ABSTRACT
One of the primary concerns for the survival of the human species is the growing demand for food brought on by an increasing global population. New developments in genome-editing technology present promising opportunities for the growth of wholesome and prolific farm animals. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. Genome editing entails modifying genetic material by removing, adding, or manipulating particular DNA sequences from a particular locus in a way that does not happen naturally. The three primary genome editors are CRISPR/Cas 9, TALENs, and ZFNs. Each of these enzymes is capable of precisely severing nuclear DNA at a predetermined location. One of the most effective inventions is base editing, which enables single base conversions without the requirement for a DNA double-strand break (DSB). As reliable methods for precise genome editing in studies involving animals, cytosine and adenine base editing are now well-established. Effective zygote editing with both cytosine and adenine base editors (ABE) has resulted in the production of animal models. Both base editors produced comparable outcomes for the precise editing of point mutations in somatic cells, advancing the field of gene therapy. This review focused on the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of ZFNs, TALENs, and CRISPR/Cas9 base editors, and prime editing in diverse lab and farm animals. Additionally, we address the methodologies that can be used for gene regulation, base editing, and epigenetic alterations, as well as the significance of genome editing in animal models to better reflect real disease. We also look at methods designed to increase the effectiveness and precision of gene editing tools. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. This review is an overview of the existing knowledge of the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of zinc finger nucleases (ZFNs), transcription-activator-like endonucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas 9), base editors and prime editing in diverse lab and farm animals, which will offer better and healthier products for the entire human race.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Livestock , Gene Editing/methods , Animals , Livestock/genetics , Disease Resistance/geneticsABSTRACT
BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite that causes severe threats to humans and livestock. Macrophages are the cell type preferentially infected by T. gondii in vivo. Protein phosphorylation is an important posttranslational modification involved in diverse cellular functions. A rapidly accelerated fibrosarcoma kinase (A-Raf) is a member of the Raf family of serine/threonine protein kinases that is necessary for MAPK activation. Our previous research found that knockout of A-Raf could reduce T. gondii-induced apoptosis in porcine alveolar macrophages (3D4/21 cells). However, limited information is available on protein phosphorylation variations and the role of A-Raf in macrophages infected with T. gondii. METHODS: We used immobilized metal affinity chromatography (IMAC) in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile changes in phosphorylation in T. gondii-infected 3D4/21 and 3D4/21-ΔAraf cells. RESULTS: A total of 1647 differentially expressed phosphorylated proteins (DEPPs) with 3876 differentially phosphorylated sites (DPSs) were identified in T. gondii-infected 3D4/21 cells (p3T group) when compared with uninfected 3D4/21 cells (pho3 group), and 959 DEPPs with 1540 DPSs were identified in the p3T group compared with infected 3D4/21-ΔAraf cells (p3KT group). Venn analysis revealed 552 DPSs corresponding to 406 DEPPs with the same phosphorylated sites when comparing p3T/pho3 versus p3T/p3KT, which were identified as DPSs and DEPPs that were directly or indirectly related to A-Raf. CONCLUSIONS: Our results revealed distinct responses of macrophages to T. gondii infection and the potential roles of A-Raf in fighting infection via phosphorylation of crucial proteins.
