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.
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.
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.
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.
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.
APOC3 deficiency can delay the formation of atherosclerosis-induced HFD in rabbits, indicating this is a novel therapeutic target to treat atherosclerosis.
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.
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.
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.
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.
Abstract
Background
Congenital hyper-homocysteinemia (HHcy) is caused by a defective cystathionine β-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
CBS
G307S
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.
Construction of point mutation rabbits using CRISPR/Cas9 Yan, Kunning; Zhang, Ting; Zha, Yiwen ...
Zhejiang da xue xue bao. Journal of Zhejiang University. Medical sciences. Yi xue ban,
04/2021, Volume:
50, Issue:
2
Journal Article
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.
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 β‑casein/CMV chimeric promoter in order to facilitate rhLF expression. This pCL25‑rhLF‑Neo 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 high‑purity (95.8%) rhLF from a cation‑exchange 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 β‑casein/CMV chimeric promoter. It represents an initial step towards the production of rhLF, potentially allowing for industrialized purification in the future.
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 (
) gene can cause the ...'double-muscling' phenotype. Although there have been many studies performed on
-mutant animals, there have been few studies that have investigated the cystine-knot motif in exon 3 of
in rabbits. In the current study, CRISPR/Cas9 sgRNA anchored exon 3 of a rabbit's
was used to disrupt the cystine-knot motif to change the
construction and cause a loss of its function. Eleven
-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
-KO rabbits displayed an obvious 'double-muscling' phenomena, with a 20-30% increase in body weight compared with WT rabbits. In the
-KO rabbits, all of the
rabbits showed teeth dislocation and tongue enlargement, and the percentage of rabbits having pelvic tilt was 0% in
, 0% in
, 77.78% in female
rabbits, and 37.50% in male
rabbits. The biomechanical mechanism of pelvic tilt and teeth dislocation in the
-KO rabbits requires further investigation.These newly generated
-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.
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