ABSTRACT
Zika virus can infect the fetus through the placental barrier, causing ZIKV congenital syndrome and even miscarriage, which can cause great harm to pregnant women and infants. Currently, there is no vaccine and drug available to combat the Zika virus. In this study, we designed a fusion protein named EDIII-Fc, including the EDIII region of Zika E protein and human IgG Fc fragment, and obtained 293T cells that stably secreted EDIII-Fc protein using the lentiviral expression system. Mice were immunized with the EDIII-Fc protein, and it was observed that viral replication was significantly inhibited in the immunized mice compared to non-immunized mice. In rhesus macaques, we found that EDIII-Fc effectively induce the secretion of neutralizing antibodies and T cell immunity. These experimental data provide valid data for further use of Zika virus E protein to prepare an effective, safe, affordable Zika vaccine.
Subject(s)
Viral Vaccines , Zika Virus Infection , Zika Virus , Female , Animals , Humans , Pregnancy , Mice , Zika Virus Infection/prevention & control , Macaca mulatta , Antibodies, Viral , Placenta , Antibodies, Neutralizing , ImmunityABSTRACT
Acute ischaemic stroke (AIS) is a complex, systemic, pathological, and physiological process. Systemic inflammatory responses and disorders of the gut microbiome contribute to increased mortality and disability following AIS. We conducted 16S high-throughput sequencing and ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry-based non-targeted metabolomic analyses of the plasma from a Tibetan miniature pig middle cerebral artery occlusion (MCAO) model. A significant decrease in the abundance of Firmicutes and a significant increase in the abundance of Actinobacteria were observed after the onset of AIS. Among the plasma metabolites, the levels of phospholipids and amino acids were considerably altered. Loading values and differential metabolite-bacterial group association analyses of the metabolome and microbiome indicated a correlation between the microbiome and metabolome of Tibetan miniature pigs after MCAO. Furthermore, significant changes were observed in the ABC transporter pathway and purine metabolism in the gut microbiome-plasma metabolome during the early stage of AIS. Kyoto Encyclopaedia of Genes and Genomes enrichment analysis showed that arginine, proline, and cyanoamino acid metabolism was upregulated while ABC transporter metabolism pathway and carbohydrate digestion and absorption were substantially downregulated. The results of this study suggest that AIS affects the gut microbiota and plasma metabolites in Tibetan miniature pigs and that faecal microbiota transplantation could be a potential therapeutic approach for AIS.
Subject(s)
Brain Ischemia , Ischemic Stroke , Stroke , Animals , Swine , Swine, Miniature/genetics , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 16S/analysis , Tibet , Brain Ischemia/genetics , Genes, rRNA , Feces/microbiology , Stroke/genetics , Metabolomics/methods , Metabolome , Biomarkers/analysis , Ischemic Stroke/genetics , ATP-Binding Cassette Transporters/geneticsABSTRACT
Gene therapy's entrance into clinical settings has made it an ever more attractive field of study for various diseases. However, relatively little progress has been made in targeting kidney diseases due to poor gene delivery efficiency in renal cells. The development of novel gene therapy vectors for medical intervention to treat kidney diseases is needed. In this study, we designed and produced a pseudotyped lentiviral vector with envelope glycoproteins of Zika virus (ZIKV), and evaluated its potential use in viral vector entry, neutralization assay, and gene delivery especially in the renal context. The lentiviral vector, simplified as ZIKV-E, is pseudotyped with Env/G-TC representing the transmembrane (TM) and cytoplasmic (CY) domains of Env replaced with the TM and CY domains of the glycoprotein (G) of the vesicular stomatitis virus. In vivo results show that ZIKV-E induced efficient transduction in tubular epithelial cells in mouse kidneys, demonstrating >100-fold higher expression of exogenous green fluorescent protein gene compared with that achieved by vesicular stomatitis virus G (VSV-G) protein pseudotyped lentiviral vector. The results also showed that the vector ZIKV-E transduced cells in a pH-independent manner and the transduction was inhibited by anti-ZIKV Env domain III antibodies. Results also show that ZIKV-E can be used as a surrogate for studies of ZIKV entry mechanisms and neutralization antibody assay. In all, this study successfully demonstrated a novel pseudotyped lentiviral vector ZIKV-E for inducing high transduction efficiency in renal tubular epithelial cells that could serve as a foundation for gene therapy for the treatment of inherited renal diseases in humans.
