[Propagation and phenotypic analysis of mutant rabbits with MSTN homozygous mutation].

Myostatin gene (MSTN) encodes a negative regulator for controlling skeletal muscle growth in animals. In this study, MSTN-/- homozygous mutants with "double muscle" phenotypic traits and stable inheritance were bred on the basis of MSTN gene editing rabbits, with the aim to establish a method for breeding homozygous progeny from primary MSTN biallelic mutant rabbits. MSTN-/- primary mutant rabbits were generated by CRISPR/Cas9 gene editing technology. The primary mutant rabbits were mated with wild type rabbits to produce F1 rabbits, whereas the F2 generation homozygous rabbits were bred by half-sibling mating or backcrossing with F1 generation rabbits of the same mutant strain. Sequence analysis of PCR products and its T vector cloning were used to screen homozygous rabbits. The MSTN mutant rabbits with 14-19 week-old were weighed and the difference of gluteus maximus tissue sections and muscle fiber cross-sectional area were calculated and analyzed. Five primary rabbits with MSTN gene mutation were obtained, among which three were used for homozygous breeding. A total of 15 homozygous rabbits (5 types of mutants) were obtained (M2-a: 3; M2-b: 2; M3-a: 2; M7-a: 6; M7-b: 2). The body weight of MSTN-/- homozygous mutant rabbits aged 14-19 weeks were significantly higher than that of MSTN+/+ wild-type rabbits of the same age ((2 718±120) g vs. (1 969±53) g, P < 0.01, a 38.0% increase). The mean cross sections of gluteus maximus muscle fiber in homozygous mutant rabbits were not only significantly higher than that of wild type rabbits ((3 512.2±439.2) µm2 vs. (1 274.8±327.3) µm2, P < 0.01), but also significantly higher than that of MSTN+/- hemizygous rabbits ((3 512.2±439.2) µm2 vs. (2 610.4±604.4) µm2, P < 0.05). In summary, five homozygous mutants rabbits of MSTN-/- gene were successfully bred, which showed a clear lean phenotype. The results showed that the primary breeds were non-chimeric mutant rabbits, and the mutant traits could be inherited from the offspring. MSTN-/- homozygous mutant rabbits of F2 generation could be obtained from F1 hemizygous rabbits by inbreeding or backcrossing. The progenies of the primary biallelic mutant rabbits were separated into two single-allelic mutants, both of which showed a "double-muscle" phenotype. Thus, this study has made progress in breeding high-quality livestock breeds with gene editing technology.

Identification, purification, and pharmacological activity analysis of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAα1) expressed in transgenic rabbit mammary glands.

Desmodus rotundus plasminogen activator alpha 1(DSPAα1) is a thrombolytic protein with advantages, such as a long half-life, high accuracy and specificity for thrombolysis, wide therapeutic window, and no neurotoxicity. To date, DSPAα1 has only been expressed in the Chinese hamster ovary, insect cells, transgenic tobacco plants, and Pichia pastoris. To the best of our knowledge, we are the first to report the expression of DSPAα1 in transgenic rabbit mammary glands, extract the product, and analyze its pharmacology activity. An efficient mammary gland-specific expression vector pCL25/DSPAα1 was transferred to prokaryotic zygotes in rabbits by microinjection to generate six DSPAα1 transgenic rabbits. The recombinant DSPAα1 (rDSPAα1) expression in transgenic rabbit milk was 1.19 ± 0.26 mg/mL. The rDSPAα1 purification protocol included pretreatment, ammonium sulfate precipitation, benzamidine affinity chromatography, cation exchange chromatography, and Cibacron blue affinity chromatography; approximately 98% purity was achieved using gel electrophoresis. According to sequencing results, the primary structure of rDSPAα1 was consistent with the theoretical design sequence, and its molecular weight was consistent with that of the natural protein. N-terminal sequencing results indicated rDSPAα1 to be a mature protein, as the goat signal peptide sequence of the expression vector was no longer detected. The fibrinolytic activity of rDSPAα1 was estimated to be 773,333 IU/mg. Fibrin-agarose plate assay and in vitro rat blood clot degradation assay showed that rDSPAα1 had strong thrombolytic activity. In conclusion, we report recombinant DSPAα1 with high thrombolytic activity expressed in transgenic rabbit mammary glands.

CRISPR/Cas9-mediated knockout of APOC3 stabilizes plasma lipids and inhibits atherosclerosis in rabbits.

BACKGROUND: High levels of apolipoprotein C3 (APOC3) can lead to hypertriglyceridemia, which increases the risk of cardiovascular disease. We aim to create APOC3-knockout (KO) rabbits and explore the effects of APOC3 deletion on the occurrence and development of atherosclerosis. METHODS: An sgRNA anchored to exon 2 of APOC3 was designed to edit embryo genomes using the CRISPR/Cas9 system. The founder rabbits were sequenced, and their lipid profile, inflammatory cytokines, and atherosclerotic plaques were analyzed. RESULTS: When given a normal chow (NC) diet, all APOC3-KO rabbits had 50% lower triglyceride (TG) levels than those of the matched age control group. Additionally, their plasma lipoprotein lipase increased. When fed a high-fat diet, APOC3 deficiency was observed to be more conducive to the maintenance of plasma TG, total cholesterol, and low-density lipoprotein cholesterol levels, and the inhibition of the inflammatory response and the protection against atherosclerosis in rabbits. CONCLUSION: APOC3 deficiency can delay the formation of atherosclerosis-induced HFD in rabbits, indicating this is a novel therapeutic target to treat atherosclerosis.

