Spontaneous severe hypercholesterolemia and atherosclerosis lesions in rabbits with deficiency of low-density lipoprotein receptor (LDLR) on exon 7

Abstract

Rabbits (Oryctolagus cuniculus) have been the very frequently used as animal models in the study of human lipid metabolism and atherosclerosis, because they have similar lipoprotein metabolism to humans. Most of hyperlipidemia and atherosclerosis rabbit models are produced by feeding rabbits a high-cholesterol diet. Gene editing or knockout (KO) offered another means of producing rabbit models for study of the metabolism of lipids and lipoproteins. Even so, apolipoprotein (Apo)E KO rabbits must be fed a high-cholesterol diet to induce hyperlipidemia.In this study, we used the CRISPR/Cas9 system anchored exon 7 of low-density lipoprotein receptor (LDLR) in an attempt to generate KO rabbits. We designed two sgRNA sequences located in E7:g.7055–7074 and E7:g.7102–7124 of rabbit LDLR gene, respectively. Seven LDLR-KO founder rabbits were generated, and all of them contained biallelic modifications. Various mutational LDLR amino acid sequences of the 7 founder rabbits were subjected to tertiary structure modeling with SWISS-MODEL, and results showed that the structure of EGF-A domain of each protein differs from the wild-type. All the founder rabbits spontaneously developed hypercholesterolemia and atherosclerosis on a normal chow (NC) diet. Analysis of their plasma lipids and lipoproteins at the age of 12 weeks revealed that all these KO rabbits exhibited markedly increased levels of plasma TC (the highest of which was 1013.15 mg/dl, 20-fold higher than wild-type rabbits), LDL-C (the highest of which was 730.00 mg/dl, 35-fold higher than wild-type rabbits) and TG accompanied by reduced HDL-C levels. Pathological examinations of a founder rabbit showed prominent aortic atherosclerosis lesions and coronary artery atherosclerosis.In conclusion, we have reported the generation LDLR-KO rabbit model for the study of spontaneous hypercholesterolemia and atherosclerosis on a NC diet. The LDLR-KO rabbits should be a useful rabbit model of human familial hypercholesterolemia (FH) for the simulations of human primary hypercholesterolemia and such models would allow more exact research into cardio-cerebrovascular disease.

Hyperlipidaemia and cardioprotection: Animal models for translational studies

“…The generation of ROS produces oxidized derivatives of cholesterol, the oxysteroles, which propagate lipid peroxidation and induce cell death and mitochondria dysfunction (Paradis, Leoni, Caccia, Berdeaux, & Morin, 2013). Particularly, oxysterols are involved in the development of atherosclerosis and in the genesis of chronic diseases (Lordan, Mackrill, & O'Brien, 2009).…”

Hyperhomocysteinemia and dyslipidemia in point mutation G307S of cystathionine β-synthase-deficient rabbit generated using CRISPR/Cas9

“…1). The detailed protocol has been described previously [41,42] Female rabbits were superovulated by intramuscular injection of follicle-stimulating hormone (Sansheng Pharmaceutical Co., Ltd., NingBo, China) twice daily. The dosage was 15 IU for the first two injections, 10 IU for the next two and 5 IU for the last two injections.…”

Hyperhomocysteinemia and Dyslipidemia in point mutation G307S of cystathionine β-synthase-deficient rabbit generated using CRISPR/Cas9

“…Three CBS-targeting sgRNAs were screened (sgRNA1 sited E8: g.6631-6650, sgRNA2 sited E8: g.6637-6656 and sgRNA3 sited E8: 6641-6660) ( Figure 1). The detailed protocol has been described previously [41,42]. Single-stranded oligodeoxynucleotide donor templates (ssODN) with silent mutations were designed and synthesized by Sangon as follows: GGCTCCATCCTGGCGGAGCCGGAGGAGCTGAACCAGACGGAGGTGACGGCCTAtGAaGTaGAaGGtATCtcCTACGACTTCATCCCCACCGTGCTCGACCGGACGGTGTGT Zygote injection with Cas9/sgRNAand embryo transfer New Zealand White rabbits (6-8 months old) were housed at the Animal Genetic Engineering Laboratory of Yangzhou University under a 12 h diurnal/nocturnal cycle, and fed twice a day with free access to water.…”