March 2006
Volume 47, Issue 3
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Retina  |   March 2006
RPE65 Gene Delivery Restores Isomerohydrolase Activity and Prevents Early Cone Loss in Rpe65−/− Mice
Author Affiliations
  • Ying Chen
    From the Department of Cell Biology, Department of Medicine, The University of Oklahoma, Health Sciences Center, Oklahoma City, Oklahoma.
  • Gennadiy Moiseyev
    From the Department of Cell Biology, Department of Medicine, The University of Oklahoma, Health Sciences Center, Oklahoma City, Oklahoma.
  • Yusuke Takahashi
    From the Department of Cell Biology, Department of Medicine, The University of Oklahoma, Health Sciences Center, Oklahoma City, Oklahoma.
  • Jian-xing Ma
    From the Department of Cell Biology, Department of Medicine, The University of Oklahoma, Health Sciences Center, Oklahoma City, Oklahoma.
Investigative Ophthalmology & Visual Science March 2006, Vol.47, 1177-1184. doi:10.1167/iovs.05-0965
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      Ying Chen, Gennadiy Moiseyev, Yusuke Takahashi, Jian-xing Ma; RPE65 Gene Delivery Restores Isomerohydrolase Activity and Prevents Early Cone Loss in Rpe65−/− Mice. Invest. Ophthalmol. Vis. Sci. 2006;47(3):1177-1184. doi: 10.1167/iovs.05-0965.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. Recent in vitro evidence has shown that RPE65 is the isomerohydrolase that converts all-trans retinyl ester to 11-cis retinal, the chromophore for visual pigments in vertebrates. Homozygous RPE65 knockout (Rpe65 −/−) mice lack 11-cis retinoids and have early cone degeneration. The purpose of this study is to determine whether RPE65 gene delivery restores the isomerohydrolase activity and normal profile of endogenous retinoids in Rpe65 −/− mice.

methods. Adenovirus-expressing RPE65 (Ad-RPE65) was injected into the subretinal space of Rpe65 −/− mice. The expression of RPE65 was determined by immunohistochemistry and Western blot analysis. The isomerohydrolase activity was measured in vitro in eyecup homogenates. Endogenous retinoid profile in the eyecups was analyzed by high-performance liquid chromatography (HPLC). Photoreceptor-specific gene expression was determined with real-time RT-PCR. Cone degeneration was determined by cone-specific staining and counting cones in flatmounted retina.

results. High levels of RPE65 expression from the Ad-RPE65 injection generated robust isomerohydrolase activity in the eyecup of Rpe65 −/− mice, at levels comparable to those in wild-type (wt) mice. Consequently, the RPE65 gene delivery resulted in substantial amounts of 11-cis retinal in Rpe65 −/− mice. The RPE65 gene delivery prevented the downregulation of cone-specific genes, including both cone opsins and cone tranducin α subunit in Rpe65 −/− mice. Moreover, the Ad-RPE65 injection also prevented massive cone degeneration at early ages of Rpe65 −/− mice.

conclusions. RPE65 gene delivery generates isomerohydrolase activity and restores retinoid profile in Rpe65 −/− mice. Regeneration of 11-cis retinal is essential for survival of cone photoreceptors.

