors.
Cell lines indicated within bar graphs were infected with the AAV vectors depicted in Figure 1A and processed for fluorescence FCM after 2-, 4-, 6-, and 8-weeks culture in the presence of G418. Mean GFP intensity (acquired as mean FL1-A) for each infectant is indicated relative to data obtained with the CMV promoter four weeks after infection (mean ± s.e.m.; n = 3). For abbreviations, refer to the legend for Figure 1. *Significantly higher than any of the other promoters and controls (p<0.001 for all pairs). **Significantly higher than any of the other promoters and controls (p = 0.004, versus hACTB; p = 0.002, versus hEF-1α; p<0.001 versus the others). ***Significantly higher than hACTB in two weeks (p = 0.01) and eight weeks (p<0.001), and than the other promoters and controls in any of four time points (p<0.001, versus all the others in any of four time points, except for the comparison of CMV versus SV40 in two weeks, p = 0.001). ****Significantly higher than CAG, CMV, SV40, HSV-TK, hACTB-R, and N.P. (p = 0.001 for hACTB versus SV40; p<0.001 for all the other pairs). p-values were determined based on Scheffe’s post hoc test.
Figure 3.
The impact of the MOI of an AAV vector on GFP expression level.
The HCT116, DLD-1, and MCF-10A cell lines were infected with various MOIs of an AAV vector expressing EGFP under the control of the CMV promoter, and GFP expression was quantified by fluorescence FCM (mean ± s.e.m.; n = 3). X-axes indicate MOIs of the AAV vector. (A) Fluorescence FCM analyses performed after two days of culture post-infection without G418. (left) Mean GFP intensities (acquired as mean FL1-A) for respective infectants are shown relative to the data obtained with the cells infected at an MOI of 3×102. (right) Percentage of GFP positive cells in each infectant. (B) Fluorescence FCM analyses performed after four weeks of culture post-infection in the presence of G418. Data are shown in the same manner with the left panel of (A).
Figure 4.
qRT-PCR analyses of CDKN2A gene expression.
The indicated cell lines were infected with an AAV vector carrying a CDKN2A gene (H83Y) downstream of the CMV or the hACTB promoter. The cells were then cultured for two days without G418 (left) or for four weeks with G418 (right), and total RNA was extracted from each infectant, converted to cDNA, and used as a template in qRT-PCR. The expression level of CDKN2A was normalized to that of the GAPDH gene in each sample and shown relative to the data of the DLD-1 cell line obtained with the CMV promoter (mean ± s.e.m.; n = 3). An AAV vector carrying no CDKN2A gene was used as V.C. p-values were determined based on Scheffe’s post hoc test and denoted in the graph.
Figure 5.
Formation of G418-resistant colonies by the infection of AAV vectors in which the NeoR gene is directed by various promoters.
(A) Schematic representation of AAV-based PIGA-targeting vectors used for assays shown in Figures 5B and 6A. PIGA 5′ and PIGA 3′ represent homology arms designed for the targeting of human PIGA gene. The diagram is not drawn to scale. (B) Numbers of G418-resistant colonies obtained by the infection of AAV vectors. The cell lines indicated in the graphs were infected with the AAV-based PIGA-targeting vectors depicted in (A) and selected with G418 until visible colonies were formed. Colonies were stained with crystal violet and then counted (mean ± s.e.m.; n = 3). For abbreviations, refer to legend for Figure 1. *Significantly higher than hEF-1α, CAG, SV40, HSV-TK, hACTB-R, and N.P. (p = 0.01 versus hEF-1α; p<0.001 versus the others). **Significantly higher than any of the other promoters and controls (p<0.001 for all pairs). ***Significantly higher than hACTB, hEF-1α, CAG, HSV-TK, hACTB-R, and N.P. (p = 0.003 versus hACTB; p = 0.02 versus hEF-1α; p<0.001 versus the others). p-values were determined based on Scheffe’s post hoc test.
Figure 6.
The impact of a promoter driving NeoR 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 HygR–5′ EGFP fusion reporter gene. The HygR–5′ EGFP reporter vector (top) was introduced into DLD-1, and a cell clone stably expressing the HygR–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 HygR–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 NeoR gene was used for V.C. For abbreviations, refer to legend for Figure 1.