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Alport syndrome is a basement membrane disorder, characterized by hereditary nephropathy that results in irreversible, progressive renal failure. In the past decades, several Alport syndrome animal models have been produced in mice. The knockout mice for Col4α3, Col4α4, and Col4α5 have been developed and well characterized. The disease progression and pharmacological therapy were shown to genetic variability in the human patients. However, in spite of variabilities displayed in the genetic background, only a few mammalian models of Alport syndrome have been established.
Recent progress in of the CRISPR/Cas9 system has been shown to be an efficient gene-editing technology in various organisms. We recently developed a novel method called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD); a in vivo genome editing system that does not require ex vivo handling of embryos. This technology has now been improved and renamed as i-GONAD for mice, and rat GONAD rGONAD for rats. In this study, we produced a novel Alport syndrome rat model utilizing the rGONAD technology.
Type IV Collagen α5 deficient rats were produced by the rGONAD method as previously described. Briefly, gene targeting strategy is designed to integrate tandem STOP codons into 27 bases after the first ATG in the rat Col4α5 gene. For the preparation of CRISPR/Cas9 reagents, the IDT CRISPR/Cas9 system was used in accordance with the manufacturer’s protocol. Approximately 2-2.5µl of the electroporation solution was injected into the oviductal lumen from up-stream of ampulla using a micropipette. Electroporation was performed using a NEPA21. All measurements of proteinuria, blood urea nitrogen (BUN), and serum creatinine (Cre) were performed according to the manufacturer’s recommended protocols.
We developed Col4α5 deficient mutant rats of the WKY, SD, and F344 strains, identical to the Col4α5 G5X mutant mice. Col4α5 deficient rats exhibited hematuria, proteinuria, high levels of BUN, and CRE, and histological and ultrastructural analyses displayed the abnormalities including parietal cell hyperplasia, mesangial sclerosis, and interstitial fibrosis. Then, we demonstrated that α3/α4/α5 (IV) and α5/α5/α6 (IV) chains of type IV collagen were disrupted in the Col4α5 deficient rats. However, we found that there were strain-specific differences in the lifespan of the Col4α5 deficient rats. In the WKY rat strain, all hemizygous mutant males died from 18 to 28 weeks of age. In contrast, in the SD and F344 rat strains, the lifespan of the hemizygous mutant males was much longer than that of the WKY males.
We first established the rGONAD method for generating genome-edited rats, suggesting that the rGONAD method will facilitate the production of rat genome engineering experiments in many laboratories. We also described the generation of a novel rat model of Alport syndrome. Col4α5 deficient rats revealed typical physiological, pathological, and also histological characteristics of Alport syndrome. Thus, Col4α5 mutant rat is a reliable candidate for an Alport syndrome model animal for underlying the mechanism of renal diseases and further identifying potential therapeutic targets for human renal diseases.