The chromophore 11-cis retinal functions in visual pigments of both rods and cones. 1 Light isomerizes 11-cis retinal to its all-trans isomer, which triggers the activation of the phototransduction pathway and initiates vision. 1 2 Regeneration of 11-cis retinal through a multistep visual cycle is essential for normal vision. 3 4 A key step in the visual cycle is the conversion of all-trans retinyl ester to 11-cis retinol in the retinal pigment epithelium (RPE). 5 This conversion is believed to be catalyzed by a single enzyme, isomerohydrolase. 5  
RPE65, a membrane-associated protein predominantly expressed in the RPE, has been shown to bind all-trans retinyl ester specifically. 6 7 Mutations in RPE65 protein are associated with inherited retinal dystrophies such as retinitis pigmentosa (RP), Leber’s congenital amaurosis (LCA), and early-onset severe retinal dystrophies. 8 9 10 11 12 Homozygous RPE65 gene knockout (Rpe65 −/−) mice lack isomerohydrolase activity and have no 11-cis retinoids, suggesting an interrupted visual cycle. 13 We have recently reported that in Rpe65 −/− mice, massive cone degeneration occurs at early ages (before 4 weeks of age), which is associated with significant downregulation of cone-specific genes such as cone opsins and cone transducin, while rods remain intact at early ages. 14  
It has been reported by several groups that RPE65 gene delivery prevents retinal degeneration and restores visual functions in dog and mouse models of RPE65 deficiency. 15 16 17 18 19 20 However, the restoration of the isomerohydrolase activity and normal profile of endogenous retinoids after RPE65 gene delivery has never been documented. The effects of RPE65 gene delivery on early downregulation of cone-specific genes have not been determined. 
Recently, we have demonstrated that RPE65 is the isomerohydrolase in the visual cycle and is essential for the generation of 11-cis retinoids. 21 In the present study, we set out to determine the isomerohydrolase activity in Rpe65 −/− mice after RPE65 gene delivery. We also determined whether a restored visual cycle by RPE65 gene delivery can prevent the decreased expression of cone opsins and transducin at early ages of Rpe65 −/− mice. 
Methods
Animals
Animals were kept in a 12-hour light–dark cycle with an ambient light intensity of 85 ± 18 lux. Rpe65 −/− mice were genotyped as described previously. 13 Wild-type (wt) C57BL/6 and 129sv mice were purchased from Jackson Laboratory (Bar Harbor, MA). Care, use, and treatment of the animals were in strict agreement with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Preparation of Recombinant Adenovirus Expressing RPE65
The human RPE65 cDNA containing a full-length coding region was subcloned into a shuttle vector under the CMV promoter (Qbiogene, Carlsbad, CA). The recombinant viral DNA was generated in BJ5183-AD1 cells (Qbiogene). Adenovirus expressing-RPE65 (Ad-RPE65) was produced in QBI-293A cells and purified by two-step cesium chloride density gradient centrifugation. The titer of Ad-RPE65 was determined using the plaque-forming assay and is expressed as plaque-forming units per milliliter (pfu/mL). 
Subretinal Injection of Ad-RPE65
Briefly, mice were anesthetized with a 50:50 mix of ketamine (100 mg/mL) and xylazine (20 mg/mL), and pupils were dilated with topical application of phenylephrine (2.5%) and tropicamide (1%). A sclerotomy was created approximately 0.5 mm posterior to the limbus with a self-made blade, and a glass injector (∼33 gauge) connected to a syringe filled with 1 μL virus (2.1 × 1010 pfu/mL) was introduced through the sclerotomy into the vitreous cavity. The tip of the injector was introduced into the subretinal space through a peripheral retinotomy, and the virus was slowly injected. A gray bubble demonstrated that retina was detached from the RPE, and the virus was delivered into the subretinal space. 
Western Blot Analysis
The eyes were enucleated from the mice, and the cornea and lenses were removed. The eyecups from each mouse were homogenized individually, and protein concentration measured by the Bradford method. 22 The same amount (50 μg) of total protein from each mouse was resolved by SDS-PAGE and electrotransferred onto a polyvinylidene fluoride (PVDF) membrane, as described previously. 22 The membrane was blotted by a polyclonal anti-RPE65 peptide antibody. 23 The signal was detected using the enhanced chemiluminescence (ECL) system (GE Healthcare, Piscataway, NJ). 
Immunohistochemistry
Frozen sections (4 μm) were cut, air dried, and washed in phosphate-buffered saline (PBS), blocked with 20% goat serum in 0.1% Triton X-100/1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO) in PBS. For RPE65 single staining, the sections were incubated with the rabbit anti-RPE65 antibody overnight at 4°C. For cone opsin and RPE65 double staining, the sections were incubated with biotin-conjugated anti-RPE65 antibody and an FITC-conjugated anti-bovine medium wave length (MWL) cone opsin antibody. The sections were rinsed several times with PBS and incubated with Texas red–conjugated goat anti-rabbit antibody (for single staining) at a dilution of 1:150 for 30 minutes or Texas red–conjugated goat streptavdin (for double staining; Jackson ImmunoResearch Laboratory, Inc., West Grove, PA). After this, the slides were rinsed in PBS and the nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO). The sections were then mounted in antifade medium (Vectashield; Vector Laboratories, Burlingame, CA) and viewed on a laser scanning confocal microscope (model LSM 510; Carl Zeiss Meditec, Inc., Jena, Germany). 
In Vitro Isomerohydrolase Activity Assay
After overnight dark adaptation, mice were killed and their eyes enucleated. The anterior portion of the globe was removed, and the remaining eyecup (including the retina and RPE) from each mouse was homogenized individually for the assay. All-trans [11,12-3H]-retinol in ethanol (1 mCi/mL, 52 Ci/mmol; Perkin Elmer, Boston, MA) was dried under argon and resuspended in the same volume of dimethyl formamide (DMF). For each reaction, 2 μL of the nondiluted all-trans [11,12-3H]-retinol in DMF and 250 μg of total protein from each homogenate were added to 200 μL of a reaction buffer (10 mM BTP [pH 8.0] 100 mM NaCl) containing 0.5% BSA and 25 μM cellular retinaldehyde-binding protein (CRALBP). 24 After 2 hours incubation in the dark at 37°C, retinoids generated were extracted by the addition of 300 μL cold methanol and 300 μL hexane. The upper organic phase was collected and analyzed by normal phase HPLC coupled to a flow scintillation analyzer (Radiomatic 610TR; Perkin Elmer), as described elsewhere. 22 Each retinoid was identified based on comparison to retention times of known retinoid standards. The activity was calculated from the area of the 11-cis [3H]-retinol peak using synthetic 11-cis [3H]-retinol as a standard for calibration. 
Analysis of Endogenous Retinoids in the Mouse Eyecup
