Preparation Of Atherosclerosis Model In Wuzhishan Minipig By Double Gene Knockout Of LDLR And ApoE

Minipigs,because the cardiovascular system and anatomical and physiological structures are quite similar to humans,are more suitable than rodents for constructing animal models of blood lipid metabolic diseases such as atherosclerosis.However,the traditional method of minipig model construction is mainly induced by high-fat feeding and surgery,which is time-consuming,complicated and costly,and cannot well simulate the occurrence and development of this type of disease.A minipig model with severe familial hypercholesterolemia and fast spontaneous atherosclerotic lesions is currently lacking.In order to cultivate a new medical pig strain with spontaneous atherosclerosis,we selected Wuzhishan minipig,which is highly sensitive to exogenous fat and prone to hypercholesterolemia,and used CRISPR/Cas9 system to simultaneously target apolipoprotein E（ApoE）and low-density lipoprotein receptor（LDLR）genes to build a model.The results showed that:（1）we obtained a total of 32 healthy cloned pigs with biallelic knockout of LDLR and ApoE.（2）Compared with the wild type,the body weight of LDLR^-/-/ApoE^-/-Wuzhishan minipigs was significantly increased(the average body weight of the wild type at 12months was 50.33kg,and that of the LDLR^-/-/ApoE^-/-Wuzhishan minipigs was 66.69kg,p<0.01),and high levels of hypercholesterolemia appeared from 2 months of age and have been maintained(the total serum cholesterol of the wild type at 2 months of age was 2.2mmol/l,and that of the LDLR^-/-/ApoE^-/-Wuzhishan minipigs was 14.28 mmol/l,p<0.0001),At the age of 6 months,the anatomic pathology showed that the aorta had obvious lipoid deposits.（3）After 6 months of high-fat feeding,the serum cholesterol level and atherosclerotic index of LDLR^-/-/ApoE^-/-Wuzhishan minipigs were significantly higher than those of high-fat feeding group(average 8.64 mmol/l for wild-type and 24.95mmol/l for LDLR^-/-/ApoE^-/-Wuzhishan minipigs,p<0.05).（4）A total of 15 offspring LDLR^-/-/ApoE^-/-Wuzhishan minipigs were obtained from F0 generation cloned pigs of LDLR^-/-/ApoE^-/-through breeding and natural mating.The LDLR^-/-/ApoE^-/-Wuzhishan minipig successfully constructed in this study,whose high serum cholesterol level accelerates the rate of atherosclerosis formation,can spontaneously develop atherosclerotic disease without high-fat feeding induction,and can be cultured as a new animal model of human cardiovascular disease,providing good research materials for further research on the mechanism of atherosclerosis and the transformation and association mechanism of related secondary diseases.