Subject(s)
Vesicular Stomatitis , Zika Virus Infection , Zika Virus , Animals , Mice , Humans , Transduction, Genetic , Zika Virus/genetics , Viral Envelope , Viral Envelope Proteins/genetics , Genetic Vectors/genetics , Kidney , Zika Virus Infection/genetics , Zika Virus Infection/therapy , Lentivirus/geneticsABSTRACT
BACKGROUND: Diabetes mellitus (DM), characterized by hyperglycemia, is one of the leading causes of death worldwide. An appropriate DM animal model to explore the underlying mechanism of DM and develop new antidiabetic drugs is still desirable. Here, we aim to provide alternatives of DM animal models for medical researches. OBJECTIVE: To establish a diabetes susceptible family in the Bama minipig (BMP) by N-ethyl-N-nitrosourea (ENU) induction. METHODS: Male BMPs with hyperglycemia were selected from G1 and bred by the inbreeding strategy. After 5 generations, parameters such as fasting plasma glucose (FPG), 2-hour plasma glucose (2hPG), intravenous glucose-tolerance test (IVGTT), and insulin resistance were determined to evaluate susceptible family members. RESULTS: The male BMP 2907 (FPG = 6.1 mmol/L, IGVTT 2hPG = 11.9 mmol/L) with hyperglycemia was selected from G1 to generate the 2907 hyperglycemic family. With the number of breeding generations, average FPG levels in BMPs increased significantly (p < 0.05). G5 displayed the characteristics of elevated FPG, insulin resistance, dyslipidemia and abnormal glucose tolerance (p < 0.05). CONCLUSION: A diabetes susceptible family has been successfully established, which might be used for further inbreeding or induced to mimic the phenotype of diabetes.
Subject(s)
Diabetes Mellitus, Type 2 , Diabetes Mellitus , Animals , Blood Glucose , Ethylnitrosourea , Fasting , Glucose Tolerance Test , Male , Swine , Swine, MiniatureABSTRACT
Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by ADAR enzymes, is an essential post-transcriptional modification. Although hundreds of thousands of RNA editing sites have been reported in mammals, brain-wide analysis of the RNA editing in the mammalian brain remains rare. Here, a genome-wide RNA-editing investigation is performed in 119 samples, representing 30 anatomically defined subregions in the pig brain. We identify a total of 682,037 A-to-I RNA editing sites of which 97% are not identified before. Within the pig brain, cerebellum and olfactory bulb are regions with most edited transcripts. The editing level of sites residing in protein-coding regions are similar across brain regions, whereas region-distinct editing is observed in repetitive sequences. Highly edited conserved recoding events in pig and human brain are found in neurotransmitter receptors, demonstrating the evolutionary importance of RNA editing in neurotransmission functions. Although potential data biases caused by age, sex or health status are not considered, this study provides a rich resource to better understand the evolutionary importance of post-transcriptional RNA editing.
Subject(s)
Brain/metabolism , RNA Editing , Swine/genetics , Adenosine/genetics , Animals , Female , Gene Expression Regulation , Inosine/genetics , MaleABSTRACT
Hereditary tyrosinemia type I (HT1) results from the loss of fumarylacetoacetate hydrolase (FAH) activity and can lead to lethal liver injury (LLI). Therapeutic options for HT1 remain limited. The FAH -/- pig, a well-characterized animal model of HT1, represents a promising candidate for testing novel therapeutic approaches to treat this condition. Here, we report an improved single-step method to establish a biallelic (FAH -/- ) mutant porcine model using CRISPR-Cas9 and cytoplasmic microinjection. We also tested the feasibility of rescuing HT1 pigs through inactivating the 4-hydroxyphenylpyruvic acid dioxygenase (HPD) gene, which functions upstream of the pathogenic pathway, rather than by directly correcting the disease-causing gene as occurs with traditional gene therapy. Direct intracytoplasmic delivery of CRISPR-Cas9 targeting HPD before intrauterine death reprogrammed the tyrosine metabolism pathway and protected pigs against FAH deficiency-induced LLI. Characterization of the F1 generation revealed consistent liver-protective features that were germline transmissible. Furthermore, HPD ablation ameliorated oxidative stress and inflammatory responses and restored the gene profile relating to liver metabolism homeostasis. Collectively, this study not only provided a novel large animal model for exploring the pathogenesis of HT1, but also demonstrated that CRISPR-Cas9-mediated HPD ablation alleviated LLI in HT1 pigs and represents a potential therapeutic option for the treatment of HT1.
