The Establishment Of Rabbit Models Of Human Inherited Diseases And Its Pathological Analysis

In this study,we mainly focused on the establishment of the rabbit models of human inherited diseases using gene-editing.--Our work can be divided into two parts.(1)The Wilson Disease was related to the gene ATB7 B,or,more exactly,caused by its point mutation.For instance,the hot spot in our country was Arg778 Leu.After NCBI blast,what we found was that there were highly homologous between Arg780 Leu of rabbit and Arg778 Leu of human,which make it possible to use rabbit for researches on point mutation of the Wilson Disease.Therefore,we applied the CRISPR/Cas9 system to create a single amino acid substituted rabbit model.On the whole,the defined point mutations in the rabbit ATP7 B gene could be derived by microinjecting synthesized RNAs and single-strand oligodeoxynucleotide(ssODN)donor sequences into zygotes.As a consequence,mutations accounted for 74.19%,while point mutation was 47.83%,which proved our success in building the WD disease model in the rabbit.Not surprisingly,the concentration of CP,TP,ALB and copper in plasma dropped separately.While there was an opposite result on the concentration of ALP,GGT,ALT,AST and copper in tissues.Pathology results showed that liver cells suffered from morphological changes,appearing with proliferation of fibrous connective tissue.Besides,with the bile ductule structure damaged,liver rope was also not clear.(2)We produced HPRT gene knock-out rabbits by recombinant Adeno-associated virus(rAAV)-mediated homologous recombination and SCNT,among which male and female gene knock-out fibroblast cell lines were obtained by different strategies.Even though only female HPRT gene knock-out rabbits could survive to birth,after mated with the wild-type rabbits,the percentage of the HPRT gene knock-out rabbits in F1 was 39.58%,including 73.68% males and 26.32% female.According to analysis of Real-time PCR,the expressions of HPRT in the different tissues of the knock-out rabbits were less than those in the wild-type rabbits.Meanwhile,the expressions of neo were detected in the different tissues of the knock-out rabbits.In addition,the expressions of EN1?Wnt1?P2Y1 were upragulated in the HPRT gene knock-out rabbits,all of which showed significant differences with the wild-type rabbits(*P<0.05).The expression of EN2 in the female HPRT gene knock-out rabbits was lower than that of the wild-type(**P<0.01).However,the result was opposite in the male HPRT gene knock-out rabbits(*P<0.05).To conclude: we generated the gene modified rabbits by CRISPR/Cas9 together with homologous recombination technology.What's more,those rabbit models may be of practical value with wide application prospect in the research or treatment of human hereditary diseases.