Production Of LDLR Knockout Rabbits And Rabbit Models For The Study Of Antherosclerosis

Atherosclerosis is the major underlying cause of cardiovascular and cerebrovascular diseases.Due to lack of ideal animal models,there are limited studies of pathogenesis and drug intervention of Atherosclerosis.Because of their unique characters,the rabbits have become the most suitable models of choice for studying mechanisms of atherogenesis and for testing antiatherogenic interventions.In this study,LDLR gene was knockouted by CRISPR/Cas9 system in New Zealand white rabbits,and successfully established spontaneous hyperlipidemia animal models.In this study,we constructed two pairs of CRISPR/Cas9 plasmids expressing sgRNAs targeting exon 2 and 7 of Ldlr gene,Following complete transcription of Cas9 mRNA,the structure of cap(ARCA)and the poly(A)tailing reaction were performed.Both the the Cas9 mRNA and sgRNA were microinjected into the rabbit embyos.In this study,804 fertilized oocytes were harvested from 34 superovulated donor rabbits.116 were injected with the mix of Cas9 mRNA and sgRNA targeting E2 of Ldlr.110 embryos were transplanted,embryo survival rate was 94.83%.6 New Zealand rabbits were transplanted,2 baby rabbits were delivered by the pregnant rabbits,pregnant rate was 33.33%.4 baby rabbits were born,all died,survival rate was 0%.209 were injected with Cas9 mRNA and sgRNA targeting E7 of Ldlr.202 embryos were transplanted,embryo survival rate was 96.65%.12 New Zealand rabbits were transplanted,6 baby rabbits were delivered by the pregnant rabbits,pregnant rate was 50%.14 baby rabbits were born,11 survived,survival rate was 78.5%.442 were injected with Cas9 mRNA and sgRNA targeting E2+E7 of Ldlr.373 embryos were transplanted,embryo survival rate was 84.39%.19 New Zealand rabbits were transplanted,11 baby rabbits were delivered by the pregnant rabbits,pregnant rate was 33.33%.19 baby rabbits were born,9 survived,survival rate was.42.11%.Due to feeding conditions(temperature,diseases etc.),16 transgenic rabbits were obtained for blood lipid tests.Sequencing of the 16 baby rabbits indicated that the 2 sites of LDLR were both mutated.The mutation rate of E7 was 100%,all 8 mutations were biallelic mutation,biallelic mutation rate was 100%.7 out of the 8 transgenic rabbits had mutations on both E2 and E7,mutation rate was 87.50%,all 7 were biallelic mutations.Increased serum cholesterol concentration is one of the most important characters of hyperlipidemia,and it is a risk factor causing atherosclerosis.We bleed gene knockout rabbits on week 5,15,25,serum CHOL,TG,LDL,HDL were tested.In comparison with control group,KO rabbits had 17-fold higher levels of serum CHOL,12-fold higher levels of LDL,3-fold higher levels of TG,and 2-fold higher levels of HDL.Data was analyzed using spss.These results suggested that hyperlipidemia model was successful constructed.Plasma lipoprotein profiles were analyzed using lipoprotein electrophoresis,results revealed that apoB(apolipoprotein B,ApoB)levels were significantly increased and apoAl(apolipoprotein A ?,ApoA ?)levels were significantly lower in LDLR KO rabbits,compare to WT rabbits.ApoB and ApoAl are associated with low density lipoprotein cholesterol(LDL-C)and high density lipoprotein cholesterol(HDL-C)levels.Lesions in the blood vessel were visualized with Sudan ? staining.Plaque formation in Ldlr gene KO rabbits were significantly increased,compare to WT rabbits.The lesions consist of a typical lipid or necrotic core covered by a fibrotic cap,a typical atherosclerotic Plaque.Thus,Ldlr gene KO rabbits can be successfully applifed as a model for atherosclerosis.After 6 months regular diet,aorta was harvested from Ldlr gene KO rabbits.HE staining showed that the intimal lesions of aortic atherosclerosis of Ldlr gene KO rabbits were thicker than those of the WT rabbits.Thrombosis rate in i gene KO rabbits was 73.48%,which was significantly higher than 37.65%in WT rabbits.Rabbits were euthanized after 16 and 24 weeks using3%sodium pentobarbital(60mg/kg),and carotid artery and aorta were harvested.Immunohistochemical analyses was performed on formalin-fixed sections of carotid artery and aorta.Macrophages were marked increased in Ldlr gene KO rabbits.Taken together,our laboratory successfully produced Ldlr gene knockout rabbit by microinjecting Cas9mRNA and sgRNA into rabbit fertilized eggs.To our knowledge,this is the first report of generating Ldlr gene KO rabbits for the study of lipid and lipoprotein metabolism.This study can provide an ideal model for studying thrombus diseases.