ABSTRACT
The Tibet minipig is a rare highland pig breed worldwide and has many applications in biomedical and agricultural research. However, Tibet minipigs are not like domesticated pigs in that their ovulation number is low, which is unfavourable for the collection of zygotes. Partly for this reason, few studies have reported the successful generation of genetically modified Tibet minipigs by zygote injection. To address this issue, we described an efficient way to generate gene-edited Tibet minipigs, the major elements of which include the utilization of synchronized oestrus instead of superovulation to obtain zygotes, optimization of the preparation strategy, and co-injection of clustered regularly interspaced short palindromic repeat sequences associated protein 9 (Cas9) mRNA and single-guide RNAs (sgRNAs) into the cytoplasm of zygotes. We successfully obtained allelic TYR gene knockout (TYR-/-) Tibet minipigs with a typical albino phenotype (i.e., red-coloured eyes with light pink-tinted irises and no pigmentation in the skin and hair) as well as TYR-/-IL2RG-/- and TYR-/-RAG1-/- Tibet minipigs with typical phenotypes of albinism and immunodeficiency, which was characterized by thymic atrophy and abnormal immunocyte proportions. The overall gene editing efficiency was 75% for the TYR single gene knockout, while for TYR-IL2RG and TYR-RAG1 dual gene editing, the values were 25% and 75%, respectively. No detectable off-target mutations were observed. By intercrossing F0 generation minipigs, targeted genetic mutations can also be transmitted to gene-edited minipigs' offspring through germ line transmission. This study is a valuable exploration for the efficient generation of gene-edited Tibet minipigs with medical research value in the future.
Subject(s)
Estrus Synchronization/physiology , Gene Editing/methods , Swine, Miniature/genetics , Animals , CRISPR-Cas Systems/genetics , Cytoplasm , Female , Male , Microinjections , Mutation , Superovulation , SwineABSTRACT
BACKGROUND: Mesenchymal stromal cells (MSCs) were originally characterized by the ability to differentiate into different mesenchymal lineages in vitro, and their immunomodulatory and trophic functions have recently aroused significant interest in the application of MSCs in cell-based regenerative medicine. However, a major problem in clinical practice is the replicative senescence of MSCs, which limits the cell proliferation potential of MSCs after large-scale expansion. Telomeric zinc finger-associated protein (TZAP), a novel specific telomere-binding protein, was recently found to stimulate telomere trimming and prevent excessive telomere elongation. The aim of this study was to elucidate the role of TZAP in regulating MSCs senescence, differentiation and proliferation. METHOD: Primary porcine mesenchymal stromal cells (pMSCs) were isolated from the bone marrow of Tibet minipigs by a noninvasive method in combination with frequent medium changes (FMCs). The deterioration of the pMSCs' proliferation capacity and their resultant entry into senescence were analyzed by using CCK8 and EdU incorporation assays, SA-ß-gal staining and comparisons of the expression levels of cellular senescence markers (p16INK14 and p21) in pMSC cell lines with TZAP overexpression or knockout. The effects of TZAP overexpression or knockout on the differentiation potential of pMSCs were assessed by alizarin red S staining after osteogenic induction or by oil red O staining after adipogenic induction. The effect of TZAP overexpression and the involvement of the p53 signaling pathway were evaluated by detecting changes in ARF, MDM2, P53 and P21 protein levels in pMSCs. RESULTS: TZAP levels were significantly elevated in late-passage pMSCs compared to those in early-passage pMSCs. We also observed significantly increased levels of the senescence markers p16INK4A and p21. Overexpression of TZAP reduced the differentiation potential of the cells, leading to premature senescence in early-passage pMSCs, while knockout of TZAP led to the opposite phenotype in late-passage pMSCs. Furthermore, overexpression of TZAP activated the P53 pathway (ARF-MDM2-P53-P21WAF/CDKN1A) in vitro. TZAP also downregulated the expression levels of PPARγ and Cebpα, two key modulators of adipogenesis. CONCLUSIONS: This study demonstrates that the level of TZAP is closely related to differentiation potential in pMSCs and affects cellular senescence outcomes via the p53 pathway. Therefore, attenuation of intracellular TZAP levels could be a new strategy for improving the efficiency of pMSCs in cell therapy and tissue engineering applications.