Two eyecups (including the retina and RPE) from each mouse were combined and homogenized in 200 μL of PBS in a glass minigrinder. Retinal extraction was performed under dim red light, by a published method. 25 After the addition of 300 μL cold methanol and 60 μL 1 M hydroxylamine in 0.2 M sodium phosphate buffer (pH 7.0), the resultant suspension was thoroughly mixed (vortexed) for 30 seconds, and 300 μL of dichloromethane were added and vortexed for another 30 seconds. The solution was centrifuged at 10,000g for 5 minutes. The lower organic layer was collected with a Pasteur pipette. The residual suspension was extracted once more with an equal volume of dichloromethane. The combined organic layer was evaporated with oxygen-free argon. The residue was dissolved in 200 μL of HPLC mobile phase and applied to the HPLC column. The HPLC separation of retinoids and peak analyses were the same as described. 22  
Quantitative Real-Time Reverse Transcription–PCR
Six mice of C57BL/6, 129sv, and Rpe65 −/−, with or without subretinal injection of Ad-RPE65 were used for real-time RT-PCR. RNA was isolated from the retinas of each mouse individually. RT reaction was performed as described previously. 26 The RT products were diluted to 1:10; 2 μL of each diluted RT product was used for PCR which included 3 pM of each primer in a final volume of 25 μL. The PCR included a denaturation and hot start at 95°C for 10 minutes, followed by 45 cycles, with melting at 95°C for 15 seconds and elongation at 60°C for 60 seconds. Fluorescence changes were monitored after each cycle (SYBR Green; Applied Biosystems, Inc., Foster City, CA). Melting curve analysis was performed (0.5°C/sec increase from 55°C to 95°C, with continuous fluorescence readings) at the end of 45 cycles, to ensure that specific PCR products were obtained. Amplicon size and reaction specificity were confirmed by single-band product in agarose gel electrophoresis. All reactions were performed in triplicate, and the results analyzed by computer (SmartCycler II software; Cepheid, Sunnyvale, CA). The average C T (threshold cycle) of fluorescence units was used for analysis. The mRNA level was normalized by the 18s rRNA level. Quantification was calculated by using the C T of the target signal relative to the 18s rRNA signal in the same RNA sample. The mRNA levels were averaged in each group and expressed as percentages of that in wt 129sv mice. 
Cone-Density Analysis
Retinas of the experimental animals were prepared as described previously. 27 Briefly, the retina–lens complex was fixed in 4% formaldehyde solution in PBS (pH 7.4). After several washes in PBS, the retina–lens complex was incubated with FITC-conjugated peanut agglutinin (PNA; Sigma-Aldrich) overnight. After several washes in PBS, the retina was detached from the lens, flatmounted, and covered by a coverslip after the application of several drops of antifade solution (Prolong; Molecular Probes, Eugene, OR). 
The samples were analyzed with a fluorescence microscope (Axioplan II; Carl Zeiss Meditec, Inc.) equipped with a digital camera. Images were captured (Spot-RT Camera, with Spot software, ver. 3.0; Diagnostic Instruments, Sterling Heights, MI) and processed (Photoshop; Adobe Systems, Mountain View, CA). 
Ten random micrographs from each retina were taken in each animal, at 400× magnification. The cones in these micrographs were counted, and the number of cones per field was averaged and analyzed by Student’s t-test. 
Results
Expression of RPE65 Resulting from RPE65 Gene Delivery and Induction of Isomerohydrolase Activity in Rpe65−/− RPE
Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. As shown by immunohistochemistry, Ad-RPE65 injection resulted in expression of RPE65 in a large area of the RPE in the Rpe65 −/− mice at 4 weeks of age, whereas untreated Rpe65 −/− mice had no detectable RPE65 signal (Fig. 1) . Western blot analysis demonstrated that expression levels of RPE65 in the eyecups injected with Ad-RPE65 were comparable to that of wt C57BL/6 mice (Fig. 1D)
The isomerohydrolase activity in the mouse eyecup homogenate was measured using all-trans [3H]-retinol as a substrate. When the same amount of eyecup homogenates was incubated with all-trans [3H]-retinol, both wt 129sv and C57BL/6 eyecup homogenates generated 11-cis [3H]-retinol, whereas Rpe65 −/− mice injected with the same titer of a control virus (Ad-β-gal and Ad-GFP) did not show any detectable isomerohydrolase activity (Fig. 2) . The isomerohydrolase activity was quantified by measuring the area of the 11-cis [3H]-retinol peak in the HPLC profile and averaged in six mice from each group. The isomerohydrolase activity in wt 129sv eyecups was significantly higher than that in wt C57BL/6 eyecups, consistent with the previously reported strain difference in isomerohydrolase activity. 22 In Rpe65 −/− mice injected with Ad-RPE65, isomerohydrolase activity in the eyecup homogenates was comparable to that in wt C57BL/6 mice but lower than in wt 129sv mice (Fig. 2E) , correlating with RPE65 expression levels in these mice. These results indicate that adenovirus-mediated RPE65 gene delivery results in efficient RPE65 expression and restores retinol isomerohydrolase activity in the RPE of Rpe65 −/− mice to the level of wt mice. 
Effect of RPE65 Gene Delivery on the Normal Profile of Endogenous Retinoids in Rpe65−/− Retina and RPE
Rpe65 −/− mice are known to lack any detectable 11-cis retinoids in the RPE and retina. 13 To test whether the Ad-RPE65 injection also restored the normal profile of endogenous retinoids in the RPE and retina in Rpe65 −/− mice, the mice were injected with Ad-RPE65 at the age of 2 weeks, and endogenous retinoids were extracted from the RPE and retina at 6 weeks of age. The endogenous retinoids were analyzed by HPLC, and each isoform of retinoids was identified based on the retention time of respective retinoid standard. The retinoid profile was compared with those from age-matched wt 129sv and C57BL/6 mice and with Rpe65 −/− mice injected with Ad-β-gal. Consistent with previous reports, 13 syn-11-cis retinal oxime and anti-11-cis retinal oxime were detected in eyecups from both the 129sv and C57BL/6 mice, whereas Rpe65 −/− mice showed only increased levels of retinyl ester and had no other forms of retinoids (Fig. 3) . In the Ad-RPE65-injected Rpe65 −/− mice, however, substantial amounts of syn-11-cis retinal oxime and anti-11-cis retinal oxime were detected (Fig. 3) . Endogenous 11-cis retinal was quantified by measuring areas of 11-cis retinal peaks in HPLC profile and averaged. The levels of 11-cis retinal were 252 ± 50 and 285 ± 43 picomoles/eye in 129sv and C57BL/6 mice, respectively. In the Ad-RPE65 injected Rpe65 −/− mice, the levels of 11-cis retinal were 38 ± 2 picomoles/eye (Fig. 3)
Effect of Ad-RPE65 Delivery on the Downregulation of Cone-Specific Genes in Rpe65–/– Mice
