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1.
Chemosphere ; 361: 142407, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38795919

RESUMO

Polymethylmethacrylate (PMMA) has been used in many products, such as acrylic glass, and is estimated to reach 5.7 million tons of production per year by 2028. Thus, nano-sized PMMA particles in the environment are highly likely due to the weathering process. However, information on the hazards of nanoplastics, including PMMA in mammals, especially reproductive toxicity and action mechanism, is scarce. Herein, we investigated the effect of PMMA nanoplastics on the female reproductive system of mice embryos during pre-implantation. The treated plastic particles in embryos (10, 100, and 1000 µg/mL) were endocytosed into the cytoplasm within 30 min, and the blastocyst development and indices of embryo quality were significantly decreased from at 100 µg/mL. Likewise, the transfer of nanoplastic-treated embryos at 100 µg/mL decreased the morula implantation rate on the oviduct of pseudopregnant mice by 70%, calculated by the pregnant individual, and 31.8% by the number of implanted embryos. The PMMA nanoplastics at 100 µg/mL significantly increased the cellular levels of reactive oxygen species in embryos, which was not related to the intrinsic oxidative potential of nanoplastics. This study highlights that the nanoplastics that enter systemic circulation can affect the early stage of embryos. Thus, suitable action mechanisms can be designed to address nanoplastic occurrence.


Assuntos
Desenvolvimento Embrionário , Estresse Oxidativo , Polimetil Metacrilato , Espécies Reativas de Oxigênio , Animais , Polimetil Metacrilato/química , Polimetil Metacrilato/toxicidade , Camundongos , Desenvolvimento Embrionário/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Feminino , Espécies Reativas de Oxigênio/metabolismo , Gravidez , Nanopartículas/toxicidade , Nanopartículas/química , Blastocisto/efeitos dos fármacos , Microplásticos/toxicidade
2.
Animals (Basel) ; 13(3)2023 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-36766312

RESUMO

The supplementation of pig diets with exogenous enzymes is widely used with the expectation that it will improve the efficiency of nutrient utilization, thereby, improving growth performance. This study aims to evaluate the effects of a 0.1% (v/v) multi-enzyme (a mixture of arazyme (2,500,000 Unit/kg), xylanase (200,000 Unit/kg) and mannanase (200,000 Unit/kg)) supplementation derived from invertebrate symbiotic bacteria on pig performance. Here, 256 growing pigs were assigned to control and treatment groups, respectively. The treatment group exhibited a significantly reduced average slaughter age; the final body weight and average daily gain increased compared with that of the control group. In the treatment group, the longissimus muscle showed a remarkable decrease in cooking loss, shear force, and color values with increased essential and non-essential amino acid concentrations. Furthermore, the concentrations of mono- and polyunsaturated fatty acids in the treatment group increased. Feed additive supplementation increased the family of Ruminococcaceae and genera Lactobacillus, Limosilactobacillus, Turicibacter, and Oscillibacter, which play a positive role in the host physiology and health. Predicted metabolic pathway analysis confirmed that operational taxonomic units and predicted amino acid biosynthesis pathways were strongly associated. The results suggest that applying exogenous enzymes derived from invertebrate symbiotic bacteria enhances animal performance.

3.
Sci Total Environ ; 848: 157735, 2022 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-35926625

RESUMO

This study evaluated the effects of supplementing feed with arazyme and dietary carbohydrolases derived from invertebrate gut-associated symbionts on the noxious gas emissions, gut microbiota, and host-microbiome interactions of pigs. Here, 270 and 260 growing pigs were assigned to control and treatment groups, respectively. The tested feed additives contained a mixture of arazyme (2,500,000 Unit/kg) and synergetic enzymes, xylanase (200,000 Unit/kg) and mannanase (200,000 Unit/kg), derived from insect gut-associated symbionts in a 7.5:1:1 ratio. The control group was fed a basal diet and the treatment group was fed the basal diet supplemented with 0.1 % enzyme mixture (v/v) for 2 months. Odorous gases were monitored in ventilated air from tested houses. Fecal samples were collected from steel plate under the cage at the completion of the experiment to determine chemical composition, odor emissions, and bacterial communities. There was a significant decrease in the concentration of NH3 (22.5 vs. 11.2 ppm; P < 0.05), H2S (7.35 vs. 3.74 ppm; P < 0.05), trimethylamine (TMA) (0.066 vs. 0.001 ppm; P < 0.05), and p-cresol (0.004 ppm vs. 0 ppm; P < 0.05) at 56 d in treatment group compared with the control group. Moreover, fecal analysis results showed that exogenous enzyme supplementation caused a reduction in VFAs and indole content with approximately >60 % and 72.7 %, respectively. The result of gas emission analysis showed that NH3 (9.9 vs. 5.3 ppm; P < 0.05) and H2S (5.8 vs. 4.1 ppm; P < 0.05) were significantly reduced in the treatment group compared to the control group. The gut microbiota of the treatment group differed significantly from that of the control group, and the treatment group altered predicted metabolic pathways, including sulfur and nitrogen related metabolism, urea degradation. The results demonstrated that supplementing feed with arazyme with dietary carbohydrolases effectively controls noxious gas emissions and improves health and meat quality of pigs.


