• Abca4 encodes an ATP-binding cassette transporter that shuttles all-trans-retinal across photoreceptor disc membranes, while Rdh8 encodes retinol dehydrogenase 8, the primary enzyme that reduces all-trans-retinal to all-trans-retinol in the visual cycle. Combined deletion of Abca4 and Rdh8 severely impairs all-trans-retinal clearance, leading to excessive accumulation of cytotoxic bisretinoid lipofuscin (such as A2E) in the retinal pigment epithelium (RPE), complement activation, and progressive photoreceptor degeneration, which can be accelerated by bright light exposure.
• The exons 3-50 of mouse Abca4 gene were knocked out in B-Abca4/Rdh8 DKO mice. The exons 2-6 of mouse Rdh8 gene were knocked out in B-Abca4/Rdh8 DKO mice.
• mRNA expression analysis: Mouse Abca4 and Rdh8 mRNA was only detectable in wild-type mice but not in homozygous B-Abca4/Rdh8 DKO mice.
• Protein expression analysis: Mouse Abca4 was only detected in the eyes of wild-type mice but not in homozygous B-Abca4/Rdh8 DKO mice.
• A2E analysis: B-Abca4/Rdh8 DKO mice had substantially higher A2E levels than wild-type mice in both the absence and presence of light exposure.
• Total retinal thickness and ONL thickness analysis: ONL thinning demonstrates that Abca4/Rdh8 double deletion exacerbates light-induced photoreceptor degeneration.
• Dark and light ERG analysis: Mouse retinal function was markedly impaired in light-exposed B-Abca4/Rdh8 DKO mice compared with control groups.
• B-Abca4/Rdh8 DKO mice are widely used for evaluating therapeutic candidates targeting retinal degenerative disorders

Gene targeting strategy for B-Abca4/Rdh8 DKO mice.

The exons 3-50 of mouse Abca4 gene were knocked out in B-Abca4/Rdh8 DKO mice.

The exons 2-6 of mouse Rdh8 gene were knocked out in B-Abca4/Rdh8 DKO mice.

Strain specific analysis of Abca4 and Rdh8 mRNA expression in wild-type C57BL/6JNifdc and B-Abca4/Rdh8 DKO mice by RT-PCR. Eye RNA were isolated from wild-type C57BL/6JNifdc (+/+) and homozygous B-Abca4/Rdh8 DKO mice (-/-), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse Abca4 and Rdh8 primers. Mouse Abca4 and Rdh8 mRNA was only detectable in wild-type mice but not in homozygous B-Abca4/Rdh8 DKO mice.

Western blot analysis of ABCA4 protein expression in homozygous B-Abca4/Rdh8 DKO mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-Abca4/Rdh8 DKO mice (-/-), and then analyzed by western blot with anti-ABCA4 antibody (Abcam, ab72955). 40 μg total proteins were loaded for western blotting analysis. Mouse Abca4 was only detected in eye of wild-type mice but not in homozygous B-Abca4/Rdh8 DKO mice.

A2E analysis in wild-type C57BL/6JNifdc and B-Abca4/Rdh8 DKO mice. Eyes were isolated from wild-type C57BL/6JNifdc (+/+) and homozygous B-Abca4/Rdh8 DKO mice (-/-). LC-MS measurements of A2E showed that B-Abca4/Rdh8 DKO mice had substantially higher A2E levels than wild-type animals in both the absence and presence of light exposure, indicating that Abca4/Rdh8 deficiency alone drives robust bis-retinoid accumulation independent of illumination. Values are expressed as mean ± SEM. Significance was determined by t-test, *P<0.05, **P<0.01, ***P<0.001.

The co-validation data were provided by Pharmalegacy.

Total retinal thickness and ONL thickness analysis in wild-type C57BL/6JNifdc and homozygous B-Abca4/Rdh8 DKO mice. Left panels: Total retinal thickness; Right panels: ONL thickness. ONL thinning demonstrates that Abca4/Rdh8 double deletion exacerbates light-induced photoreceptor degeneration. Values are expressed as mean ± SEM. Significance was determined by t-test, *P<0.05, **P<0.01, ***P<0.001.

The co-validation data were provided by Pharmalegacy.

Dark-adapted ERG analysis of rod-mediated retinal function in B-Abca4/Rdh8 DKO and C57BL/6JNifdc mice after light exposure. Dark ERG recordings were performed at Day 0, Day 7 and Day 14 in control and light-exposed B-Abca4/Rdh8 DKO and C57BL/6JNifdc mice. a-wave amplitude, a-wave latency, b-wave amplitude and b-wave latency were quantified. Rod-mediated retinal function was markedly impaired in light-exposed B-Abca4/Rdh8 DKO mice compared with control groups. Values are expressed as mean ± SEM. Significance was determined by t-test, *P<0.05, **P<0.01, ***P<0.001.

The co-validation data were provided by Pharmalegacy.

Light-adapted ERG analysis of cone-mediated retinal function in B-Abca4/Rdh8 DKO and C57BL/6JNifdc mice after light exposure. Light-adapted ERG recordings were performed at Day 0, Day 7 and Day 14 in control and light-exposed B-Abca4/Rdh8 DKO and C57BL/6JNifdc mice. a-wave amplitude, a-wave latency, b-wave amplitude and b-wave latency were quantified. Cone-mediated retinal function was markedly impaired in light-exposed B-Abca4/Rdh8 DKO mice compared with control groups. Values are expressed as mean ± SEM. Significance was determined by t-test, *P<0.05, **P<0.01, ***P<0.001.

The co-validation data were provided by Pharmalegacy.