Previously, we found that cone opsins and cone transducin are downregulated at early ages (4 weeks) of Rpe65 −/− mice, because of the lack of 11-cis retinal. 14 To assess the effects of Ad-RPE65 delivery on cone opsin expression in vivo, we quantified mRNA levels of a subset of photoreceptor-specific genes including short wave length (SWL) and MWL cone opsins, rhodopsin, and rod and cone transducin α-subunits (GNAT1 and GNAT2, respectively) using quantitative real-time RT-PCR analysis and normalized against 18s rRNA levels. The results demonstrated that at 4 weeks of age, Rpe65 −/− mice showed significantly decreased levels of SWL and MWL cone opsins and GNAT2 mRNAs, compared with that in age-matched wt mice, whereas the rhodopsin and rod transducin mRNAs remained unchanged at this age, confirming our previous studies. 14 Ad-RPE65 injection into Rpe65 −/− mice at 2 weeks of age resulted in significant increases in mRNA levels of MWL and SWL cone opsins over the Rpe65 −/− mice injected with a control virus (Fig. 4) , as tested at 4 weeks of age. Similarly, GNAT2 mRNA levels were also significantly increased after Ad-RPE65 subretinal delivery into the Rpe65 −/− mice (Fig. 4C) . As expected, the Ad-RPE65 delivery did not result in a significant change in mRNA levels of GNAT1 and rhodopsin in Rpe65 −/− mice (Figs. 4D 4E)
Effect of Ad-RPE65 Injection on Cone Degeneration
To determine the effect of RPE65 on cone degeneration, the ocular cross sections from wt C57BL/6 mice and from Rpe65 −/− mice, with and without a subretinal injection of Ad-RPE65, were labeled with an anti-MWL cone opsin antibody. The noninjected Rpe65 −/− mice lacked MWL cone outer segments at 6 weeks of age, consistent with our previous observation. 14 Ad-RPE65 injection at 2 weeks of age generated MWL cone outer segments in Rpe65 −/− mice, 4 weeks after the injection (Fig. 5) . The cone outer segments were only detected in the area that showed high levels of RPE65 expression, but not in the area lacking RPE65 expression, suggesting that the cone-rescuing effect correlates with the RPE65 expression levels (Fig. 5D)
To quantify the total cone numbers, both cones were stained with fluorescent PNA which labels both MWL and SWL cones in the retina. 28 In the retina flatmount from Rpe65 −/− mice at age of 4 weeks, significantly fewer cone numbers were observed, especially in the center and ventral retina, consistent with our previous studies. 14 In the age-matched Rpe65 −/− mice injected with Ad-RPE65 at age of 2 weeks, apparent increases of cone density were detected, especially in the central retina (Fig. 6) . The cones were counted in 10 randomly selected fields in each retina, and the average number of cones increased significantly in the Ad-RPE65-injected mice over the untreated Rpe65 −/− mice (P < 0.01). 
Effect of RPE65 Mutants from LCA Patients on Isomerohydrolase Activity and Cone Rescue in Rpe65−/− Mice
We also compared the effects of adenovirus-expressing wtRPE65 (Ad-wtRPE65), with those expressing two-point mutants of RPE65 from patients with LCA, R91W (Ad-R81W), and Y368H (Ad-Y368H), on isomerohydrolase activity and cone rescue. Ad-R91W; Ad-Y368H; a negative control, Ad-GFP; and a positive control, Ad-wtRPE65, were separately injected into the subretinal space of Rpe65 −/− mice at the age of 2 weeks at the same titer. Two weeks after the injection, an in vitro isomerohydrolase activity assay using the eyecups showed that isomerohydrolase activity was restored only in the mice injected with Ad-wtRPE65 but not in those injected with Ad-R91W, Ad-Y368H, or Ad-GFP. Consistent with the isomerohydrolase activity, the mutants R91W and Y368H and Ad-GFP did not rescue cone outer segments in Rpe65 −/− mice (Fig. 7)
Discussion
RPE65 mutations are associated with inherited retinal dystrophies. 10 11 12 29 Recently, our in vitro evidence has indicated that RPE65 is the isomerohydrolase essential for the regeneration of 11-cis retinoid in the visual cycle and for normal vision. 21 In the present study, RPE65 gene delivery was shown for the first time to generate robust isomerohydrolase activity in the eyecups of Rpe65 −/− mice and to restore partially the normal profile of endogenous retinoids in Rpe65 −/− retina and RPE. The restored generation of 11-cis retinal correlated with prevention of early cone degeneration and downregulation of cone opsin and transducin genes in Rpe65 −/− mice. 
Rpe65 −/− mice are commonly considered a model for vitamin A deficiency. 13 However, this model showed only cone degeneration, whereas rods are intact at early ages. 14 Our previous studies showed that cone degeneration starts at 2 weeks of age in Rpe65 −/− mice. By 4 weeks of age, massive cone loss was observed, and a large area of central retina becomes cone free. 14 In agreement, the expression of cone opsins and cone transducin was decreased significantly at early ages of the knockout mice, compared with age-matched wt mice. In contrast, the expression of rhodopsin and rod transducin was not significantly changed until 8 weeks of age. Furthermore, we have shown that the lack of 11-cis retinal is the cause of the early cone loss. 14 The present study further confirmed the early cone degeneration in this mouse model. This observation suggests that cones are more sensitive to the absence of chromophore for its pigments than are rods. It remains to be determined whether the downregulation of cone opsins and transducin is the cause or consequence of cone degeneration in Rpe65 −/− mice. 
Several previous studies have shown that RPE65 gene delivery restores photopic and scotopic ERG in dogs with RPE65 mutations. 15 30 Similar effects were observed in Rpe65 −/− mice. 18 20 Moreover, a recent study showed that RPE65 gene delivery increases total photoreceptor nuclear layers and also increases the number of UV cones in Rpe65 −/− mice at 8 months of age, compared with the age-matched, untreated Rpe65 −/− mice. 18 As our previous study showed that massive cone loss and significant downregulation of cone opsin and transducin genes occurs in Rpe65 −/− mice before 4 weeks of age, the present study determined the rescuing effect of RPE65 expression on Rpe65 −/− cones at early ages. The results showed that RPE65 expression in Rpe65 −/− prevented both cone opsin downregulation and cone photoreceptor degeneration at early ages in Rpe65 −/− mice. Although the intent of this study was not to develop a gene therapy, this observation, together with those in the previous report, 18 suggests that early RPE65 gene delivery can preserve cone structure and function in patients with retinal dystrophies associated with RPE65 mutations. 
Although its mutations are associated with retinal dystrophies, RPE65 function was not identified until we recently demonstrated that recombinant RPE65 has isomerohydrolase activity in vitro. 21 The effects of RPE65 gene delivery on retinoid profile in Rpe65 −/− mice and dogs have not been documented previously. As a result, the mechanism for the beneficial effects of RPE65 gene delivery on the ERG was not certain. Based on our in vitro evidence indicating that RPE65 is the isomerohydrolase, we have determined whether RPE65 gene delivery restores the isomerohydrolase activity in Rpe65 −/− mice. Our data showed that RPE65 delivery indeed generated isomerohydrolase activity in the eyecups of Rpe65 −/− mice to levels comparable to that in wt mice, confirming our in vitro results and indicating that RPE65 is the isomerohydrolase in the retinoid visual cycle. 21 Further, RPE65 gene expression also generated a significant amount of endogenous 11-cis retinal in Rpe65 −/− mice. This restored retinoid profile correlates with the effect on cone-specific gene expression and cone viability, suggesting that the cone-rescuing effect of RPE65 gene delivery is likely through the restoration of 11-cis retinal levels for the cone visual pigments. 
 