Subject(s)
Cell Self Renewal , Cellular Senescence , DNA-Binding Proteins/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Signal Transduction , Tumor Suppressor Protein p53/metabolism , Animals , Cell Differentiation , Cell Proliferation , Humans , SwineABSTRACT
Unexpectedly, we found that c-Myc-expressing porcine embryonic fibroblasts (PEFs) subcutaneously implanted into nude mice formed cartilage-like tissues in vivo, while previous studies revealed the direct conversion of mouse and human somatic cells into chondrocytes by the combined use of several defined factors, including c-Myc, which prompted us to explore whether PEFs can be reprogrammed to become pig induced chondrocyte-like cells (piCLCs) via ectopic expression of c-Myc alone. In this study, c-Myc-expressing PEFs, designated piCLCs, which exhibited a significantly enhanced proliferation ability in vitro, displayed a chondrogenic phenotypes in vitro, as shown by the cell morphology, toluidine blue staining, alcian blue staining and chondrocyte marker gene expression. Additionally, piCLCs with a polygonal chondrocyte-like morphology were readily and efficiently converted from PEFs by enforced c-Myc expression within 10 days, while piCLCs maintained the chondrocytic phenotype and normal karyotype during long-term subculture. piCLC-derived single clones with a chondrogenic phenotype in vitro exhibited homogeneity in cell morphology and staining intensity compared with mixed piCLCs. Although the mixtures of cartilaginous tissues and tumorous tissues accounted for ~12% (6/51) of all xenografts (51), piCLCs generated stable, homogenous, hyaline cartilage-like tissues without tumour formation at 45 out of the 51 injected sites when subcutaneously injected into nude mice. The hyaline cartilage-like tissues remained for at least 16 weeks. Taken together, these findings demonstrate for the first time the direct induction of chondrocyte-like cells from PEFs with only c-Myc.
ABSTRACT
In this study, the gut microbiota was characterized in four age strata of Tibetan minipigs. Results indicated that the fecal bacteria of 7-, 28-, 56-, and 180-day-old minipigs did not significantly differ in terms of phylogenetic diversity (i.e., PD whole tree) or the Shannon index (both, p > 0.05). Findings of a principal coordinate analysis demonstrated that fecal bacteria of 180-day-old minipigs were discernable from those of the other three age groups. From ages seven to 56 days, the abundance of Bacteroidetes or Firmicutes appeared to vary. Regarding genera, the populations of Bacteroides and Akkermansia decreased with increasing age.
