hRHO-R135W

Fig.1 Detection of RHO expression in eyeball by RT-PCR. Eyeball RNA was extracted from 18-week-old male wild-type C57BL/6 (WT)(n=1), 14-week-old male homozygous hRHO knockin mice (HO)(n=1) and 18-week-old male homozygous hRHO-R135W knockin mice (HO)(n=2), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mRNA primers. Mouse Rho mRNA (339 bp) was detectable only in wild-type C57BL/6 mice. Human RHO mRNA (445 bp) was detectable only in homozygous hRHO-R135W knockin mice but not in wild-type mice. 

Abbr. M, marker; HO, homozygous; WT, wild type.

Fig.2 mRNA sequencing of hRHO-R135W mice. Sequencing data of RT-PCR products confirmed the presence of target mutations in hRHO-R135W knockin mice. 

Abbr. HO, homozygous; WT, wild type.

Fig.3 Representative images of retinal morphology in WT C57BL/6 mice, hRHO mice, and homozygous hRHO-R135W mice. The results showed that the homozygous hRHO mice (n=2, 14 weeks, male) had no defect in morphology of fundus and retinal layers compared to the WT C57BL/6 mice. Compared with WT C57BL/6 and hRHO mice, the outer nuclear layer (ONL) of hRHO-R135W (n=2, 18 weeks, male) mice was thinner. 

Abbr. FFA, fundus fluorescein angiography; OCT, optical coherence tomography.

Fig.4 Representative images of HE staining in WT C57BL/6 mice, hRHO mice, and homozygous hRHO-R135W mice. The results indicated that the photoreceptor outer segments (OS), outer nuclear layers (ONL), and inner nuclear layer (INL) became thinner in hRHO-R135W mice (n=2, 18 weeks, male) compared with WT C57BL/6 and hRHO mice (n=2, 14 weeks, male). Scale bar, 50 μm. 

Abbr. OS, outer segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. 

Fig.5 Representative images of immunohistochemistry staining in WT C57BL/6 mice, hRHO mice, and homozygous hRHO-R135W mice. The results indicated that the outer nuclear layers (ONL) of homozygous hRHO-R135W mice (n=2, 18 weeks, male) was thinner, and the expression of rhodopsin protein on the retina was abnormal compared with WT C57BL/6 and hRHO mice (n=2, 14 weeks, male). Black arrow represents the positive area of rhodopsin protein. Scale bar, 50 μm.

Abbr. OS, outer segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. 

Fig.6 Electroretinogram responses in WT C57BL/6 mice, hRHO mice, and homozygous hRHO-R135W mice. ERG measurements showed that the amplitudes scotopic a-wave (A), scotopic b-wave (B), photopic a-wave (C), and photopic b-wave (D) significantly decreased in hRHO-R135W mice (n=2, 18 weeks, male) compared with WT C57BL/6 and hRHO mice (n=2, 14 weeks, male).