Supporting Information

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1 Supporting Information Deng et al /pnas SI Text Animals and Subretinal Injections. Tr / mice were a gift from Dr. Janis Lem. GNAT2 cpfl3, rd7 and wildtype ALR/LtJ mice were purchased from Jackson Laboratory. All mice were maintained in the University of Florida Health Science Center Animal Care facilities under a 2/2 h light/dark cycle under shade dark cloth because of their albino background to avoid retinal light damage. Animals were handled in accordance with the ARVO statement for Use of Animals in Ophthalmic and Vision Research and the animal use policies of the University of Florida College of Medicine and Washington University School of Medicine. The procedures for subretinal injections were described in ref.. Only right eyes were injected, with the left eyes serving as untreated controls. Eyes were inspected for cataracts and corneal scarring before ERG to rule out an optical basis for differences in sensitivity. Ex Vivo Electrophysiology. Mice were maintained in 2/2 h light/dark cycle and dark-adapted overnight before experiments. After euthanasia, the eyes were removed under dim red light. All subsequent manipulations were done under infrared light. For single-cell recordings retina was finely chopped with a razor blade, and a small piece was placed in a recording chamber fit to an inverted microscope and perfused at C. Membrane current was recorded from the outer segment of a single rod photoreceptor protruding from the retina and drawn into the electrode as described in ref. 2. For transretinal ERG recordings, a quarter of the isolated retina was mounted on filter paper, photoreceptor-side up, and placed on the recording chamber with an electrode connected to the bottom. A second electrode was placed above the retina. The perfusion Locke solution, heated to C, contained 2 mm L-glutamic acid to block higher order component of photoresponse (3). The electrode solution under the retina contained, in addition, 0 mm BaCl 2 to suppress glial components (4, ). For both single-cell and whole retina ERG recordings, 0-ms test-flashes at 00 nm were delivered from a calibrated light source. Flash intensity was set by a combination of neutral density filters. Intensity-response data were fit by the Eq. R Ik R max I k k [] I o where R is the transient-peak amplitude of response, R max is maximal response amplitude, I is flash intensity, I o is flash intensity to generate half-maximal response. The rescue of light sensitivity in individual rods was initially determined by using nonattenuated flashes of light (.7 0 photons m 2 at 00 nm). Normalized flash sensitivity was calculated as the ratio of dim-flash response amplitude and flash intensity normalized by the corresponding saturating response. Cone components from whole retinal responses were isolated using an initial photons m 2 00-nm flash to suppress the rod component. The second cone-only flash was delivered 700 ms after the first flash.. Timmers AM, Zhang H, Squitieri A, Gonzalez-Pola C (200) Subretinal injections in rodent eyes: Effects on electrophysiology and histology of rat retina. Mol Vis 7: Shi G, Yau KW, Chen J, Kefalov VJ (2007) Signaling properties of a short-wave cone visual pigment and its role in phototransduction. J Neurosci 27: Sillman AJ, Ito H, Tomita T (969) Studies on mass receptor potential of isolated frog retina.. General properties of response. Vision Res 9: Bolnick DA, Walter AE, Sillman AJ (979) Barium suppresses slow piii in perfused bullfrog retina. Vision Res 9:7 9.. Nymark S, Heikkinen H, Haldin C, Donner K, Koskelainen A (200) Light responses and light adaptation in rat retinal rods at different temperatures. J Phys-London 67: of6

2 A. rd7 untreated B. rd7 treated with rod Tα C. rd7 untreated D. rd7 treated with cone Tα Fig. S. Immunostaining of rd7 retinal whole mounts showed nonuniform photoreceptor transduction in AAV-CBA-rod T or AAV-CBA-cone T treated retinas. Retinas from one eye of three different mice were analyzed for each condition. (A) Untreated rd7 retinas had no detectable rod T. (B) rd7 retinas treated with AAV-CBA-rod T showed higher levels of rod T expression near the vector injection site (arrow) than more distally (arrowhead). (C) Untreated rd7 retinas showed low levels of GNAT2 cpfl3 mutant cone T.(D) rd7 retinas treated with AAV-CBA-cone T showed a higher density of cone T expression near the vector injection site (arrow) than more distally (arrowhead). Most vector treated eyes had 40 70% of the retinal area transduced at a detectable level. All images were taken using the same microscope and camera settings to directly compare intensities of immunostaining. (Scale bar, 00 m.) 2of6

