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.

ObjectiveTo generate novel rabbit models with a large-fragment deletion of either LDL receptor (LDLR) and/or apolipoprotein (apoE) genes for the study of hyperlipidemic and atherosclerosis.MethodsCRISPR/Cas9 system directed by a multiple sgRNAs system was used in rabbit embryos to edit their LDLR and apoE genes. The LDLR and apoE genes of founder rabbits were sequenced, and their plasma lipids and lipoprotein profiles on a normal chow diet were analyzed, western blotting was also performed to evaluate the expression of apolipoprotein. Sudan IV and HE staining of aortic were performed to confirm the formation of atherosclerosis.ResultsSix knockout (KO) rabbits by injection of both LDLR and apoE sgRNAs were obtained, including four LDLR KO rabbits and two LDLR/apoE double- KO rabbits. Sequence analysis of these KO rabbits revealed that they contained multiple mutations including indels, deletions, and substitutions, as well as two rabbit lines containing biallelic large fragment deletion in the LDLR region. Analysis of their plasma lipids and lipoprotein profiles of these rabbits fed on a normal chow diet revealed that all of these KO rabbits exhibited remarkable hyperlipidemia with total cholesterol levels increased by up to 10-fold over those of wild-type rabbits. Pathological examinations of two founder rabbits showed that KO rabbits developed prominent aortic and coronary atherosclerosis.ConclusionLarge fragment deletions can be achieved in rabbits using Cas9 mRNA and multiple sgRNAs. LDLR KO along with LDLR/apoE double KO rabbits should provide a novel means for translational investigations of human hyperlipidemia and atherosclerosis.

Background In recent years, an increasing number of studies have proved that circulating miRNAs could be used for the early diagnosis of cardiovascular diseases and even play vital roles in the evaluation of therapeutic effects or prognosis. This study was conducted to examine the correlation between serum microRNAs and hyperlipidemia to provide a theoretical basis for the early screening and intervention of atherosclerotic cardiovascular diseases (ASCVD). Methods The serum samples and clinical data of 122 patients with hyperlipidemia and 168 healthy subjects were collected. Related clinical information was statistically analyzed for the two groups. Expression of circulating miRNAs was detected by miRNA microarray analysis and further verified by reverse transcription-quantitative PCR (RT-qPCR). Results Statistical analysis of clinical information revealed a significant difference in the incidence of ASCVD between the two groups. The MiRNA microarray analysis ( n = 10) showed 22 miRNAs with significantly different expression, among which 12 showed upregulation, and the others showed downregulation. Those possessing obvious differences and stable expression in the miRNA microarray, including miRNA-191-3p, miRNA-933, and miRNA-425-3p, were chosen for further investigation using RT-qPCR. The results demonstrated that several miRNAs were related to lipid metabolism disorders, especially miRNA-933. The area under the curve (AUC) of miRNA-933 in distinguishing the hyperlipidemia and ASCVD patients was 0.739 (95% CI, 0.682–0.795; P < 0.01) and 0.703 (95% CI, 0.643–0.763, P < 0.01), respectively. Conclusions In conclusion, miRNA-191-3p, miRNA-933, and miRNA-425-3p may be depressed in the peripheral circulation of patients with lipid metabolism disorders (mainly LDL). Circulating miRNA-933 could be a feasible predictor for ASCVD at the early stage. Electronic supplementary material The online version of this article (10.1186/s12944-019-1046-z) contains supplementary material, which is available to authorized users.

Gene mutations at different gene sites will produce totally different phenotypes or biological functions in gene-edited animals. An allelic series of mutations in the myostatin (MSTN) gene can cause the ‘double-muscling’ phenotype. Although there have been many studies performed on MSTN-mutant animals, there have been few studies that have investigated the cystine-knot motif in exon 3 of MSTN in rabbits. In the current study, CRISPR/Cas9 sgRNA anchored exon 3 of a rabbit’s MSTN was used to disrupt the cystine-knot motif to change the MSTN construction and cause a loss of its function. Eleven MSTN-KO founder rabbits were generated, and all of them contained biallelic modifications. Various mutational MSTN amino acid sequences of the 11 founder rabbits were modeled to the tertiary structure using the SWISS-MODEL, and the results showed that the structure of the cystine-knot motif of each protein in the founder rabbits differed from the wild-type (WT). The MSTN-KO rabbits displayed an obvious ‘double-muscling’ phenomena, with a 20−30% increase in body weight compared with WT rabbits. In the MSTN-KO rabbits, all of the MSTN−/− rabbits showed teeth dislocation and tongue enlargement, and the percentage of rabbits having pelvic tilt was 0% in MSTN+/+, 0% in MSTN+/−, 77.78% in female MSTN−/− rabbits, and 37.50% in male MSTN−/− rabbits. The biomechanical mechanism of pelvic tilt and teeth dislocation in the MSTN-KO rabbits requires further investigation. These newly generated MSTN-KO rabbits will serve as an important animal model, not only for studying skeletal muscle development, but also for biomedical studies in pelvic tilt correction and craniofacial research.