Subject(s)
Fibrosarcoma , Toxoplasma , Toxoplasmosis , Humans , Animals , Swine , Phosphorylation , Chromatography, Liquid , Tandem Mass Spectrometry , Toxoplasmosis/parasitology , Toxoplasma/physiology , Macrophages/metabolismABSTRACT
Introduction: The increasing industrial and biomedical utilization of graphene oxide silver nanoparticles (GO-AgNPs) raises the concern of nanosafety: exposure to the AgNPs or GO-AgNPs increases the generation of reactive oxygen species (ROS), causes DNA damage and alters the expression of whole transcriptome including mRNA, miRNA, tRNA, lncRNA, circRNA and others. Although the roles of different RNAs in epigenetic toxicity are being studied during the last decade, but still we have little knowledge about the role of circle RNAs (circRNAs) in epigenetic toxicity. Methods: Rabbit fetal fibroblast cells (RFFCs) were treated with 0, 8, 16, 24, 32 and 48 µg/mL GO-AgNPs to test the cell viability and 24 µg/mL GO-AgNPs was selected as the experimental dose. After 24 h treatment with 24 µg/mL GO-AgNPs, the level of ROS, malondialdehyde (MDA), superoxide dismutase (SOD), intracellular ATP, glutathione peroxidase (GPx), and glutathione reductase (Gr) were measured in the RFFCs. High-throughput whole transcriptome sequencing was performed to compare the expression of circRNAs, long non-coding RNAs (lncRNA) and mRNA between 24 µg/mL GO-AgNPs-treated RFFCs and control cells. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to validate the accuracy of circRNA sequencing data. Bioinformatics analyses were performed to reveal the potential functional roles and related pathways of differentially expressed circRNAs, lncRNA and mRNA and to construct a circRNA-miRNA-mRNA interaction network. Results: We found that 57 circRNAs, 75 lncRNAs, and 444 mRNAs were upregulated while 35 circRNAs, 21 lncRNAs, and 186 mRNAs were downregulated. These differentially expressed genes are mainly involved in the transcriptional mis-regulation of cancer through several pathways: MAPK signaling pathway (circRNAs), non-homologous end-joining (lncRNAs), as well as PPAR and TGF-beta signaling pathways (mRNAs). Conclusion: These data revealed the potential roles of circRNAs in the GO-AgNPs induced toxicity through oxidative damage, which would be the basis for further research to determine their roles in the regulation of different biological processes.
Subject(s)
Metal Nanoparticles , MicroRNAs , RNA, Long Noncoding , Animals , Rabbits , RNA, Circular/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Silver/toxicity , Silver/metabolism , Reactive Oxygen Species/metabolism , Metal Nanoparticles/toxicity , Gene Expression Profiling , RNA, Messenger/genetics , RNA, Messenger/metabolism , MicroRNAs/genetics , Oxidative Stress , Epigenesis, GeneticABSTRACT
Exosomes are nanovesicles with a wide range of chemical compositions used in many different applications. Mesenchymal stem cell-derived exosomes (MSCs-EXOs) are spherical vesicles that have been shown to mediate tissue regeneration in a variety of diseases, including neurological, autoimmune and inflammatory, cancer, ischemic heart disease, lung injury, and liver fibrosis. They can modulate the immune response by interacting with immune effector cells due to the presence of anti-inflammatory compounds and are involved in intercellular communication through various types of cargo. MSCs-EXOs exhibit cytokine storm-mitigating properties in response to COVID-19. This review discussed the potential function of MSCs-EXOs in a variety of diseases including neurological, notably epileptic encephalopathy and Parkinson's disease, cancer, angiogenesis, autoimmune and inflammatory diseases. We provided an overview of exosome biogenesis and factors that regulate exosome biogenesis. Additionally, we highlight the functions and potential use of MSCs-EXOs in the treatment of the inflammatory disease COVID-19. Finally, we covered a strategies and challenges of MSCs-EXOs. Finally, we discuss conclusion and future perspectives of MSCs-EXOs.
Subject(s)
COVID-19 , Exosomes , Mesenchymal Stem Cells , Humans , COVID-19/therapy , Cell CommunicationABSTRACT
The widespread use of graphene oxide-silver nanoparticle nanocomposites (GO-AgNPs) in biomedical sciences is increasing the chances of human and animal exposure to its chronic non-toxic doses. Exposure to AgNPs-related nanomaterials may result in the negative effect on the dam, fetus and offspring. However, there are only little available information for profound understanding of the epigenetic alteration in the cells and animals caused by low-dose chronic exposure of GO-AgNPs. The present study investigated the effect of 0.5 µg/mL GO-AgNPs for 10 weeks on the differential expression of circular RNAs (circRNAs) in caprine fetal fibroblast cells (CFFCs), and this dose of GO-AgNPs did not affect cell viability and ROS level. We predicted the functions of those differentially expressed (DE) circRNAs in CFFCs by bioinformatics analysis. Furthermore, we validated the expression of ten DE circRNAs using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) to ensure the reliability of the sequencing data. Our results showed that the DE circRNAs may potentially regulate the GO-AgNPs-inducing epigenetic toxicity through a regulatory network consisted of circRNAs, miRNAs and messenger RNAs (mRNAs). Therefore, the epigenetics toxicity is essential to assess the biosafety level of GO-AgNPs.
ABSTRACT
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.
