| Objective:Hyperlipidemia is a kind of metabolic disease caused by genetic and environmental factors,and it often causes atherosclerosis and in further leads to chronic cardiovascular and cerebrovascular diseases,which is one of the major diseases threatening human health.A suitable animal model is an important tool for the study of this disease.In this paper,CRISPR/cas9 technology was used to establish an ApoE-deficiency golden hamster model with hyperlipidemia and the phenotypes of this model were analyzed.Method:Apolipoprotein E?ApoE?is a key gene involved in lipid metabolism.two pairs of sg RNA was designed?sg RNA 1,sg RNA2?for CRISPR/Cas9 gene editing technology targeting the fourth exon of ApoE gene in golden hamster.To construct sg RNA plasmid vectors,the synthesized sg RNA sequence was ligated to PX458plasmid by Cleavage assay and ligated reaction.Sanger sequencing and Cleavage assay were used to validate the plasmid vectors.To check the efficiency of sg RNA,BHK-21 cells were transfected with sg RNA plasmid vectors and the editing efficiency of sg RNA was estimated based on fluorescence intensity and results of Cleavage assay.The sg RNA with higher efficiency were used for the microinjection for zygotes.The injected zygotes eggs were transplanted into the surrogate golden hamsters and the offspring?F0?was born about 2 weeks later.Each F0 individual was labeled by cutting their toes at 2 weeks old.The toe tissues were collected for genomic DNA extraction,PCR amplification and vector cloning.Sanger sequencing was performed to detect the genotype of F0 golden hamster.Potential off-targeted sites were predicted using software Benching,and the first 8 sites with the highest score were selected to design PCR primers.For each potential off-targeted site,PCR amplification and Sanger sequencing was conducted to check if off-target occurred.When F0 hamster grows to 3 weeks old,the male and female were separated and each individual was raised in single cage.At the age of 10 weeks,F1 golden hamster was bred by mating the mutant F0 with wild-type golden hamster.After genome extraction,sequencing was performed to identify the genotype of F1 golden hamsters.According to the frequency of each genotype occurred in F1,the heritability of the mutant genotype in F0 was analyzed.The male and female F1 golden hamsters carrying the same mutant were mated,and the F2 golden hamster with homozygous mutation(ApoE-/-)was selected for propagation.After fasting overnight,blood was collected from the retrobulbar vein of 8 weeks old golden hamster and serum was separated.The blood lipid of golden hamster with different genotypes,i.e.wide-type,phenotype of ApoE-deficiency golden hamster model under different dietary conditions,we picked WT and ApoE-/-golden hamster at 8-10 weeks old and both the WT and ApoE-/-individuals were divided into control group and experimental group,respectively,6-8 golden hamsters in each group.The control group fed with normal diet,and the experimental group fed with high-fat diet?containing 0.5%cholesterol and 15%fat?.Blood samples were collected at the 4th and 8th week and plasma lipids were measured.Results:We have designed two pairs of sg RNAs?sg RNA1 and sg RNA2?and constructed sg RNA-plasmid vectors,which were confirmed by Sanger sequencing and cleavage assay.However,we found that sg RNA2 showed relative higher gene-editing efficiency than sg RNA1 in vitro cell experiments,thus sg RNA2-plasmid vector was used for microinjection of zygotes,which were transplanted into golden hamsters.Finally,six golden hamsters were born and five of them carried ApoE mutations,with editing efficiency of 83.3%.The types of mutations included deletion and single base insertion.No off-targeted effect was detected for the 5 knock-out hamsters.According to the genotype and reproductive characteristics,the individuals with single base insertion?derived from F0-5?were selected for propagation,and finally the ApoE-/-hamster model was established.The level of blood lipids of ApoE-/-hamster fed with normal diet was detected,which was significantly higher than that of WT hamster at the age of 8 weeks.For example,the total cholesterol?TC?level was nearly 1.75 times higher than that of WT hamster?461.81±107.10 mg/d L vs 167.74±21.64 mg/d L,P<0.01?,triglyceride?TG?was 53.36%higher than that of WT hamster?251.16±20.08 mg/d L vs 196.09±23.38 mg/d L,P<0.01?,but there was no significant change in HDL-C.This result indicated that hyperlipidemia could occur by natural in the ApoE-/-hamster.In addition,no obvious difference about blood lipid level was observed between WT and promote the dyslipidemia of ApoE-/-golden hamster.After fed with high-fat diet for 4weeks,the levels of blood lipids were 3730.21±416.72 mg/d L,1759.60±153.53mg/d L and 588.84±90.18 mg/d L respectively TC,TG and HDL-C,which were significantly higher than that of WT group fed with high-fat diet?861.09±88.11mg/d L,836.50±124.96 mg/d L and 121.36±21.50 mg/d L,respectively,P<0.01?.After fed with high-fat diet for 8 weeks,the levels of TC,TG and HDL-C in ApoE-/-golden hamster were further increased to 4214.67±341.96 mg/d L,2066.92±211.36 mg/d L and 666.04±67.16 mg/d L,respectively,with significantly higher than that of the WT-H group?961.45±69.45 mg/d L,925.00±139.08 mg/d L and 129.85±15.84,respectively,P<0.0001?.At the same time,we compared the blood lipid indexes of each group at the 8th week with those at the 4th week,and found that the TG level in indicated that ApoE-/-golden hamster model with hyperlipidemia were successfully established.Conclusion:We have successfully constructed an ApoE-deficiency golden hamster model with hyperlipidemia,and confirmed that this model can present obviously increased plasma lipid level under different dietary conditions.This provides a novel animal model for the study of hyperlipidemia and cardiovascular disease. |
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