Assuntos
Microbioma Gastrointestinal , Ração Animal/análise , Animais , Dieta/veterinária , Gases/metabolismo , Indóis , Nitrogênio/metabolismo , Odorantes/análise , Aço , Enxofre , Suínos , Ureia
4.
Anim Biosci ; 35(1): 126-137, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34293843

RESUMO

OBJECTIVE: Efficient gene editing technology is critical for successful knock-in in domestic animals. RAD51 recombinase (RAD51) gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by checkpoint kinase 1 (CHK1) and CHK2 genes, which are upstream elements of RAD51 recombinase (RAD51). In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine ß-casein gene locus can be increased by suppressing DNA MMR-related genes (MSH2, MSH3, MSH6, MLH1, and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2). METHODS: Bovine mammary epithelial (MAC-T) cells were transfected with a knock-in vector, RAD51, CHK1, or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine ß-casein gene locus, followed by treatment of the cells with CdCl2 for 24 hours. After 3 days of CdCl2 treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative real-time PCR (RT-qPCR). RESULTS: Treatment with CdCl2 decreased the mRNA expression of RAD51 and MMR-related genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMR-related genes and failed to increase the knock-in efficiency. CONCLUSION: Treatment with CdCl2 inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in various cell types or species.

5.
Genes (Basel) ; 12(7)2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34356062

RESUMO

Genetically engineered (GE) pigs with various combinations of genetic profiles have been developed using heterologous promoters. This study aimed to identify autologous promoters for high and ubiquitous expression of xenotransplantation relevant genes in GE pigs. A 1.4 kb upstream regulatory sequence of porcine elongation factor 1α (pEF1α) gene was selected and isolated for use as a promoter. Activity of the pEF1α promoter was subsequently compared with that of the cytomegalovirus (CMV) promoter, CMV enhancer/chicken ß-actin (CAG) promoter, and human EF1α (hEF1α) promoter in different types of pig-derived cells. Comparative analysis of luciferase and mutant human leukocyte antigen class E-F2A-ß-2 microglobulin (HLA-E) expression driven by pEF1α, CMV, CAG, and hEF1α promoters revealed the pEF1α promoter mediated comparable expression levels with those of the CAG promoter in porcine ear skin fibroblasts (PEFs) and porcine kidney-15 (PK-15) cells, but lower than those of the CAG promoter in porcine aortic endothelial cells (PAECs). The pEF1α promoter provided long-term stable HLA-E expression in PEFs, but the CAG promoter failed to sustain those levels of expression. For xenogeneic serum-induced cytotoxicity assays, the cells were cultured for several hours in growth medium supplemented with primate serum. Notably, the pEF1α promoter induced significant increases in luciferase and HLA-E expression in response to primate serum in PAECs compared with those driven by the CAG promoter, suggesting the pEF1α promoter could regulate temporal expression of heterologous genes under xenogeneic-cytotoxic conditions. These results suggest the pEF1α promoter may be valuable for development of GE pigs spatiotemporally and stably expressing immunomodulatory genes for xenotransplantation.