3 A α-tubulin Tα rd7 untreated sham-injected uninjected rod Tα rd7 cone Tα B transducin levels (% control) rd7 untreated uninjected P < 0.0 rd7- rod Tα rd7-cone Tα Fig. S2. Western blot analysis of T expression in the retinas of AAV-CBA-rod or cone T treated rd7 retinas and wild type controls. (A) Three eyecups from untreated rd7, CBA-rod T, CBA-cone T treated rd7 mice, and age matched normal controls (uninjected and sham-injected), were pooled into separate groups and aliquots extracts containing equal amounts of protein were electrophoresed, transferred, and probed with a primary antibody against a conserved epitope (KENLKDCGLF) in both rod and cone T subunits (GenWay Biotech, Inc.). The same blot was also probed with an antibody against -tubulin (Santa Cruz Biotechnology, sc-46) as an internal loading control. The signals were detected by using an infrared dye-conjugated secondary antibody (IRDye 800, Rockland Immunochemicals). Visualization and quantification of the specific bands were performed using the Odyssey Infrared Fluorescence Imaging System (Li-Cor). (B) Qualitative analysis showed that total levels of T were similar in CBA-rod T and CBA-cone T treated eyes, at 30% of wild-type (P 0.0). No statistical difference of T levels was found between the untreated and treated rd7 eyes. T levels were normalized to those of -tubulin and the densitometric value from each AAV-treated sample was expressed as a percentage relative to age matched wild-type controls. Before performing the final quantitative analysis, serial dilutions of retinal extracts were performed to ensure that the analysis remained within the linear range. There were variations in the Western blot analysis from eye to eye because of subretinal injection variability that resulted in somewhat different retinal areas exposed to the vector in each treated eye. These individual variations were averaged from pooled eyecups of each treatment group to obtain the final result. Results of Western blot analysis from three independent samples are shown in a bar graph representing the mean SEM. Statistical analysis was performed with one-way ANOVA plus Bonferroni post hoc test. 3of6

4 A B C Relative fluorescence in the ROS, percent of (ROS+RIS) rd7, CBA-rod Tα rd7, CBA-cone Tα light dark Fig. S3. Quantification of the ratio of T within rod OS (ROS) / (ROS RIS) from untreated wild-type mice and rd7 mice treated with AAV-CBA-rod or cone T under light- and dark-adapted conditions. (A and B) Representative retina images of untreated wild-type mice (A) and rd7 mice treated with CBA-rod T (B) showing the areas used to determine the fraction of fluorescence in the ROS vs. total fluorescence from ROS RIS expressed as percentage. For wild-type mice, a single rod or several rods when adjacent and the corresponding OS were highlighted as examples, and for rd7 mice treated with rod or cone T, a single rod and its corresponding OS were highlighted as an example. Although connecting cilia were not visible in the images, small errors in defining IS and OS are expected to cancel out over the multiple areas selected. Fluorescence intensity of rod or cone T immunostaining was determined from confocal-projected twodimensional images using ImageJ software (National Institutes of Health). Total fluorescence for each enclosed area was obtained by multiplying the area by the average fluorescence density within the area. From every retina, four separate retinal regions were selected and data collected from one rod in each region from three animals in each treatment group. The ROS / (ROS RIS) T ratio was determined as the average from a total of 2 rods for each experimental group and plotted as shown in (C). The imaging parameters were kept constant and set so that none of the areas within each field analyzed was saturated. Statistical analysis using one-way ANOVA showed there was no significant difference among untreated wild-type mice and rd7 mice treated with either rod T or cone T under either light or dark-adapted conditions (P 0.9). 4of6

5 rod Trα -/- untreat A B C 20µV Response D Response 0µV Response 20µV Response 0µV 2ms Gnat2 cpfl3 -untreat contralateral rod Tα- treated 0. cd.s.m ms Time rd7 dark-adapted ERG untreated rod Tα-treated cone Tα-treated 2ms Time Time contralateral cone Tα- treated cd.s.m rd7 light-adapted ERG untreated rod Tα-treated cone Tα-treated 20ms Time cd.s.m cd.s.m Fig. S4. Representative in vivo rod and cone ERG recordings from Tr /, GNAT2 cpfl3, and rd7 mice showing rescue by treatment with rod or cone T vectors. (A) The dark-adapted, rod-driven ERG response to a 0.0 cd.s.m 2 test flash (arrow) in a Tr / eye was partially restored after treatment with AAV-mOP-cone T (Right). Rod-mediated ERG response was undetectable in an untreated eye (Left). (B) The light-adapted, cone-mediated ERG b-wave in response to a 0 cd.s.m 2 test flash was not detectable in an untreated GNAT2 cpfl3 eye (Left) but was restored by AAV-PR2.-rod T treatment in the fellow eye (Right). (C) Dark-adapted, rod-mediated ERG responses were partially restored in rd7 mice treated with either AAV-CBA-rod T or cone T vectors. The rod-driven b-wave response to a 0.0 cd.s.m 2 test flash (arrow), undetectable in untreated eyes, was rescued in representative rod T - or cone T - treated rd7 eyes. (D) The light-adapted, cone-mediated ERG responses, undetectable in untreated eyes, were rescued in representative rod T - or cone T - treated rd7 eyes. of6

6 Table S. Summary of experiment results from each AAV vector In vivo vectored T translocation In vivo ERG Ex vivo transretinal ERG AAV vector Cell targeted In rods In cones Rod Cone Rod Cone Rod single-cell recordings CBA-rod T Rod/cone As wild type As wild type Rescued Rescued Rescued Rescued CBA-cone T Rod/cone As wild type As wild type Rescued Rescued Rescued Rescued PR2.-rod T Cone Rescued Rescued PR2.-cone T Cone Rescued Rescued mop-cone T Rod Rescued 6of6