Subject(s)
Bacteria/classification , Feces/microbiology , Gastrointestinal Microbiome , Age Factors , Animals , Bacteroidetes/genetics , Bacteroidetes/isolation & purification , Gastrointestinal Tract/microbiology , Phylogeny , RNA, Ribosomal, 16S/genetics , Swine , Swine, Miniature/microbiologyABSTRACT
BACKGROUND: Radiation-induced kidney damage can severely affect renal function, and have a serious impact on glucose reabsorption. Fluoro-2-deoxyglucose positron emission tomography (FDG-PET) is routinely utilized for metabolic imaging of glucose utilization. In this study, we are trying to assess the diagnostic value of 18F-FDG-PET/CT on measuring hyperacute effect of total body irradiation (TBI) on the kidneys. METHODS: Forty-eight Tibet minipigs were treated by TBI of different dosages using an 8-MV X-ray linear accelerator. Whole-body 18F-FDG-PET/CT was performed at 6, 24 and 72 h followed by histologic examination, blood samples' and renal function analysis. RESULTS: The uptake of 18F-FDG was significantly different between 11/14 Gy dose groups and control group, the standard Uptake Values reached a maximal level at 72 h after 14-Gy TBI treatment. At doses over 8 Gy, histological observation showed formation of tube casts, degeneration, necrosis of tubular cells, inflammatory cell infiltration and dilatation of the mitochondria of tubule cells. Renal function analysis confirmed the changes in blood urea nitrogen and creatinine levels at various dosages and time intervals. Immunohistochemistry and western blot results indicate that the expression levels of IL-10 and TNF-α proteins were positively correlated with radiation dose up to 8 Gy. CONCLUSIONS: 18F-FDG PET/CT can reflect pathological changes in kidneys and it may be a useful tool for rapid and non-invasive assessment in cases of suspected radiation-induced kidney damage.
Subject(s)
Fluorodeoxyglucose F18/chemistry , Kidney/diagnostic imaging , Positron Emission Tomography Computed Tomography , Radiation Injuries/diagnostic imaging , Radiation Injuries/diagnosis , Animals , Interleukin-10/metabolism , Kidney/pathology , Kidney/physiopathology , Kidney/ultrastructure , Kidney Function Tests , Reference Standards , Swine , Swine, Miniature , Time Factors , Tumor Necrosis Factor-alpha/metabolism , Whole-Body IrradiationABSTRACT
Clinical evidence suggests that there exists a strong correlation between Zika virus (ZIKV) infection and abnormal development of the nervous system. However, the underlying mechanisms remain to be elusive. In this study, recombinant lentiviral vectors coding for ZIKV structural proteins and truncations (prM-Env, M-Env and Env) were transduced into PC12 cells. Envelope (Env) overexpression induced significant inhibition of proliferation and triggered G2/M cell cycle arrest and apoptosis in PC12 cells. Flow cytometry and western blot analysis showed that the apoptosis was associated with up-regulation of both p53 and p21Cip1/Waf1 and down-regulation of cyclin B1. Presence of aberrant nuclei clusters were confirmed by immunofluorescence staining analysis. The data indicate that overexpression of prM-Env, M-Env or Env led to apoptosis via an intrinsic cell death signaling pathway that is dependent on the activation of caspase-9 and caspase-3 and accompanied by an increased ratio of Bax to Bcl-2 in transduced PC12 cells. In summary, our results suggest that ZIKV Env protein causes apoptosis in PC12 cells via an intrinsic cell death signaling pathway, which may contribute to ZIKV-induced abnormal development of the nervous system.
Subject(s)
Apoptosis/genetics , Cell Cycle Checkpoints/genetics , Viral Envelope Proteins/metabolism , Viral Envelope Proteins/physiology , Zika Virus/metabolism , Animals , Apoptosis/physiology , Caspase 3/genetics , Caspase 3/metabolism , Caspase 9/genetics , Caspase 9/metabolism , Cell Cycle Checkpoints/physiology , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Genetic Vectors/genetics , Lentivirus/genetics , PC12 Cells , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Signal Transduction/genetics , Signal Transduction/physiology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Viral Envelope Proteins/genetics , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