Brain injury has been proposed as the major cause of the poor outcomes associated with intracerebral hemorrhage (ICH). Emerging evidence indicates that the nuclear receptor, peroxisome proliferator-activated receptor β/δ (PPAR-β/δ), plays a crucial role in the pathological process of central nervous impairment. The present study was undertaken to evaluate the protective effects of PPAR-β/δ activation using a selective PPAR-β/δ agonist, GW0742, against brain injury after ICH in a mouse model. ICH was induced by intravenous injection of collagenase into the right caudate putamen. To examine the protective effect of PPAR-β/δ activation against ICH-induced brain injury, mice were either intraperitoneally injected with GW0742 (3 mg/kg, body weight) or saline (control group) 30 min before inducing ICH. Behavioral dysfunction was evaluated 24 and 72 h after injury. Then, all mice were killed to assess hematoma volume, brain water content, and blood-brain barrier (BBB) permeability. TUNEL and Nissl staining were performed to quantify the brain injury. The expression of PPAR-β/δ, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, Bcl-2-related X-protein (Bax), and B-cell lymphoma 2 (Bcl-2) in the perihematomal area was examined by immunohistochemistry and western blotting analysis. Mice treated with GW0742 showed significantly less severe behavioral deficits compared to the control group, accompanied by increased expression of PPAR-β/δ and Bcl-2, and increased expression of IL-1β, TNF-α, and Bax decreased simultaneously in the GW0742-treated group. Furthermore, the GW0742-pretreated group showed significantly less brain edema and BBB leakage. Neuronal loss was attenuated, and the number of apoptotic neuronal cells in perihematomal tissues reduced, in the GW0742-pretreated group compared to the control group. However, the hematoma volume did not decrease significantly on day 3 after ICH. These results suggest that the activation of PPAR-β/δ exerts a neuroprotective effect on ICH-induced brain injury, possibly through anti-inflammatory and anti-apoptotic pathways.

Poor expression is the key factor hampering the large-scale application of transgenic animal mammary gland bioreactors. A very different approach would be to evaluate the secretion of recombinant proteins into milk in response to a cleavable signal peptide of highly secreted lactoproteins. We previously reported rabbits harboring mammary gland-specific expression vector containing a fusion cDNA (goat β-lactoglobulin (BLG) signal peptide and recombinant human plasminogen activator (rhPA) coding sequences) expressed rhPA in the milk, but we did not realize the signal peptide contributed to the high rhPA concentration and did not mention it at that time. And the molecular structure and biological characteristics still remain unknown. So, rhPA in the milk was purified and characterized in the present study. rhPA was purified from the milk, and the purity of the recovered product was 98% with no loss of biological activity. Analysis of the N-terminal sequence, C-terminal sequence, and the molecular mass of purified rhPA revealed that they matched the theoretical design requirements. The active systemic anaphylaxis (ASA) reactions of the purified rhPA were negative. Taken together, these results indicated that the goat BLG signal peptide can efficiently mediate rhPA secretion into milk and was accurately cleaved off from rhPA by endogenous rabbit signal peptidase. We have reinforced the importance of a rhPA coding region fused to a cleavable heterologous signal peptide from highly secreted goat BLG to improve recombinant protein expression. It is anticipated that these findings will be widely applied to high-yield production of medically important recombinant proteins.

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Advancements in genome editing make possible to exploit the functions of enzymes for efficient DNA modifications with tremendous potential to treat human genetic diseases. Several nuclease genome editing strategies including Meganucleases (MNs), Zinc Finger Nucleases (ZFNs), Transcription Activator-like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas) have been developed for the correction of genetic mutations. CRISPR-Cas has further been engineered to create nickase genome editing tools including Base editors and Prime editors with much precision and efficacy. In this review, we summarized recent improvements in nuclease and nickase genome editing approaches for the treatment of genetic diseases. We also highlighted some limitations for the translation of these approaches into clinical applications.