Subject(s)
CRISPR-Cas Systems , Myostatin , Animals , CRISPR-Cas Systems/genetics , Gene Editing , Muscle, Skeletal/metabolism , Mutation , Myostatin/genetics , Myostatin/metabolism , Phenotype , RabbitsABSTRACT
Graphene oxide-silver nanoparticle (GO-AgNPs) nanocomposites have drawn much attention for their potential in biomedical uses. However, the potential toxicity of GO-AgNPs in animals and humans remains unknown, particularly in the developing fetus. Here, we reported the GO-AgNP-mediated cytotoxicity and epigenetic alteration status in caprine fetal fibroblast cells (CFFCs). In brief, the proliferation and apoptosis rate of GO-AgNP-treated CFFCs (4 and 8 µg/mL of GO-AgNPs) were measured using the cell-counting kit (CCK-8) assay and the annexin V/propidium iodide (PI) assay, respectively. In addition, the oxidative stress induced by GO-AgNPs and detailed mechanisms were studied by evaluating the generation of reactive oxygen species (ROS), superoxide dismutase (SOD), lactate dehydrogenase (LDH), malondialdehyde (MDA), and caspase-3 and abnormal methylation. The expression of pro- and anti-apoptotic genes and DNA methyltransferases was measured using reverse transcription followed by RT-qPCR. Our data indicated that GO-AgNPs cause cytotoxicity in a dose-dependent manner. GO-AgNPs induced significant cytotoxicity by the loss of cell viability, production of ROS, increasing leakage of LDH and level of MDA, increasing expression of pro-apoptotic genes, and decreasing expression of anti-apoptotic genes. GO-AgNPs incited DNA hypomethylation and the decreased expression of DNMT3A. Taken together, this study showed that GO-AgNPs increase the generation of ROS and cause apoptosis and DNA hypomethylation in CFFCs. Therefore, the potential applications of GO-AgNPs in biomedicine should be re-evaluated.
Subject(s)
Fibroblasts/metabolism , Metal Nanoparticles , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolism , Silver/pharmacology , Cell Survival/drug effects , Humans , Malondialdehyde/metabolism , Methylation/drug effects , Silver/metabolismABSTRACT
Ribosomal proteins (RPs) are mostly derived from the energy-consuming enzyme families such as ATP-dependent RNA helicases, AAA-ATPases, GTPases and kinases, and are important structural components of the ribosome, which is a supramolecular ribonucleoprotein complex, composed of Ribosomal RNA (rRNA) and RPs, coordinates the translation and synthesis of proteins with the help of transfer RNA (tRNA) and other factors. Not all RPs are indispensable; in other words, the ribosome could be functional and could continue the translation of proteins instead of lacking in some of the RPs. However, the lack of many RPs could result in severe defects in the biogenesis of ribosomes, which could directly influence the overall translation processes and global expression of the proteins leading to the emergence of different diseases including cancer. While microRNAs (miRNAs) are small non-coding RNAs and one of the potent regulators of the post-transcriptional gene expression, miRNAs regulate gene expression by targeting the 3' untranslated region and/or coding region of the messenger RNAs (mRNAs), and by interacting with the 5' untranslated region, and eventually finetune the expression of approximately one-third of all mammalian genes. Herein, we highlighted the significance of miRNAs mediated regulation of RPs coding mRNAs in the global protein translation.
Subject(s)
MicroRNAs/metabolism , Protein Biosynthesis , Ribosomal Proteins/metabolism , Animals , Disease Progression , Humans , Ribosomes/metabolismABSTRACT
Kisspeptin (Kp), a multifunctional neuropeptide critical for initiating puberty and regulating ovulation, was reported to be expressed in mammalian ovaries. Fibronectin (FN), a major secretory product of granulosa cells, provided the extracellular environment for the cumulus cells during maturation. In the current study, we aimed to investigate the potential interplay between FN and Kp in bovine preantral follicles in the context of follicular development and quality. The results showed that Kp significantly reduced the follicular diameters after 14 days in culture, and this was prevented by the addition of FN. Follicles treated with Kp in the presence of FN showed lower levels of apoptotic cells compared to the Kp-treated group. The immunofluorescence analysis showed high levels of cyclooxygenase-2 (COX2), nuclear factor kappa B (NF-κB), and caspase 3, and low levels of sirtuin 1 (Sirt1) and Poly ADP-Ribose Polymerase 1 (PARP1) in the Kp-treated group compared to the control and FN-Kp co-treated groups. The protein expression levels of phosphoinositide 3 kinase (PI3K) increased significantly in the FN and FN-Kp combination treatment groups. Finally, we examined the signal pathway affecting the follicular development after Kp treatment. We detected a significant decrease in the mRNA levels of B-cell lymphoma 2 (BCL2), Sirt1, and PI3K, but the mRNA levels of NF-κB, Caspase3, COX2, P21, and P53 were significantly higher than in the control. Taken together, our results showed the importance of FN for preantral follicle developmental, and, for the first time, we reported that FN could neutralize the deleterious consequences of Kp, suggesting a potential role in the regulation of PI3K/Sirt1 signaling in bovine preantral follicle development.
