hRHO-P23H

Fig.1 Detection of RHO expression in eyeball by RT-PCR. 

Wild type: only one band at 247 bp with primers F1/R1(mRho);

Heterozygous: one band at 247 bp with primers F1/R1(mRho) and one band at 695 bp with primers F2/R2(hRHO).

Abbr. M, DNA marker; HE, heterozygous; WT, wild type.

Fig.2 RNA sequencing analysis of RT-PCR products.

Abbr. HO, homozygous; HE, heterozygous.

Fig.3 Representative images of retinal morphology in WT C57BL/6 mice, hRHO mice, and heterozygous hRHO-P23H 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-P23H (n=2, 15 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 heterozygous hRHO-P23H mice. 

The results indicated that the photoreceptor outer segments, outer nuclear layers, and inner nuclear layer became thinner in hRHO-P23H mice (n=2, 15 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 heterozygous hRHO-P23H mice. 

The results indicated that the outer nuclear layers (ONL) of heterozygous hRHO-P23H mice (n=2, 15 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 heterozygous hRHO-P23H mice. 

ERG measurements showed that the amplitudes scotopic a-wave (A), scotopic b-wave (B), and photopic b-wave (D) significantly decreased in hRHO-P23H mice compared with WT C57BL/6 and hRHO mice. (n=2). Blue，WT C57BL/6 mice; orange, hRHO mice; purple, hRHO-D190Y mice.