BACKGROUND: Hairless mice have been widely applied in skin-related researches, while hairless pigs will be an ideal model for skin-related study and other biomedical researches because of the similarity of skin structure with humans. The previous study revealed that hairlessness phenotype in nude mice is caused by insufficient expression of phospholipase C-delta 1 (PLCD1), an essential molecule downstream of Foxn1, which encouraged us to generate PLCD1-deficient pigs. In this study, we plan to firstly produce PLCD1 knockout (KO) mice by CRISPR/Cas9 technology, which will lay a solid foundation for the generation of hairless PLCD1 KO pigs. METHODS: Generation of PLCD1 sgRNAs and Cas 9 mRNA was performed as described (Shao in Nat Protoc 9:2493-2512, 2014). PLCD1-modified mice (F0) were generated via co-microinjection of PLCD1-sgRNA and Cas9 mRNA into the cytoplasm of C57BL/6J zygotes. Homozygous PLCD1-deficient mice (F1) were obtained by intercrossing of F0 mice with the similar mutation. RESULTS: PLCD1-modified mice (F0) showed progressive hair loss after birth and the genotype of CRISPR/Cas9-induced mutations in exon 2 of PLCD1 locus, suggesting the sgRNA is effective to cause mutations that lead to hair growth defect. Homozygous PLCD1-deficient mice (F1) displayed baldness in abdomen and hair sparse in dorsa. Histological abnormalities of the reduced number of hair follicles, irregularly arranged and curved hair follicles, epidermal hyperplasia and disturbed differentiation of epidermis were observed in the PLCD1-deficient mice. Moreover, the expression level of PLCD1 was significantly decreased, while the expression levels of other genes (i.e., Krt1, Krt5, Krt13, loricrin and involucrin) involved in the differentiation of hair follicle were remarkerably increased in skin tissues of PLCD1-deficient mice. CONCLUSIONS: In conclusion, we achieve PLCD1 KO mice by CRISPR/Cas9 technology, which provide a new animal model for hair development research, although homozygotes don't display completely hairless phenotype as expected.
Subject(s)
CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems/genetics , Hair/pathology , Phospholipase C delta/deficiency , Skin/pathology , Animals , Base Sequence , Gene Expression Regulation , Mice, Inbred C57BL , Mice, Knockout , Phospholipase C delta/metabolism , RNA, Guide, Kinetoplastida/geneticsABSTRACT
Obesity is a public health problem in both developed and developing countries, and the negative effects of obesity on reproductive physiology have been highlighted recently. We evaluated the effects of porcine obesity index, sex hormones, and peptide hormones on litter size in various breeds of minipigs. Blood samples were collected from sedated 8-,10-, and 12-mo-old minipigs to measure preovulatory levels of sex hormones (follicle-stimulating hormone, luteinizing hormone, estradiol, progesterone, testosterone, and prolactin) and peptide hormones (insulin-like growth factor, glucagon, cortisol, growth hormone, free thyroxine, free triiodothyronine, insulin, and leptin). We also measured weight, abdominal circumference, neck circumference, and body length and then calculated the porcine obesity index. Data were analyzed by one-way ANOVA, and means were compared by least significance difference testing. Pearson correlation between parameters and litter size was analyzed. Prepregnancy porcine obesity index and litter size were negatively correlated in primiparous minipigs. Litter size was influenced by luteinizing hormone, estradiol, progesterone, testosterone, prolactin, follicle-stimulating hormone, cortisol, insulin-like growth factor 1, growth hormone, free thyroxine, insulin, and leptin. In conclusion, prepregnancy obesity reduces litter size in primiparous minipigs.
Subject(s)
Litter Size/physiology , Obesity/veterinary , Pregnancy, Animal , Swine Diseases/physiopathology , Swine, Miniature , Animals , Female , Follicle Stimulating Hormone , Humans , Luteinizing Hormone , Obesity/pathology , Obesity/physiopathology , Parity , Pregnancy , Swine , Swine Diseases/pathologyABSTRACT
Hairless mice have been widely applied in skin-related researches, while hairless pigs will be a useful model for skin-related study and other biomedical researches. Dickkopf-related protein 1 (DKK1) is inhibitor of Wnt signaling pathway. Transgenic mice expressing DKK1 transgene under control of a human keratin 14 (K14) promoter display hairless phenotype, which encouraged us to generate transgenic minipigs expressing pig DKK1 transgene under control of K14 promoter and finally achieve hairless minipigs. To generate transgenic cloned pigs, we constructed the lentiviral expression vector pERKDZG which contains two independent expression cassettes, the transcription of Tibet minipig DKK1 and EGFP genes are driven by K14 promoter, while mRFP is regulated under the control of Ef-1α promoter. Prior to generating the transgenic pig, the functionality of pERKDZG construct was verified by fluorescence assay and via checking pDKK1 expression. Subsequently, lentiviruses harboring ERKDZG transgene infected porcine embryonic fibroblasts (PEFs), followed by sorting RFP-positive PEFs by flow cytometry to obtain the purified PEFs carrying ERKDZG, designated DKK1-PEFs as donor cells used for somatic cell nuclear transfer (SCNT). Finally, we obtained 3 DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 and EGFP transgenes, but unfortunately, DKK1 transgenic cloned pigs don't display hairless phenotype as expected. Taken together, we achieve DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 transgene which provide a pig model for exploring DKK1 gene functions in pigs.