Subject(s)
Fibronectins , Kisspeptins , Animals , Cattle , Female , Granulosa Cells , Kisspeptins/genetics , Kisspeptins/pharmacology , Ovarian Follicle , Phosphatidylinositol 3-KinasesABSTRACT
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.
Subject(s)
CRISPR-Cas Systems , Cystathionine beta-Synthase/genetics , Dyslipidemias/genetics , Hyperhomocysteinemia/genetics , Animals , Betaine/pharmacology , Body Weight/genetics , Disease Models, Animal , Female , Gene Knockout Techniques , Hyperlipidemias/drug therapy , Hyperlipidemias/genetics , Liver/pathology , Male , Point Mutation , Rabbits , Vitamin B Complex/pharmacologyABSTRACT
Increased oxidative stress is one of the main causes of poorly developed embryos in assisted reproductive technologies. Nicotinamide (NAM) has been shown to suppress reactive oxygen species (ROS) production through its potent antioxidative and anti-senescent effects. In the present study, we explored the effects of short-term NAM-treatment (3 and 5 h) during in vitro fertilization (IVF) on the development of bovine embryos. Treatment with 10 mM NAM for 3 h significantly increased the blastocyst formation but extending the treatment to 5 h did not enhance the benefits any further. Immunofluorescence analysis demonstrated that treatment with 10 mM NAM for 3 h decreased the expression of intracellular ROS, 8-oxo-7,8-dihydroguanine, caspase-3, and increased the expression of Sirt1, and incorporation of bromodeoxyuridine in one-cell stage embryos. Similarly, the level of H3K56ac significantly increased in the NAM-treated (3 and 5 h) one-cell stage embryos. Contrastingly, the treatment with 10 mM NAM for 5 h increased the caspase-9 level in blastocysts. Collectively, these findings suggest that NAM possesses antioxidant activity and supplementation of IVF medium with 10 mM NAM for 3 h improves the in vitro developmental competence of bovine embryos.
Subject(s)
Embryonic Development/drug effects , Fertilization in Vitro , Niacinamide/pharmacology , Animals , Antioxidants/pharmacology , Cattle/embryology , Cells, Cultured , Culture Media/chemistry , Culture Media/pharmacology , Embryo Culture Techniques/methods , Embryo Culture Techniques/veterinary , Embryo, Mammalian , Female , Fertilization in Vitro/methods , Fertilization in Vitro/veterinary , Male , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolismABSTRACT
Cell cycle of mouse embryo could be delayed by nicotinamide (NAM). Histone H3 lysine 56 (H3K56ac) acetylation plays an important role in mammalian genomic stability and the function of this modification in mouse embryos is not known. Hence, we designed to study the effects of NAM-induced oxidative stress on the developmental ability of mouse embryos, on the acetylation of H3K56ac and the possible functions of this modification related to mouse embryo development. Treatment with NAM (10, 20, or 40 mmol/L for 24 or 48 hr) during in vitro culture significantly decreased developmental rate of blastocyst (24 hr: 90.2 vs. 81.2, 43.2, and 18.2, with p > .05, p < .01, respectively; 48 hr: 89.3 vs. 53.2%, 12.1%, and 0% with p < .05, respectively). NAM treatment (20 mmol/L) for 6 and 31 hr resulted in increased intracellular reactive oxygen species levels in two-cell embryos, and apoptotic cell numbers in blastocysts. Resveratrol (RSV) and I-CBP112 rescued the 20 mmol/L NAM-induced embryo developmental defects. RSV and I-CBP112 increased the level of Sirt1 and decreased the level of H3K56ac induced by NAM in two-cell embryos (p < .05). These data suggest that NAM treatment decreases the expression of Sirt1, which induces high levels of H3K56 acetylation that may be involved in oxidative stress-induced mouse embryo defects, which can be rescued by RSV and I-CBP112.