Figure 1.
 
RPE65 expression after subretinal injection of Ad-RPE65. (A– C) Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. RPE65 expression was examined by immunohistochemistry using an anti-RPE65 antibody in ocular cross sections from C57BL/6 mice (A), untreated Rpe65 −/− mice (B), and Rpe65 −/− mice 2 weeks after the Ad-RPE65 injection (C). Scale bar, 10 μm. (D) The RPE65 expression levels were compared by using Western blot analysis with 100 μg proteins from each eyecup. No RPE65 expression was found in the eyecup of untreated Rpe65 −/− mice (lane 2), whereas RPE65 expression was detected in Rpe65 −/− mice after the subretinal injection of Ad-RPE65 (lane 1) to a level comparable to that of C57BL/6 mice (lane 3).
Figure 1.
 
RPE65 expression after subretinal injection of Ad-RPE65. (A– C) Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. RPE65 expression was examined by immunohistochemistry using an anti-RPE65 antibody in ocular cross sections from C57BL/6 mice (A), untreated Rpe65 −/− mice (B), and Rpe65 −/− mice 2 weeks after the Ad-RPE65 injection (C). Scale bar, 10 μm. (D) The RPE65 expression levels were compared by using Western blot analysis with 100 μg proteins from each eyecup. No RPE65 expression was found in the eyecup of untreated Rpe65 −/− mice (lane 2), whereas RPE65 expression was detected in Rpe65 −/− mice after the subretinal injection of Ad-RPE65 (lane 1) to a level comparable to that of C57BL/6 mice (lane 3).
Figure 2.
 
Isomerohydrolase activity in the Rpe65 −/− eyecup after subretinal injection of Ad-RPE65. Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. At 4 weeks of age, the eyecups were homogenized, and the same amount (250 μg) of eyecup protein was incubated with all-trans [3H]-retinol for 1.5 hours. The generated retinoids were analyzed by HPLC. A representative HPLC profile from each group is shown. (A, B) 129sv wt and C57BL/6 mice, respectively. (C) Rpe65 −/− mice injected with a control virus, Ad-β-gal; (D) Rpe65 −/− mice injected with Ad-RPE65. Peak 1, retinyl esters; 2, all-trans retinal; 3, 11-cis retinol; 4, 13-cis retinol; and 5, all-trans retinol. Arrow: peaks corresponding to 11-cis retinol. (E) The 11-cis retinol generated in the in vitro isomerohydrolase activity assay was quantified by measuring the area of the 11-cis retinol peak in the HPLC profile and was averaged in the same group (mean ± SD, n = 4).
Figure 2.
 
Isomerohydrolase activity in the Rpe65 −/− eyecup after subretinal injection of Ad-RPE65. Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. At 4 weeks of age, the eyecups were homogenized, and the same amount (250 μg) of eyecup protein was incubated with all-trans [3H]-retinol for 1.5 hours. The generated retinoids were analyzed by HPLC. A representative HPLC profile from each group is shown. (A, B) 129sv wt and C57BL/6 mice, respectively. (C) Rpe65 −/− mice injected with a control virus, Ad-β-gal; (D) Rpe65 −/− mice injected with Ad-RPE65. Peak 1, retinyl esters; 2, all-trans retinal; 3, 11-cis retinol; 4, 13-cis retinol; and 5, all-trans retinol. Arrow: peaks corresponding to 11-cis retinol. (E) The 11-cis retinol generated in the in vitro isomerohydrolase activity assay was quantified by measuring the area of the 11-cis retinol peak in the HPLC profile and was averaged in the same group (mean ± SD, n = 4).
Figure 3.
 
Endogenous retinoid profile in the mouse RPE and retina. Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. At 6 weeks of age, the eyecup was homogenized, and endogenous retinoids were extracted and analyzed by HPLC. Each panel is a representative chromatogram from wt 129sv (A), C57BL/6 mice (B) and Rpe65 −/− mice injected with β-gal (C), and Rpe65 −/− mice injected with Ad-RPE65 (D). Peaks were identified by comparison to known standards. Peak 1, retinyl ester; 2, syn-11-cis retinal oxime; 3, syn-all-trans retinal oxime; 4, anti-13-cis retinal oxime; 5, anti-11-cis retinal oxime; and 6, anti-all-trans retinal oxime. (E) Amounts of 11-cis retinal were quantified by measuring the peak areas and were averaged within the group (mean ± SD, n = 4).
Figure 3.
 
Endogenous retinoid profile in the mouse RPE and retina. Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. At 6 weeks of age, the eyecup was homogenized, and endogenous retinoids were extracted and analyzed by HPLC. Each panel is a representative chromatogram from wt 129sv (A), C57BL/6 mice (B) and Rpe65 −/− mice injected with β-gal (C), and Rpe65 −/− mice injected with Ad-RPE65 (D). Peaks were identified by comparison to known standards. Peak 1, retinyl ester; 2, syn-11-cis retinal oxime; 3, syn-all-trans retinal oxime; 4, anti-13-cis retinal oxime; 5, anti-11-cis retinal oxime; and 6, anti-all-trans retinal oxime. (E) Amounts of 11-cis retinal were quantified by measuring the peak areas and were averaged within the group (mean ± SD, n = 4).
Figure 4.
 
Effect of Ad-RPE65 delivery on the expression of rod- and cone-specific genes in Rpe65 −/− mice. Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. Rod- and cone-specific mRNA levels in the retina were compared by real-time RT-PCR using equal amounts of mRNA from the Rpe65 −/− mice, with and without Ad-RPE65 injection at 4 weeks of age and age-matched wt 129sv and C57BL/6 mice. Representative real-time RT-PCR amplicons for the MWL cone opsin (A), SWL cone opsin (B), cone transducin α-subunit (GNAT2; C), rhodopsin (D) and rods transducin α-subunit (GNAT1; E). The relative mRNA levels were averaged, and each mRNA level in Rpe65 −/− mice was expressed as a percentage of that in wt 129sv mice (mean ± SD, n = 6).
Figure 4.
 
Effect of Ad-RPE65 delivery on the expression of rod- and cone-specific genes in Rpe65 −/− mice. Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. Rod- and cone-specific mRNA levels in the retina were compared by real-time RT-PCR using equal amounts of mRNA from the Rpe65 −/− mice, with and without Ad-RPE65 injection at 4 weeks of age and age-matched wt 129sv and C57BL/6 mice. Representative real-time RT-PCR amplicons for the MWL cone opsin (A), SWL cone opsin (B), cone transducin α-subunit (GNAT2; C), rhodopsin (D) and rods transducin α-subunit (GNAT1; E). The relative mRNA levels were averaged, and each mRNA level in Rpe65 −/− mice was expressed as a percentage of that in wt 129sv mice (mean ± SD, n = 6).
Figure 5.
 
