Study On The Preparation Of Apolipoprotein C3 Gene Knockout Rabbit Model By CRISPR/Cas9 Gene Editing Technology

Object:The formation of atherosclerosis is the common pathological basis of most cardiovascular and cerebrovascular diseases,which seriously endanger people's health.A large number of clinical data show that the loss function of Apolipoprotein C3(APOC3)can reduce the risk of atherosclerosis and cardiovascular disease.Therefore,this experiment aims to prepare APOC3 gene knockout rabbits using gene editing techniques of clustered,regularly interspaced,short palindromic repeats,CRISPR-associated protein 9(CRISPR/Cas9)and provide an anthropomorphic animal model for the study of the importance of APOC3 in the formation mechanism of atherosclerosis,and analyze the impact of APOC3 gene knockout(APOC3-/-)on the occurrence of atherosclerosis.Method:In this experiment,CRISPR/cas9 gene editing technology was used to complete the editing of APOC3-/-rabbits.The first step is to screen and determine the predicted knockout site of APOC3 and to construct a single guide RNA(sgRNA)that complements the DNA sequence of knockout site,and then transcribed in vitro to obtain sgRNA and Cas9mRNA,Furethermore,they were both injected into the fertilized egg by micromanipulation technology.The fertilized egg in good condition after microinjection was transplanted into the recipient fallopian tube by embryo transfer operation.The founder generation rabbit was born naturally 30 days after surrogacy.Genotype identification and phenotype analysis were carried out on the founder generation rabbit,five pairs of primers were used to detect the possible of off-target sites.In addition,the rabbits were placed in the normal diet environment until the age of 12 weeks,the blood lipid concentration and triglyceride metabolism rate were measured by peripheral vein blood sampling at that time.Apolipoprotein E(APOE),apolipoprotein AI(APOAI),Proprotein convertase subtilisin/kexin-9(PCSK9)were detected by western blotting,The contents of plasma lipoprotein were determined by agarose gel electrophoresis,in order to observe the effect of plasma lipids after APOC3-/-Beyond that,the changes of plasma lipid,the activities of lipoprotein lipase and liver lipase were detected under the condition of high-fat diet.The changes of inflammatory factors in plasma were detected by enzyme linked immunosorbent assay(ELISA).Ultimately,through 12 weeks of high-fat diet feeding,the aorta was dissected and stained with Sudan ?,HE,Masson,smooth muscle cells and macrophage immunohistochemistry to observe and analyze whether atherosclerotic lesions.Through the analysis of the above methods,the effects of APOC3-/-on lipid metabolism and atherosclerosis were observed.Result:We have successfully obtained three APOC3-/-rabbits by CRISPR/cas9 gene editing technology.The genotypes of APOC3-/-rabbits are different,and there are some forms of deletion and substitution of bases,and the off-target phenomenon is not be found.The results showed that compared with WT group,the plasma lipid level of APOC3-/-rabbits was lower than that of WT.The blood lipid of APOC3-/-rabbit increased slightly under the intervention of high fat diet.After 12 weeks of high-fat diet,the ratio of plaque formation in the aorta of APOC3-/-rabbits was 3%,while the plaque formation ratio in group WT was 21%and showed typical atherosclerotic pathological features:neointimal formation,intimal foam cell aggregation,smooth muscle cell proliferation and migration to the intima,typical fibrous plaques.The results showed that APOC3-/-may significantly reduce the risk of atherosclerotic plaque formation under the condition of high fat diet.Conclusion:We successfully prepared APOC3-/-rabbit model by using CRISPR/Cas9 technology.The phenotype is satisfied our needs and stable heredity.It is suggested that the establishment of this model provides an ideal animal model for the study of the mechanism of APOC3 in reducing the risk of atherosclerosis and cardiovascular disease.