Subject(s)
Embryonic Development/drug effects , Niacinamide/pharmacology , Oxazepines/pharmacology , Piperidines/pharmacology , Resveratrol/pharmacology , Animals , Cells, Cultured , Cytoprotection/drug effects , Cytoprotection/genetics , Embryo Culture Techniques , Embryo, Mammalian/cytology , Embryo, Mammalian/drug effects , Embryo, Mammalian/metabolism , Embryonic Development/genetics , Gene Expression Regulation, Developmental/drug effects , Histones/drug effects , Histones/metabolism , Mice , Oxidative Stress/drug effects , Oxidative Stress/genetics , Reactive Oxygen Species/metabolism , Sirtuin 1/genetics , Sirtuin 1/metabolismABSTRACT
Successful implantation is closely linked to the expression of MMP-2 and MMP-9, which greatly influence the ability of an embryo to degrade the basement membrane of the uterine epithelium, mainly composed of type IV collagen, and invade the uterine stroma. The objective of this study was to determine the effect of MMP-2 and MMP-9 co-transfer with embryos on reproductive performance in mice. Using invasion assay, we tested the effect of MMP-2 and MMP-9 for their ability to support trophoblastic invasion in vitro. We performed co-transfer of MMP-2 and MMP-9 with mouse embryos to 2.5 days post-coitum (dpc) pseudo-pregnant uteri using nonsurgical embryo transfer (NSET) technique and evaluated the pregnancy outcomes. Uterine tissue samples were collected to determine collagen content by Masson's trichrome staining. Our results showed that in vitro treatment of MMP-2 and MMP-9 significantly promoted both spreading and invasion of mouse trophoblastic cells compared to the non-treated blastocysts. Moreover, embryo transfer results showed that MMP-9 co-transfer enhanced pregnancy outcome inform of live pup rate by degrading the extracellular matrix, collagen, and facilitate embryo implantation. Taken together our findings imply that MMP-9 can regulate trophoblastic cell invasion during preimplantation, which may have important consequences on embryo implantation, and shed the light on new strategies to avoid miscarriage and provides a platform for successful human embryo transfer technologies.
Subject(s)
Embryo Implantation/physiology , Gene Expression Regulation, Developmental/physiology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Trophoblasts/physiology , Animals , Embryo, Mammalian/metabolism , Female , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/genetics , Mice , PregnancyABSTRACT
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.
Subject(s)
Animals, Genetically Modified/genetics , Lactoferrin/genetics , Promoter Regions, Genetic , Transgenes/genetics , Animals , Animals, Genetically Modified/metabolism , Bioreactors , Caseins/genetics , Caseins/metabolism , Cytomegalovirus/genetics , Fibroblasts/metabolism , Goats/genetics , Humans , Lactoferrin/biosynthesis , Lactoferrin/pharmacology , Microinjections , Milk/chemistry , Nuclear Transfer TechniquesABSTRACT
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.