Construction of point mutation rabbits using CRISPR/Cas9.

To establish a rabbit model of proprotein convertase subtilisin/kexin type9 () point mutation with CRISPR/Cas9 gene editing technique. According to the PubMed gene protein data, the PCSK9 protein functional regions of human and rabbit were analyzed by Blast. The 386S (Ser) amino acid functional region of human gene was homologous to the 485S of rabbit gene. Three small guide RNAs and one single-stranded donor oligonucleotide were designed according to the 485S base substitution position and sequence analysis of rabbit gene. The synthetic small guide RNAs, Cas9 mRNA and single-stranded donor oligonucleotide were co-injected into the cytoplasm of rabbit fertilized eggs and the embryos were transferred into the pregnant rabbits. PCR, TA cloning and off-target analysis were performed on the F0 rabbits to identify whether the PCSK9 mutation was successful. Fifteen F0 rabbits were obtained. The sequencing results showed that one of them was PCSK9 point mutation homozygote and two of them were PCSK9 point mutation heterozygotes, and the mutation could be stably inherited. The rabbit model of PCSK9 point mutation was successfully constructed by CRISPR/Cas9 technique, which provides an animal model for exploring the molecular mechanism of impaired PCSK9 function and developing reliable and effective diagnosis and treatment measures.

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

BACKGROUND: Congenital hyper-homocysteinemia (HHcy) is caused by a defective cystathionine ß-synthase (CBS) gene, and is frequently associated with dyslipdemia. The aim of this study was to further elucidate the effect of mutated CBS gene on circulating lipids using a rabbit model harboring a homozygous G307S point mutation in CBS. METHODS: CRISPR/Cas9 system was used to edit the CBS gene in rabbit embryos. The founder rabbits were sequenced, and their plasma homocysteine (Hcy) and lipid profile were analyzed. RESULTS: Six CBS-knockout (CBS-KO) founder lines with biallelic modifications were obtained. Mutation in CBS caused significant growth retardation and high mortality rates within 6 weeks after birth. In addition, the 6-week old CBS-KO rabbits showed higher plasma levels of Hcy, triglycerides (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) compared to the age-matched wild-type (WT) controls. Histological analysis of the mutants showed accumulation of micro-vesicular cytoplasmic lipid droplets in the hepatocytes. However, gastric infusion of vitamin B and betaine complex significantly decreased the plasma levels of TG, TC and LDL-C in the CBS-KO rabbits, and alleviated hepatic steatosis compared to the untreated animals. CONCLUSION: A CBSG307S rabbit model was generated that exhibited severe dyslipidemia when fed on a normal diet, indicating that G307S mutation in the CBS gene is a causative factor for dyslipidemia.

Activation of PPAR-ß/δ Attenuates Brain Injury by Suppressing Inflammation and Apoptosis in a Collagenase-Induced Intracerebral Hemorrhage Mouse Model.

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 ß/δ (PPAR-ß/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-ß/δ activation using a selective PPAR-ß/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-ß/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-ß/δ, interleukin (IL)-1ß, 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-ß/δ and Bcl-2, and increased expression of IL-1ß, 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-ß/δ exerts a neuroprotective effect on ICH-induced brain injury, possibly through anti-inflammatory and anti-apoptotic pathways.

The goat ß­casein/CMV chimeric promoter drives the expression of hLF in transgenic goats produced by cell transgene microinjection.

There is growing interest in the application of lactoferrin (LF) as a drug or food additive for animals and humans. The objective of this study was to produce transgenic cloned goats that would serve as living bioreactors, expressing high levels of recombinant human LF (rhLF) in their milk. We designed a pCL25 expression vector containing goat ß­casein/CMV chimeric promoter in order to facilitate rhLF expression. This pCL25­rhLF­Neo vector was microinjected into goat fetal fibroblasts. G418 selection and PCR analysis were used to identify transgenic donor cells suitable for somatic cell nuclear transfer (SCNT). After SCNT and embryo transplantation, goats harboring the hLF gene were produced, as confirmed via PCR and southern blotting. The average rhLF concentration in milk from this transgenic goat was 3.89 mg/ml as determined via ELISA. We also used an optimized buffer in order to effectively elute high­purity (95.8%) rhLF from a cation­exchange column, with the recovered rhLF exhibiting high biological activity. Findings from this study demonstrated that it is possible to generate a transgenic goat harboring the hLF transgene driven by the goat ß­casein/CMV chimeric promoter. It represents an initial step towards the production of rhLF, potentially allowing for industrialized purification in the future.

Accurately cleavable goat ß-lactoglobulin signal peptide efficiently guided translation of a recombinant human plasminogen activator in transgenic rabbit mammary gland.

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 ß-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.

'Double-muscling' and pelvic tilt phenomena in rabbits with the cystine-knot motif deficiency of myostatin on exon 3.

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.

Generation of hyperlipidemic rabbit models using multiple sgRNAs targeted CRISPR/Cas9 gene editing system.

OBJECTIVE: To 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. METHODS: CRISPR/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. RESULTS: Six 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. CONCLUSION: Large 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.