ABSTRACT
Background: Zika virus (ZIKV) infection has become a major public health problem all around the world. Early diagnosis of Zika infection is important for better management of the disease. Non-structural protein 1 (NS1) is a potential biomarker for ZIKV infections. The purpose of this study was to produce the ZIKV NS1 protein for establishing serological diagnostic methods for ZIKV. Methods: The cDNA fragment encoding a chimeric protein composed of murine Igκ signal peptide, NS1 and histidine tag was synthesized and cloned into the lentiviral expression vector pLV-eGFP. The resulting expression vector pLV-eGFP-ZIKV-NS1 was packaged and transduced into human embryonic kidney (HEK) 293T cells and clonal cell lines with NS1 gene were generated from the tranduced cells by limiting dilution. Over expressed recombination NS1 (rNS1) fusion protein was purified by nickel affinity chromatography. Mice immunization and enzyme-linked immunosorbent assay (ELISA) were carried out to evaluate the immunogenicity of rNS1. Results: Western blot analysis revealed that the reconstituted cells stably expressed and secreted high levels of approximately 45-kDa NS1, and no significant changes were observed in green fluorescent protein (GFP) fluorescence ratio and fluorescence intensity. The scanned gels showed that the purity of the purified rNS1 was 99.42%. BALB/c mice were then immunized with purified rNS1 and a high level of antibodies against NS1 was elicited in the mice. Conclusion: Overall, recombinant NS1 proteins were successfully purified and their antigenicity was assessed. Immunization of mice with recombinant proteins demonstrated the immunogenicity of the NS1 protein. Thus, the generated recombinant NS1 can be potentially used in the development of serological diagnostic methods for ZIKV.
Subject(s)
Viral Nonstructural Proteins/metabolism , Zika Virus/genetics , Animals , Enzyme-Linked Immunosorbent Assay , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Lentivirus/genetics , Mice, Inbred BALB C , Mosquito Vectors/genetics , Viral Nonstructural Proteins/genetics , Zika Virus/physiologyABSTRACT
N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.
Subject(s)
Ethylnitrosourea/metabolism , Genetic Association Studies/methods , Mutagenesis , Mutagens/metabolism , Swine/genetics , Animals , China , Pilot ProjectsABSTRACT
Obesity, diabetes and fatty liver disease are extremely common in leptin-resistant patients. Dysfunction of leptin or its receptor is associated with obesity. The present study aimed to assess the effects of intramuscular injection of exogenous leptin or its receptor on fat deposition and leptin-insulin feedback regulation. Forty-five 40-day old female Sprague Dawley (SD) rats were injected thrice with leptin or its receptor intramuscularly. Adiposity and fat deposition were assessed by assessing the Lee's index, body weight, food intake, and total cholesterol, high density lipoprotein, low density lipoprotein, and triglyceride levels, as well as histological properties (liver and adipose tissue). Serum glucose, leptin, and insulin amounts were evaluated, and glucose tolerance assessed to monitor glucose metabolism in SD rats; pancreas specimens were analyzed immunohistochemically. Hypothalamic phosphorylated Janus kinase 2 (p-JAK2), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), and phosphatidylinositol-3-kinase (PI3K) signaling, and hepatic sterol regulatory element binding protein-1 (SREBP-1) were qualified by Western blotting. Leptin receptor immunogen reduced fat deposition, increased appetite, and lowered serum leptin levels, enhancing STAT3 signaling in hypothalamus and down-regulating hepatic SREBP-1. In contrast, SD rats administered leptin immunogen displayed significantly increased body weight and fat deposition, with up-regulated SREBP-1, indicating adiposity occurrence. SD rats administered leptin immunogen also showed glucose intolerance, ß- cell reduction in the pancreas, and deregulation of JAK2-STAT3/PI3K signaling, indicating that Lep rats were at risk of diabetes. In conclusion, intramuscular injection of exogenous leptin or its receptor, a novel rat model approach, can be used in obesity pathogenesis and therapeutic studies.