Ad-RPE65 delivery generated cone outer segments in Rpe65 −/− mice. Rpe65 −/− mice received a subretinal injection of Ad-RPE65 at 2 weeks of age. Four weeks after the injection, ocular cross sections from the injected mice and age-matched controls (untreated Rpe65 −/− mice) were double stained using the anti-RPE65 antibody (green) and an anti-MWL cone opsin antibody (red). RPE65 and MWL cone outer segments were detected in C57BL/6 mice (A), but not in Rpe65 −/− mice (B). The Ad-RPE65 injection generated RPE65 and MWL cone outer segments in Rpe65 −/− mice (C). The cone outer segments were detected only in the area that has high levels of RPE65 expression but not in the area lacking the RPE65 signal (D). Scale bar, 20 μm.
Figure 5.
 
Ad-RPE65 delivery generated cone outer segments in Rpe65 −/− mice. Rpe65 −/− mice received a subretinal injection of Ad-RPE65 at 2 weeks of age. Four weeks after the injection, ocular cross sections from the injected mice and age-matched controls (untreated Rpe65 −/− mice) were double stained using the anti-RPE65 antibody (green) and an anti-MWL cone opsin antibody (red). RPE65 and MWL cone outer segments were detected in C57BL/6 mice (A), but not in Rpe65 −/− mice (B). The Ad-RPE65 injection generated RPE65 and MWL cone outer segments in Rpe65 −/− mice (C). The cone outer segments were detected only in the area that has high levels of RPE65 expression but not in the area lacking the RPE65 signal (D). Scale bar, 20 μm.
Figure 6.
 
Ad-RPE65 subretinal delivery prevented cone loss in the Rpe65 −/− retina. Rpe65 −/− mice received an injection of Ad-RPE65 at the age of 2 weeks, and cones were visualized at 4 weeks of age by staining of FITC-PNA in flatmounted retinas. Images are representative of the central retina from wt C57BL/6 mice (A); Rpe65 −/− mice without injection (B); and Rpe65 −/− mice injected with Ad-RPE65 (C). Scale bar, 10 μm. (D) Cones were counted in the flatmounted retina in 10 random fields per retina, averaged in 10 mice from each group, and expressed as cones per field (mean ± SD, n = 10).
Figure 6.
 
Ad-RPE65 subretinal delivery prevented cone loss in the Rpe65 −/− retina. Rpe65 −/− mice received an injection of Ad-RPE65 at the age of 2 weeks, and cones were visualized at 4 weeks of age by staining of FITC-PNA in flatmounted retinas. Images are representative of the central retina from wt C57BL/6 mice (A); Rpe65 −/− mice without injection (B); and Rpe65 −/− mice injected with Ad-RPE65 (C). Scale bar, 10 μm. (D) Cones were counted in the flatmounted retina in 10 random fields per retina, averaged in 10 mice from each group, and expressed as cones per field (mean ± SD, n = 10).
Figure 7.
 
The LCA mutants R91W and Y368H did not have a rescuing effect in Rpe65 −/− mice. Ad- R91W, Ad-Y368H, and Ad-GFP were injected into the subretinal space of Rpe65 −/− mice at the age of 2 weeks, with the same titer of Ad-wtRPE65 injection as the positive control. Isomerohydrolase activity assay and cone staining were performed 2 weeks after the injection. (A) Isomerohydrolase activity was only detected in the Rpe65 −/− mice injected by Ad-wtRPE65 but not in those injected with Ad-R91W, Ad-Y368H, or Ad-GFP. Peak 1, retinyl esters; 2, all-trans retinal; 3, 11-cis retinol (black arrow); 4, 13-cis retinol; and 5, all-trans retinol. (BF) The mouse eye sections were double labeled by an anti-RPE65 (green) and anti-MWL cone opsin (red) antibodies. (B, C) Phase contrast and double labeling images, respectively, of the same area from the Rpe65 −/− eye injected with Ad-R91W. (D, E) Phase contrast and double-labeling images, respectively, of the same area from the Rpe65 −/− eye injected with Ad-Y368H. (F) Double-labeling image of the Rpe65 −/− eye injected with Ad-wtRPE65 as the positive control. (G) Sections from Rpe65 −/− mice injected with Ad-GFP were labeled by the anti-MWL cone opsin antibody (red) as the negative control. (G, green) GFP expression. Scale bar, 20 μm.
Figure 7.
 
The LCA mutants R91W and Y368H did not have a rescuing effect in Rpe65 −/− mice. Ad- R91W, Ad-Y368H, and Ad-GFP were injected into the subretinal space of Rpe65 −/− mice at the age of 2 weeks, with the same titer of Ad-wtRPE65 injection as the positive control. Isomerohydrolase activity assay and cone staining were performed 2 weeks after the injection. (A) Isomerohydrolase activity was only detected in the Rpe65 −/− mice injected by Ad-wtRPE65 but not in those injected with Ad-R91W, Ad-Y368H, or Ad-GFP. Peak 1, retinyl esters; 2, all-trans retinal; 3, 11-cis retinol (black arrow); 4, 13-cis retinol; and 5, all-trans retinol. (BF) The mouse eye sections were double labeled by an anti-RPE65 (green) and anti-MWL cone opsin (red) antibodies. (B, C) Phase contrast and double labeling images, respectively, of the same area from the Rpe65 −/− eye injected with Ad-R91W. (D, E) Phase contrast and double-labeling images, respectively, of the same area from the Rpe65 −/− eye injected with Ad-Y368H. (F) Double-labeling image of the Rpe65 −/− eye injected with Ad-wtRPE65 as the positive control. (G) Sections from Rpe65 −/− mice injected with Ad-GFP were labeled by the anti-MWL cone opsin antibody (red) as the negative control. (G, green) GFP expression. Scale bar, 20 μm.
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Figure 1.
 