Assuntos
Citomegalovirus/genética , Elementos Facilitadores Genéticos , Fator 1 de Elongação de Peptídeos/metabolismo , Regiões Promotoras Genéticas , Transgenes , Transplante Heterólogo/métodos , Animais , Animais Geneticamente Modificados , Células Cultivadas , Vetores Genéticos , Humanos , Fator 1 de Elongação de Peptídeos/genética , Primatas , Suínos , Ativação Transcricional
6.
Asian-Australas J Anim Sci ; 33(6): 1023-1033, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32054213

RESUMO

OBJECTIVE: The efficiency of the knock-in process is very important to successful gene editing in domestic animals. Recently, it was reported that transient loosening of the nucleosomal folding of transcriptionally inactive chromatin might have the potential to enhance homologous recombination efficiency. The objective of this study was to determine whether histone deacetylases (HDAC) inhibitor and RAD51 recombinase (RAD51) expression were associated with increased knock-in efficiency on the ß-casein (bCSN2) gene locus in mammary alveolar-large T antigen (MAC-T) cells using the transcription activator-like effector nucleases (TALEN) system. METHODS: MAC-T cells were treated with HDAC inhibitors, valproic acid, trichostatin A, or sodium butyrate for 24 h, then transfected with a knock-in vector, RAD51 expression vector and TALEN to target the bCSN2 gene. After 3 days of transfection, the knock-in efficiency was confirmed by polymerase chain reaction and DNA sequencing of the target site. RESULTS: The level of HDAC 2 protein in MAC-T cells was decreased by treatment with HDAC inhibitors. The knock-in efficiency in MAC-T cells treated with HDAC inhibitors was higher than in cells not treated with inhibitors. However, the length of the homologous arm of the knock-in vector made no difference in the knock-in efficiency. Furthermore, DNA sequencing confirmed that the precision of the knock-in was more efficient in MAC-T cells treated with sodium butyrate. CONCLUSION: These results indicate that chromatin modification by HDAC inhibition and RAD51 expression enhanced the homologous recombination efficiency on the bCSN2 gene locus in MAC-T cells.

7.
Mol Nutr Food Res ; 63(14): e1801347, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31034714

RESUMO

SCOPE: Obesity and diabetes are major public health problems and are emerging as pandemics. Considerable evidence suggests that pear fruit consumption is associated with a lower risk of obesity-related complications. Thus, the present study is conducted to investigate the therapeutic potential of pear extract (PE) for reversing obesity and associated metabolic complications in high-fat diet-induced obese mice. METHODS AND RESULTS: Obesity is induced in male C57BL/6 mice fed a high-fat diet for 11 weeks. After the first 6 weeks on the diet, obese mice are administered vehicle or PE for 5 weeks. PE treatment decreases body weight gain, expands white adipose tissue (WAT), and causes hepatic steatosis in obese mice, as well as inhibits adipogenesis and lipogenesis. Impaired glucose tolerance and insulin resistance are improved by PE. In addition, PE reduces macrophage infiltration and expression of pro-inflammatory genes and deactivates mitogen-activated protein kinases in WAT. Finally, malaxinic acid is identified as an active component responsible for the anti-obesity effects of PE in mice. CONCLUSION: The results demonstrate that PE supplementation ameliorates diet-induced obesity and associated metabolic complications and suggest the health-beneficial effects of both pear fruits and malaxinic acid in counteracting these diseases.


Assuntos
Fármacos Antiobesidade/uso terapêutico , Benzoatos/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Obesidade/dietoterapia , Paniculite/dietoterapia , Extratos Vegetais/farmacologia , Piranos/uso terapêutico , Pyrus/química , Adipogenia/efeitos dos fármacos , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/patologia , Animais , Fármacos Antiobesidade/farmacologia , Benzoatos/farmacologia , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Obesidade/etiologia , Paniculite/etiologia , Paniculite/patologia , Extratos Vegetais/análise , Polifenóis/análise , Piranos/farmacologia , Aumento de Peso/efeitos dos fármacos
8.
Sci Rep ; 8(1): 10130, 2018 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-29973637

RESUMO

Mito-TEMPO is a well-known mitochondria-specific superoxide scavenger. However, the effect of Mito-TEMPO on porcine embryo development, to our knowledge, has not been studied yet. In the present study, porcine embryos were classified into two groups (G1 and G2) based on the cytoplasm lipid contents at the zygote stage. The development of blastocysts derived from G2 zygotes was reduced (G2:16.2 ± 7.9% vs G1: 26.5 ± 5.9%; 1.6-fold, p < 0.05) compared to those from G1 zygotes. In G2 embryos, the proportion of TUNEL-positive cells was also higher than that of G1 embryos. Superoxide in G2 embryos was significantly increased compared to that in G1 embryos. Mitochondrial membrane potential and ATP production were lower in G2 embryos than in G1 embryos. Phosphorylation of Drp1 at Ser 616 increased in G1 embryos during the cleavage stages compared to that in the zygote but was not significantly different in G2 embryos. Then, the effects of Mito-TEMPO were investigated in G2 embryos. Blastocyst formation rate (G2: 19.1 ± 5.1% vs G2 + Mito-TEMPO: 28.8 ± 4.0%; 1.5-fold, p < 0.05) and mitochondrial aggregation were recovered after superoxide reduction by Mito-TEMPO treatment. Thus, we showed that Mito-TEMPO improves blastocyst development by superoxide reduction in porcine embryos in vitro.