Subject(s)
Animals, Genetically Modified/genetics , Lactoglobulins/genetics , Mammary Glands, Animal/metabolism , Plasminogen Activators/genetics , Protein Sorting Signals/genetics , Rabbits/genetics , Animals , Female , Goats/genetics , Humans , Protein Biosynthesis , Recombinant Proteins/geneticsABSTRACT
The mammalian Sirtuin family of seven enzymes, members of the NAD+-dependent histone deacetylase family that modify histones via direct deacetylation, is involved in the regulation of many antioxidant and oxidative stresses. In the present study, we explored the effects of nicotinamide (NAM)-induced oxidative stress on the in vitro development of bovine embryos, on the acetylation of histone H3 lysine 56 (H3K56ac) and on expression of apoptosis-related genes. Treatment with NAM (10, 20 or 40â¯mM for 24, 48 or 196â¯h) during IVC resulted in significantly decreased blastocyst formation (24â¯h: 38.8 vs. 33.1, 27.3 and 10.2%, with Pâ¯>â¯0.05, Pâ¯<â¯0.05 and Pâ¯<â¯0.01, respectively; 48â¯h: 37.5 vs. 28.2, 13.4 and 0%, with Pâ¯<â¯0.05 and Pâ¯<â¯0.01, respectively; 196â¯h: 35.8 vs. 23.4, 0 and 0%, with Pâ¯<â¯0.05, respectively). Treatment with NAM (20 and 40â¯mM for 24â¯h) resulted in increased intracellular reactive oxygen species (ROS) levels in 2-cell and blastocysts, and apoptotic cell numbers in blastocysts and decreased mitochondrial membrane potential (ΔΨ) in 2-cell embryos (Pâ¯<â¯0.05). Polydatin (PD) and I-CBP112 rescued the 20â¯mM NAM-induced embryo developmental defects and reduced ROS levels and apoptotic cell numbers in blastocysts (Pâ¯<â¯0.05). The gene expression of NF-κB, COX2 and p53 was significantly increased in the NAM-treated group. Immunofluorescence analysis confirmed that the protein levels of nuclear factor-kappa B (NF-κB) decreased significantly after PD and I-CBP112 treatment compared with the control (Pâ¯<â¯0.05). High level of H3K56ac induced by NAM was decreased after PD and I-CBP112 treatment (Pâ¯<â¯0.05). These findings suggest that NAM treatment induces high levels of H3K56 acetylation that may be involved in oxidative stress-induced bovine developmental defects, which can be tolerated by PD and I-CBP112 treatment.
Subject(s)
Cattle/embryology , Embryonic Development/drug effects , Glucosides/pharmacology , Oxazepines/pharmacology , Piperidines/pharmacology , Stilbenes/pharmacology , Acetylation , Animals , Apoptosis/genetics , Cyclooxygenase 2/metabolism , Embryo Culture Techniques/veterinary , Histones/metabolism , Membrane Potential, Mitochondrial/drug effects , NF-kappa B/genetics , NF-kappa B/metabolism , Niacinamide/pharmacology , Oxidative Stress , Reactive Oxygen Species/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
Predator Stress Can Exert Detrimental Effects on Female Mammals, Leading to Disrupted Reproduction. Although Many Studies Have Addressed the Effects of Predator Stress on Reproductive Output in Rodents, Few Studies Have Focused on the Effect of Visual or Auditory Stress on Pregnant Females. in This Study, We Investigated the Possible Effect of Predator Stress, Either Visual Only or Combined Visual and Auditory (visual+auditory), on the Reproductive Performance of Female Mice After Nonsurgical Embryo Transfer. Reproductive Performance Was Assessed As Pregnancy Rate, Implantation Rate, Gestation Length, Live Pup Rate, and Neonatal Birth Weight. Moreover, Serum Cortisol and Progesterone Levels in Dams Were Measured by Using Electrochemiluminescence Immunoassay. Exposure to Predator (cat) Stress Did Not Lead to a Significant Change in Pregnancy Rates in the Tested Mice. However, the Stressed Mice Showed Significantly Decreased Implantation Rates Compared with the Control Group. Similarly, the Live Pup Rate and Neonatal Birth Weight Were Significantly Lower in the Group Exposed to Preda- Tor Stress Than in the Control Group. Furthermore, Mice Exposed to Visual+auditory Stress Showed a Significant Reduction in Gestation Length Compared with the Control Mice. Our Data Showed That Predator Visual+auditory Stress As Combined Stimuli Significantly Increased Serum Cortisol Level. in Contrast, Progesterone Levels Did Not Significantly Vary Among the Experimental Groups. Taken Together, Our Findings Imply That Predator Stress Adversely Affects the Reproductive Efficiency of Pregnant Mice By Decreasing the Implantation Rate, Live Birth Rate, and Neonatal Birth Weight and by Prolonging Gestation Length.
Subject(s)
Embryo Transfer/veterinary , Mice/physiology , Predatory Behavior , Pregnancy Outcome , Reproduction , Animals , Female , Hydrocortisone/blood , Laboratory Animal Science , Pregnancy , Progesterone/blood , Reproduction/drug effects , Sound , Stress, PsychologicalABSTRACT
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 12â¯weeks revealed that all these KO rabbits exhibited markedly increased levels of plasma TC (the highest of which was 1013.15â¯mg/dl, 20-fold higher than wild-type rabbits), LDL-C (the highest of which was 730.00â¯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.
