Description

Background:

• The human RPE65 gene is located at 1p31 and encodes the retinal pigment epithelium-specific 65 kDa protein (RPE65). As a key enzyme of the carotenoid oxygenase/lyase superfamily, it plays an essential role in retinal vitamin A metabolism.
• RPE65 is a membrane-bound protein featuring an N-terminal signal peptide and a C-terminal esterase domain. Its three-dimensional structure comprises multiple transmembrane helices and extracellular loops. RPE65 primarily catalyzes the hydrolysis and isomerization of all-trans retinyl esters in the visual cycle, facilitating the regeneration of 11-cis retinol (the chromophore of rod and cone opsins).

Targeting strategy:

• The exons 3-15 of mouse Rpe65 gene were knocked out in B-Rpe65 KO mice.

Verification: 

• Mouse RPE65 protein was only detected in the eye of C57BL/6JNifdc mice, but not in homozygous B-Rpe65 KO mice.  
• Mouse Rpe65 mRNA was only detectable in wild-type mice, but not in homozygous B-Rpe65 KO mice.

Application:

• This product can be utilized to assess the pharmacodynamic effects of therapies targeting autosomal recessive retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA).

Targeting strategy

Gene targeting strategy for B-Rpe65 KO mice. The exons 3-15 of mouse Rpe65 gene were knocked out in B-Rpe65 KO mice. As a result, mouse RPE65 protein will not be expressed anymore.

Protein expression analysis

Western blot analysis of RPE65 protein expression in homozygous B-Rpe65 KO mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-Rpe65 KO mice (-/-), and then analyzed by western blot with anti-mouse RPE65 antibody (Abcam, ab231782). 40 μg total proteins were loaded for western blotting analysis. Mouse RPE65 was only detected in the eye of C57BL/6JNifdc mice.

mRNA expression analysis

Strain specific analysis of mRpe65 mRNA expression in wild-type C57BL/6JNifdc mice and B-Rpe65 KO mice by RT-PCR. Eye RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-Rpe65 KO mice (-/-), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse Rpe65 primers. Mouse Rpe65 mRNA was only detectable in wild-type mice but not in homozygous B-Rpe65 KO mice.