Subject(s)
Adiposity/drug effects , Fatty Liver/metabolism , Glucose Intolerance/metabolism , Janus Kinase 2/metabolism , Leptin/administration & dosage , Leptin/adverse effects , Phosphatidylinositol 3-Kinase/metabolism , STAT3 Transcription Factor/metabolism , Adipose Tissue/metabolism , Animals , Body Weight/drug effects , Disease Models, Animal , Eating/drug effects , Female , Glucose Intolerance/blood , Hyperglycemia/blood , Hyperglycemia/complications , Hyperglycemia/metabolism , Immunity/drug effects , Injections, Intramuscular , Insulin/metabolism , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Leptin/blood , Lipids/blood , Liver/drug effects , Liver/metabolism , Phosphorylation/drug effects , Rats, Sprague-Dawley , Receptors, Leptin/metabolism , Signal Transduction/drug effects , Sterol Regulatory Element Binding Protein 1/metabolismABSTRACT
Intramuscular fat (IMF) content has been generally recognized as a desirable trait in pork meat because of its positive effect on eating quality. An effective approach to enhance IMF content in pork is the generation of transgenic pigs. In this study, we used somatic cell nuclear transfer (SCNT) to generate cloned pigs exhibiting ectopic expression of phosphoenolpyruvate carboxykinase (PEPCK-C) driven by an α-skeletal-actin gene promoter, which was specifically expressed in skeletal muscle. Using qRT-PCR and Western blot analysis, we demonstrated that PEPCK-C was functionally expressed and had a significant effect on total fatty acid content in the skeletal muscle of the transgenic pigs, while the n-6/n-3 polyunsaturated fatty acid (PUFA) ratio showed no difference between transgenic and control pigs. Thus, genetically engineered PEPCK-Cmus pigs may be an effective solution for the production of IMF-enriched pork.
Subject(s)
Adipose Tissue , Gene Expression , Meat , Muscle, Skeletal/metabolism , Phosphoenolpyruvate Carboxykinase (ATP)/genetics , Animals , Animals, Genetically Modified , Cytosol , Gene Order , Genetic Vectors/genetics , Meat/standards , Phosphoenolpyruvate Carboxykinase (ATP)/metabolism , Protein Transport , SwineABSTRACT
Patients with heart failure (HF) have high mortality and mobility. Xinmailong (XML) injection, a Chinese Medicine, is clinically effective in treating HF. However, the mechanism of XML's effectiveness on HF was unclear, and thus, was the target of the present study. We created a mouse model of pressure-overload-induced HF with transverse aortic constriction (TAC) surgery and compared among 4 study groups: SHAM (n = 10), TAC (n = 12), MET (metoprolol, positive drug treatment, n = 7) and XML (XML treatment, n = 14). Dynamic changes in cardiac structure and function were evaluated with echocardiography in vivo. In addition, H9C2 rat cardiomyocytes were cultured in vitro and the phosphorylation of ERK1/2, AKT, GSK3ß and protein expression of GATA4 in nucleus were detected with Western blot experiment. The results showed that XML reduced diastolic thickness of left ventricular posterior wall, increased ejection fraction and fraction shortening, so as to inhibit HF at 2 weeks after TAC. Moreover, XML inhibited the phosphorylation of ERK1/2, AKT and GSK3ß, subsequently inhibiting protein expression of GATA4 in nucleus (P < 0.001). Together, our data demonstrated that XML inhibited the TAC-induced HF via inactivating the ERK1/2, AKT/GSK3ß, and GATA4 signaling pathway.