1. Volume 3, Issue 5, Pages 688-696 (May 2001)
Recombinant AAV-Mediated Delivery of a Tet-Inducible Reporter Gene to the Rat Retina 
L.H. McGee Sanftner, K.G. Rendahl, D. Quiroz, M. Coyne, M. Ladner, W.C. Manning, J.G. Flannery 
Molecular Therapy 
Volume 3, Issue 5, Pages (May 2001)
DOI: /mthe
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
(A) Schematic diagram of the inducible GFP (O7-GFP) and the siiencer/activator (tTSkid-rtTA) virai vectors. (tetO)7, inducible promoter; β-globin, β-globin intron; GFP, enhanced green fluorescent protein; bgh pA, bovine growth hormone poly(A); ITR, inverted terminal repeat sequences from AAV-2; LTR, long terminal repeat from murine Moloney virus; CI, pCI intron; tTSkid, transcriptional silencer; Syn pA, synthetic poly(A); CMV, minimal cytomegalovirus enhancer/promoter; Intron A, modified intron A; rtTA, transcriptional activator. (B) Inducible promoter's response to dox. Circled A, activator; boxed S, silencer.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
In vitro transduction of 293 cells. Total GFP fluorescence was measured in cells transduced with O7-GFP + tTSkid-rtTA viruses at ratios of 1:1 and 1:5. Fold induction as well as GFP expression level increases when the ratio of inducible vector to silencer/activator vector is increased. Total GFP fluorescence in dox-treated cells compared to cells receiving only medium was significantly different in both the 1:1 and the 1:5 groups (P < 0.01). Total GFP fluorescence measurements in uninduced cells in both the 1:1 and the 1:5 groups were similar to controls transduced with only inducible GFP virus (P > 0.05). Error bars show the standard deviation. n = 3 for each group.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
GFP expression in cryosections of O7-GFP + tTSkld-rtTA-injected retinas at 9 weeks postinjection. Cross section through the injection sites in (A) an animal receiving dox and (B) an animal receiving control water. ONL, outer nuclear layer; INL, inner nuclear layer; RPE, retinal pigment epithelium. Scale bar, 25 μm.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
(A) Western blot analysis of total cell lysate from AAV-transfected retinas probed for GFP. Protein standard marker, lanes 1 and 10. Recombinant GFP protein control, lane 2. Retinal lysates from animals injected with CMV-GFP (0.01×), lane 3; CMV-GFP (0.1×), lane 4; CMV-GFP (1×), lane 5; O7-GFP + tTSkid-rtTA (1:8) and administered dox, lane 6; O7-GFP + tTSkid-rtTA (1:8) and not administered dox, lane 7; O7-GFP control, lane 8; and tTSkid-rtTA control, lane 9. (B) Reprobing with a peripherin antibody showed that equal amounts of retinal lysate were loaded in each lane.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Representative fundus images of rats injected with O7-GFP + tTSkid-rtTA at a ratio of 1:8 in switch-on/switch-off experiment. Retinas were imaged at specified times to look for GFP expression. All dox-treated animals were given 200 μM dox in drinking water. (A) Representative animal from switch-off group. The switch-off group received dox treatment between 1 and 5 weeks postinjection and at 5 weeks was then removed from dox treatment. By 7 weeks, no GFP was detectable. (B) Representative animal from switch-on group. No GFP expression was detected in the switch-on group before induction, but was highly visible 2 weeks after the initiation of dox treatment. Scale bar, 500 μm.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
Mean percentage retinal area GFP positive in O7-GFP + tTSkid-rtTA-injected rats. All animals began drinking water treatment at 1 week postinjection. Animals from the switch-off group initially received 200 μM dox in their drinking water followed by dox removal at 5 weeks postinjection, after imaging (white bars). Animals from the switch-on group initially did not receive dox in their drinking water, but began receiving 200 μM dox at 5 weeks postinjection (black bars). Mean percentage area GFP positive data in dox-treated animals compared to animals only receiving sucrose-water were significantly different at all time points (P < 0.05). Error bars show the standard deviation. n = 8 for each group.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

8. FIG. 7
Dose response to various dox concentrations in O7-GFP + tTSkid-rtTA-injected rats. Five different groups were each given 10-fold lower concentrations of dox, 200, 20, 2, 0.2, and 0 μM, in their drinking water. (A) Images were analyzed and mean percentage area GFP positive for each group was determined. The data groups 200, 20, 2, and 0.2 μM dox were significantly different from one another (P < 0.003). Mean percentage area GFP positive data from animals receiving 0.2 μM dox were not significantly different compared with animals not receiving dox (P > 0.05). Error bars show the standard deviation. n = 8 for each group. (B) Fundus images show a representative eye from each group.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

9. FIG. 8
A comparison of reporter gene expression level produced from tet-inducible and constitutive promoter vectors at 7 weeks postinjection. All animals continuously received 200 μM dox in their drinking water. Mean percentage area GFP positive measurements in dox-treated animals were significantly larger than measurements in animals receiving only sucrose water in both the 1:1 and the 1:8 groups (P < 0.01). The GFP expression level in animals injected with inducible GFP virus and silencer/activator virus at 1:8 ratio was similar to that of those injected with CMV-GFP at a 0.01 × dilution (P > 0.05). Mean percentage area GFP positive measurements in uninduced animals in both the 1:1 and the 1:8 groups were similar to those of control animals transduced with only inducible GFP virus (P > 0.05). O7-GFP + tTSkid-rtTA at 1:1 – dox, O7-GFP + tTSkid-rtTA 1:8 – dox, O7-GFP ± dox, and tTSkid-rtTA ± dox were negative for GFP upon visual inspection. Error bars show the standard deviation. n = 6 for each column.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

10. FIG. 9
Averaged amplitudes of electroretinogram a-waves and b-waves from O7-GFP + tTSkid-rtTA-injected and uninjected rats. Scotopic ERG recordings from rats injected with O7-GFP + tTSkid-rtTA vectors, receiving dox, and uninjected controls. Stimuli were presented at intensity of log candela s/m2. Error bars show the standard deviation among averaged amplitudes from 20 eyes. Experimental average a- and b-wave amplitudes were not significantly different from those of uninjected controls (P >0.05).
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions