RNAi-based Suppression Of Human RHO In A Mouse Model Of Autosomal Dominant Retinitis Pigmentosa

Objective1. To design a mouse model of autosomal dominant retinitis pigmentosa who carry by human mutant RHO through gene targeting. To identify pathological character in different age.2. To construct the LV-GFP virus which could express the specific siRNA targeted to hRHO and evaluate its inhibitory effect in vivo. Mice treat with LV-GFP-shRNA were injected subretinally. To investigate the hRHO degeneration effect by using hRHO RNAi.Methods1. To construct mRHOp-hRHO-SV40A-PBS, targeting inject in ertilized egg. To identify RP mice by using RT-PCR and western-blot.2. Eighteen RP mice were sacrificed at different time namely postnatal days 15 and 30,60. The eyeball were immediately enuleated and fixed in 10% formaldehyde or 2.5% glutaral solution.Routine pathologic procedure were performed and sections were stained by hematoxylin and eosin(HE) and terminal deoxytransferase-mediated dUTP nicked labeling(TUNEL).The eyeball fixed with glutaral solution were examined by electron-microscopy.3. In our prophase study, we had successfully inhibited the expression of mutant hRHO genes effectively in cultured rat rod cells by applying chemical synthetic siRNA, and sieved out one high-performance RNA interference chisequence which gene silencing efficiency is 79.14%. To construct the LV-GFP virus which could express the specific siRNA targeted to mutant hRHO.4. Eighty RP mice were used in this research (postnatal day 15). LV-GFP-shRNA was injected on in the right eyes, and left eyes were neither injected with saline or with control virus as blank control. Mice were euthanized by pentobarbital sodium after injection day15,30,45,60,and then eyes were removed. To investigate the hRHO degeneration effect by using hRHO RNAi. To confirm the transfection efficiency after injected subretinally in different time. To evaluate the range of transfection efficiency through 3 point (0:none; 1: sporadic masccline; 2:part of masccline connected; 3:consecutive masccline).To observe the layer number of outer nuclear layer by immunofluorescence.Results1. We had constructed the mRHOp-hRHO-SV40-PBS and made the amplification and depuration of it. mRHOp-hRHO-SV40-PBS were microinjected in amphicytula, and breed RP mice who carry by human mutant RHO successfully.2. The layer number of retinal outer nuclear layer cells was gradually decreased from day 15 (7.05±0.25) to day 60 (2.04±0.28). At the day 30, (5.29±0.24) layer were observed. Compared with RP mice, the layer number of retinal outer nuclear layer cells in wild type mice were observed (9.30±0.37) stable in all the time. The apoptosis of outer layer cells were seen on day 15 and increased deeply in the day 60. External limiting membrane were discontinuation on day 30, vacuole occurred in the outer segmen disk. Degeneration in bioblast and endocytoplasmic reticulum were altered and the apoptosis of outer layer cells were seen by the electro-microscopy in RP mice.3. We had constructed the LV-GFP for expressing siRNA and made the amplification and depuration of it. The tite of LV-GFP-shRNA was 1.0×1010 particles per ml.4. Our in vivo test showed 15 and 60 days after LV-GFP-shRNA injected subretinally, the GFP could be detected in retina sections,which meant that LV-GFP-shRNA had successfully transduced into rod cells and the shRNA being expressed. RT-PCR test showed the recombinant virus vector could efficiently inhibited the RHO mRNA in rod cells after injected 45 days (P<0.05).Western blot test also showed the recombinant virus vector could efficiently inhibited the RHO protein in rod cells after injected 45 days (P< 0.05) compared with control.Conclutions1.We created a mouse model of autosomal dominant retinitis pigmentosa who carry by human mutant RHO through gene targeting successfully and provide RP animal model for the further research.2. We found the process of histopathology in the RP model were similar with human who suffered from RP.3. We had constructed the LV-GFP for expressing si RNA and made the amplification and depuration of it. The tite of LV-GFP-shRNA was 1.0×1010 particles per ml.4. After injected subretinally, LV-GFP-shRNA could depress of the expression of RHO either mRNA or protein in short period.