Subject(s)
Atherosclerosis/genetics , Atherosclerosis/pathology , Exons , Hypercholesterolemia/genetics , Receptors, LDL/deficiency , Animals , Animals, Genetically Modified , Atherosclerosis/metabolism , Biomarkers , CRISPR-Cas Systems , Coronary Artery Disease/genetics , Coronary Artery Disease/metabolism , Coronary Artery Disease/pathology , Disease Models, Animal , Female , Gene Targeting , Genotype , Hypercholesterolemia/diagnosis , Hypercholesterolemia/metabolism , Inflammation Mediators/blood , Leukocyte Count , Lipids/blood , Male , Plaque, Atherosclerotic/pathology , Rabbits , Sequence DeletionABSTRACT
Expression efficacy of recombinant protein in current expression systems is generally low. Therefore, the expression levels of recombinant proteins in the breast milk of transgenic animals are typically low. In view of this, the present study aimed to construct homozygous transgenic rabbits with a high expression level of recombinant human plasminogen activator (rhPA) during the entire lactation period. Homozygous transgenic rabbits were obtained using an effective rhPA mammaryspecific expression vector PCL25/rhPA. The expression level and thrombolytic ability of rhPA in the milk of both homozygous and hemizygous rabbits were detected by enzymelinked immunosorbent and fibrin agarose plate assays. It was observed that the expression of rhPA was constant during the entire lactation period in homozygous rabbits, while the expression of rhPA declined slowly in hemizygote rhPA transgenic rabbits during the lactation period. In addition, the expression of rhPA in homozygous transgenic rabbit was ~950 µg/ml, which was markedly higher in comparison with that in hemizygote rabbits. Furthermore, increased gene copy number was observed to increase the expression level of rhPA at the same integration vector.
Subject(s)
Animals, Genetically Modified , Gene Expression , Homozygote , Lactation , Mammary Glands, Animal/metabolism , Plasminogen Activators , Animals , Animals, Genetically Modified/genetics , Animals, Genetically Modified/metabolism , Female , Genetic Vectors , Humans , Plasminogen Activators/biosynthesis , Plasminogen Activators/genetics , Rabbits , Recombinant Proteins/biosynthesis , Recombinant Proteins/geneticsABSTRACT
Silver nanoparticles (AgNPs) are widely used metal nanoparticles in health care industries, particularly due to its unique physical, chemical, optical, and biological properties. It is used as an antibacterial, antiviral, antifungal, and anticancer agent. Camptothecin (CPT) and its derivatives function as inhibitors of topoisomerase and as potent anticancer agents against a variety of cancers. Nevertheless, the combined actions of CPT and AgNPs in apoptosis in human cervical cancer cells (HeLa) have not been elucidated. Hence, we investigated the synergistic combinatorial effect of CPT and AgNPs in human cervical cancer cells. We synthesized AgNPs using sinigrin as a reducing and stabilizing agent. The synthesized AgNPs were characterized using various analytical techniques. The anticancer effects of a combined treatment with CPT and AgNPs were evaluated using a series of cellular and biochemical assays. The expression of pro- and antiapoptotic genes was measured using real-time reverse transcription polymerase chain reaction. The findings from this study revealed that the combination of CPT and AgNPs treatment significantly inhibited cell viability and proliferation of HeLa cells. Moreover, the combination effect significantly increases the levels of oxidative stress markers and decreases antioxidative stress markers compared to single treatment. Further, the combined treatment upregulate various proapoptotic gene expression and downregulate antiapoptotic gene expression. Interestingly, the combined treatment modulates various cellular signaling molecules involved in cell survival, cytotoxicity, and apoptosis. Overall, these results suggest that CPT and AgNPs cause cell death by inducing the mitochondrial membrane permeability change and activation of caspase 9, 6, and 3. The synergistic cytotoxicity and apoptosis effect seems to be associated with increased ROS formation and depletion of antioxidant. Certainly, a combination of CPT and AgNPs could provide a beneficial effect in the treatment of cervical cancer compared with monotherapy.