RPE65 expression after subretinal injection of Ad-RPE65. (A– C) Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. RPE65 expression was examined by immunohistochemistry using an anti-RPE65 antibody in ocular cross sections from C57BL/6 mice (A), untreated Rpe65 −/− mice (B), and Rpe65 −/− mice 2 weeks after the Ad-RPE65 injection (C). Scale bar, 10 μm. (D) The RPE65 expression levels were compared by using Western blot analysis with 100 μg proteins from each eyecup. No RPE65 expression was found in the eyecup of untreated Rpe65 −/− mice (lane 2), whereas RPE65 expression was detected in Rpe65 −/− mice after the subretinal injection of Ad-RPE65 (lane 1) to a level comparable to that of C57BL/6 mice (lane 3).
Figure 1.
 
RPE65 expression after subretinal injection of Ad-RPE65. (A– C) Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. RPE65 expression was examined by immunohistochemistry using an anti-RPE65 antibody in ocular cross sections from C57BL/6 mice (A), untreated Rpe65 −/− mice (B), and Rpe65 −/− mice 2 weeks after the Ad-RPE65 injection (C). Scale bar, 10 μm. (D) The RPE65 expression levels were compared by using Western blot analysis with 100 μg proteins from each eyecup. No RPE65 expression was found in the eyecup of untreated Rpe65 −/− mice (lane 2), whereas RPE65 expression was detected in Rpe65 −/− mice after the subretinal injection of Ad-RPE65 (lane 1) to a level comparable to that of C57BL/6 mice (lane 3).
Figure 2.
 
Isomerohydrolase activity in the Rpe65 −/− eyecup after subretinal injection of Ad-RPE65. Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. At 4 weeks of age, the eyecups were homogenized, and the same amount (250 μg) of eyecup protein was incubated with all-trans [3H]-retinol for 1.5 hours. The generated retinoids were analyzed by HPLC. A representative HPLC profile from each group is shown. (A, B) 129sv wt and C57BL/6 mice, respectively. (C) Rpe65 −/− mice injected with a control virus, Ad-β-gal; (D) Rpe65 −/− mice injected with Ad-RPE65. Peak 1, retinyl esters; 2, all-trans retinal; 3, 11-cis retinol; 4, 13-cis retinol; and 5, all-trans retinol. Arrow: peaks corresponding to 11-cis retinol. (E) The 11-cis retinol generated in the in vitro isomerohydrolase activity assay was quantified by measuring the area of the 11-cis retinol peak in the HPLC profile and was averaged in the same group (mean ± SD, n = 4).
Figure 2.
 
Isomerohydrolase activity in the Rpe65 −/− eyecup after subretinal injection of Ad-RPE65. Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. At 4 weeks of age, the eyecups were homogenized, and the same amount (250 μg) of eyecup protein was incubated with all-trans [3H]-retinol for 1.5 hours. The generated retinoids were analyzed by HPLC. A representative HPLC profile from each group is shown. (A, B) 129sv wt and C57BL/6 mice, respectively. (C) Rpe65 −/− mice injected with a control virus, Ad-β-gal; (D) Rpe65 −/− mice injected with Ad-RPE65. Peak 1, retinyl esters; 2, all-trans retinal; 3, 11-cis retinol; 4, 13-cis retinol; and 5, all-trans retinol. Arrow: peaks corresponding to 11-cis retinol. (E) The 11-cis retinol generated in the in vitro isomerohydrolase activity assay was quantified by measuring the area of the 11-cis retinol peak in the HPLC profile and was averaged in the same group (mean ± SD, n = 4).
Figure 3.
 
Endogenous retinoid profile in the mouse RPE and retina. Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. At 6 weeks of age, the eyecup was homogenized, and endogenous retinoids were extracted and analyzed by HPLC. Each panel is a representative chromatogram from wt 129sv (A), C57BL/6 mice (B) and Rpe65 −/− mice injected with β-gal (C), and Rpe65 −/− mice injected with Ad-RPE65 (D). Peaks were identified by comparison to known standards. Peak 1, retinyl ester; 2, syn-11-cis retinal oxime; 3, syn-all-trans retinal oxime; 4, anti-13-cis retinal oxime; 5, anti-11-cis retinal oxime; and 6, anti-all-trans retinal oxime. (E) Amounts of 11-cis retinal were quantified by measuring the peak areas and were averaged within the group (mean ± SD, n = 4).
Figure 3.
 
Endogenous retinoid profile in the mouse RPE and retina. Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. At 6 weeks of age, the eyecup was homogenized, and endogenous retinoids were extracted and analyzed by HPLC. Each panel is a representative chromatogram from wt 129sv (A), C57BL/6 mice (B) and Rpe65 −/− mice injected with β-gal (C), and Rpe65 −/− mice injected with Ad-RPE65 (D). Peaks were identified by comparison to known standards. Peak 1, retinyl ester; 2, syn-11-cis retinal oxime; 3, syn-all-trans retinal oxime; 4, anti-13-cis retinal oxime; 5, anti-11-cis retinal oxime; and 6, anti-all-trans retinal oxime. (E) Amounts of 11-cis retinal were quantified by measuring the peak areas and were averaged within the group (mean ± SD, n = 4).
Figure 4.
 
Effect of Ad-RPE65 delivery on the expression of rod- and cone-specific genes in Rpe65 −/− mice. Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. Rod- and cone-specific mRNA levels in the retina were compared by real-time RT-PCR using equal amounts of mRNA from the Rpe65 −/− mice, with and without Ad-RPE65 injection at 4 weeks of age and age-matched wt 129sv and C57BL/6 mice. Representative real-time RT-PCR amplicons for the MWL cone opsin (A), SWL cone opsin (B), cone transducin α-subunit (GNAT2; C), rhodopsin (D) and rods transducin α-subunit (GNAT1; E). The relative mRNA levels were averaged, and each mRNA level in Rpe65 −/− mice was expressed as a percentage of that in wt 129sv mice (mean ± SD, n = 6).
Figure 4.
 
