Reproduction：Homozygote x Homozygote
Leber's congenital amaurosis (LCA) is a group of inherited retinal diseases with severe visual impairment. The main symptoms of LCA are loss of visual acuity, nystagmus, and decreased or absent light reflexes in the optic rods and cones at birth or several months after birth. Rpe65 is an allosteric hydrolase, mainly found in the retinal pigment epithelium (RPE), whose main role is to catalyze the conversion of all-trans retinyl ester to 11-cis retinol. Mutations in the Rpe65 gene not only disrupt ocular circulation but also cause further degeneration of the neural retina and RPE cells, resulting in irreversible blindness. Mutations in the Rpe65 allele have been found to destroy optic cells and lead to clinical manifestations of Leber's congenital amaurosis type 2 (LCA2) and early-onset retinal dystrophy, eventually leading to complete blindness[1-2].
This model was constructed by introducing the Rpe65 R44X point mutation into the mouse gene by gene editing techniques, resulting in a C to T base substitution at nucleotide 130 of the gene encoding the Rpe65 protein, leading to a stop codon at amino acid position 44 instead of arginine (p.R44*). It has been reported that the eyes of homozygous mice carrying this mutation will not express the RPE65 protein. This model can be used in studies of retinitis pigmentosa 20 (RP20), Leber's congenital amaurosis type 2 (LCA 2), and the visual cycle.
The mouse Rpe65 gene contains 15 exons. A p.R44* (CGA to TGA) point mutation was introduced into the Rpe65 gene by gene editing techniques, resulting in premature termination of protein translation.
● Retinitis pigmentosa 20 (RP20) research.
● Leber's congenital amaurosis type 2 (LCA2) research.
● The visual cycle research.
1.Optical coherence tomography (OCT)
Figure 1. Optical coherence tomography (OCT) result of 4-week-old B6-Rpe65 R44X mice and C57BL/6J wild-type mice. Compared to the wild-type mice, B6-Rpe65 R44X mice have a distinct retinopathy phenotype.
Figure 2. The H&E and IF staining of ocular tissues from 6-week-old B6-Rpe65 R44X mice and C57BL/6J wild-type mice. Compared to wild-type mice, B6-Rpe65 R44X mice have a significant deletion of Rpe65 protein expression due to the presence of the mutation.
3.Electroretinogram (ERG) testing
Figure 3. Electroretinogram (ERG) results of B6-Rpe65 R44X mice (REP65) and C57BL/6J wild-type mice (WT). The disappearance in Scotopic and Photopic ERG a- and b- waves were detected in B6-Rpe65 R44X mice.
The above results indicated that B6-Rpe65 R44X mice had symptoms of disrupted RPE cell function, apoptosis of photoreceptor cells, disorganized outer segmental disc arrangement of optic rod cells, disorganized outer segmental disc arrangement of optic rod cells, and extinguished optic rod cell wave-forms, which were consistent with the typical clinical manifestations of Leber's congenital amaurosis.