Effect of Ad-RPE65 delivery on the expression of rod- and cone-specific genes in Rpe65 −/− mice. Ad-RPE65 was injected into the subretinal space of Rpe65 −/− mice at 2 weeks of age. Rod- and cone-specific mRNA levels in the retina were compared by real-time RT-PCR using equal amounts of mRNA from the Rpe65 −/− mice, with and without Ad-RPE65 injection at 4 weeks of age and age-matched wt 129sv and C57BL/6 mice. Representative real-time RT-PCR amplicons for the MWL cone opsin (A), SWL cone opsin (B), cone transducin α-subunit (GNAT2; C), rhodopsin (D) and rods transducin α-subunit (GNAT1; E). The relative mRNA levels were averaged, and each mRNA level in Rpe65 −/− mice was expressed as a percentage of that in wt 129sv mice (mean ± SD, n = 6).
Figure 5.
 
Ad-RPE65 delivery generated cone outer segments in Rpe65 −/− mice. Rpe65 −/− mice received a subretinal injection of Ad-RPE65 at 2 weeks of age. Four weeks after the injection, ocular cross sections from the injected mice and age-matched controls (untreated Rpe65 −/− mice) were double stained using the anti-RPE65 antibody (green) and an anti-MWL cone opsin antibody (red). RPE65 and MWL cone outer segments were detected in C57BL/6 mice (A), but not in Rpe65 −/− mice (B). The Ad-RPE65 injection generated RPE65 and MWL cone outer segments in Rpe65 −/− mice (C). The cone outer segments were detected only in the area that has high levels of RPE65 expression but not in the area lacking the RPE65 signal (D). Scale bar, 20 μm.
Figure 5.
 
Ad-RPE65 delivery generated cone outer segments in Rpe65 −/− mice. Rpe65 −/− mice received a subretinal injection of Ad-RPE65 at 2 weeks of age. Four weeks after the injection, ocular cross sections from the injected mice and age-matched controls (untreated Rpe65 −/− mice) were double stained using the anti-RPE65 antibody (green) and an anti-MWL cone opsin antibody (red). RPE65 and MWL cone outer segments were detected in C57BL/6 mice (A), but not in Rpe65 −/− mice (B). The Ad-RPE65 injection generated RPE65 and MWL cone outer segments in Rpe65 −/− mice (C). The cone outer segments were detected only in the area that has high levels of RPE65 expression but not in the area lacking the RPE65 signal (D). Scale bar, 20 μm.
Figure 6.
 
Ad-RPE65 subretinal delivery prevented cone loss in the Rpe65 −/− retina. Rpe65 −/− mice received an injection of Ad-RPE65 at the age of 2 weeks, and cones were visualized at 4 weeks of age by staining of FITC-PNA in flatmounted retinas. Images are representative of the central retina from wt C57BL/6 mice (A); Rpe65 −/− mice without injection (B); and Rpe65 −/− mice injected with Ad-RPE65 (C). Scale bar, 10 μm. (D) Cones were counted in the flatmounted retina in 10 random fields per retina, averaged in 10 mice from each group, and expressed as cones per field (mean ± SD, n = 10).
Figure 6.
 
Ad-RPE65 subretinal delivery prevented cone loss in the Rpe65 −/− retina. Rpe65 −/− mice received an injection of Ad-RPE65 at the age of 2 weeks, and cones were visualized at 4 weeks of age by staining of FITC-PNA in flatmounted retinas. Images are representative of the central retina from wt C57BL/6 mice (A); Rpe65 −/− mice without injection (B); and Rpe65 −/− mice injected with Ad-RPE65 (C). Scale bar, 10 μm. (D) Cones were counted in the flatmounted retina in 10 random fields per retina, averaged in 10 mice from each group, and expressed as cones per field (mean ± SD, n = 10).
Figure 7.
 
The LCA mutants R91W and Y368H did not have a rescuing effect in Rpe65 −/− mice. Ad- R91W, Ad-Y368H, and Ad-GFP were injected into the subretinal space of Rpe65 −/− mice at the age of 2 weeks, with the same titer of Ad-wtRPE65 injection as the positive control. Isomerohydrolase activity assay and cone staining were performed 2 weeks after the injection. (A) Isomerohydrolase activity was only detected in the Rpe65 −/− mice injected by Ad-wtRPE65 but not in those injected with Ad-R91W, Ad-Y368H, or Ad-GFP. Peak 1, retinyl esters; 2, all-trans retinal; 3, 11-cis retinol (black arrow); 4, 13-cis retinol; and 5, all-trans retinol. (BF) The mouse eye sections were double labeled by an anti-RPE65 (green) and anti-MWL cone opsin (red) antibodies. (B, C) Phase contrast and double labeling images, respectively, of the same area from the Rpe65 −/− eye injected with Ad-R91W. (D, E) Phase contrast and double-labeling images, respectively, of the same area from the Rpe65 −/− eye injected with Ad-Y368H. (F) Double-labeling image of the Rpe65 −/− eye injected with Ad-wtRPE65 as the positive control. (G) Sections from Rpe65 −/− mice injected with Ad-GFP were labeled by the anti-MWL cone opsin antibody (red) as the negative control. (G, green) GFP expression. Scale bar, 20 μm.
Figure 7.
 
The LCA mutants R91W and Y368H did not have a rescuing effect in Rpe65 −/− mice. Ad- R91W, Ad-Y368H, and Ad-GFP were injected into the subretinal space of Rpe65 −/− mice at the age of 2 weeks, with the same titer of Ad-wtRPE65 injection as the positive control. Isomerohydrolase activity assay and cone staining were performed 2 weeks after the injection. (A) Isomerohydrolase activity was only detected in the Rpe65 −/− mice injected by Ad-wtRPE65 but not in those injected with Ad-R91W, Ad-Y368H, or Ad-GFP. Peak 1, retinyl esters; 2, all-trans retinal; 3, 11-cis retinol (black arrow); 4, 13-cis retinol; and 5, all-trans retinol. (BF) The mouse eye sections were double labeled by an anti-RPE65 (green) and anti-MWL cone opsin (red) antibodies. (B, C) Phase contrast and double labeling images, respectively, of the same area from the Rpe65 −/− eye injected with Ad-R91W. (D, E) Phase contrast and double-labeling images, respectively, of the same area from the Rpe65 −/− eye injected with Ad-Y368H. (F) Double-labeling image of the Rpe65 −/− eye injected with Ad-wtRPE65 as the positive control. (G) Sections from Rpe65 −/− mice injected with Ad-GFP were labeled by the anti-MWL cone opsin antibody (red) as the negative control. (G, green) GFP expression. Scale bar, 20 μm.
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