Assuntos
Blastocisto/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Compostos Organofosforados/farmacologia , Piperidinas/farmacologia , Animais , Células Cultivadas , Feminino , Potencial da Membrana Mitocondrial , Superóxidos/metabolismo , Suínos
9.
Mol Cells ; 41(5): 454-464, 2018 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-29754475

RESUMO

Crosstalk between G-protein signaling and glutamatergic transmission within the brain reward circuits is critical for long-term emotional effects (depression and anxiety), cravings, and negative withdrawal symptoms associated with opioid addiction. A previous study showed that Regulator of G-protein signaling 4 (RGS4) may be implicated in opiate action in the nucleus accumbens (NAc). However, the mechanism of the NAc-specific RGS4 actions that induce the behavioral responses to opiates remains largely unknown. The present study used a short hairpin RNA (shRNA)-mediated knock-down of RGS4 in the NAc of the mouse brain to investigate the relationship between the activation of ionotropic glutamate receptors and RGS4 in the NAc during morphine reward. Additionally, the shRNA-mediated RGS4 knock-down was implemented in NAc/striatal primary-cultured neurons to investigate the role that striatal neurons have in the morphine-induced activation of ionotropic glutamate receptors. The results of this study show that the NAc-specific knockdown of RGS4 significantly increased the behaviors associated with morphine and did so by phosphorylation of the GluR1 (Ser831) and NR2A (Tyr1325) glutamate receptors in the NAc. Furthermore, the knock-down of RGS4 enhanced the phosphorylation of the GluR1 and NR2A glutamate receptors in the primary NAc/striatal neurons during spontaneous morphine withdrawal. These findings show a novel molecular mechanism of RGS4 in glutamatergic transmission that underlies the negative symptoms associated with morphine administration.


Assuntos
Ácido Glutâmico/fisiologia , Morfina/farmacologia , Proteínas do Tecido Nervoso/fisiologia , Núcleo Accumbens/fisiologia , Proteínas RGS/fisiologia , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Recompensa , Animais , Células Cultivadas , Corpo Estriado/citologia , Comportamento Exploratório/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Núcleo Accumbens/citologia , Núcleo Accumbens/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas RGS/antagonistas & inibidores , Proteínas RGS/genética , Interferência de RNA , RNA Interferente Pequeno/genética
10.
J Pineal Res ; 64(2)2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29149522

RESUMO

Under endoplasmic reticulum (ER)-stress conditions, the unfolded protein response (UPR) generates a defense mechanism in mammalian cells. The regulation of UPR signaling is important in oocyte maturation, embryo development, and female reproduction of pigs. Recent studies have shown that melatonin plays an important role as an antioxidant to improve pig oocyte maturation. However, there is no report on the role of melatonin in the regulation of UPR signaling and ER-stress during in vitro maturation (IVM) of porcine oocytes. Therefore, the objective of this study was to investigate the antioxidative effects of melatonin on porcine oocyte maturation through the regulation of ER-stress and UPR signaling. We investigated the changes in the mRNA/protein expression levels of three UPR signal genes (Bip/Grp78, ATF4, P90/50ATF6, sXbp1, and CHOP) on oocytes, cumulus cells, and cumulus-oocyte complexes (COCs) during IVM (metaphase I; 22 hours and metaphase II; 44 hours) by Western blot and reverse transcription-polymerase chain reaction analysis. Treatment with the ER-stress inducer, tunicamycin (Tm), significantly increased expression of UPR markers. Additionally, cumulus cell expansion and meiotic maturation of oocytes were reduced in COCs of Tm-treated groups (1, 5, and 10 µg/mL). We confirmed the reducing effects of melatonin (0.1 µmol/L) on ER-stress after pretreatment with Tm (5 µg/mL; 22 hours) in maturing COCs. Addition of melatonin (0.1 µmol/L) to Tm-pretreated COCs recovered meiotic maturation rates and expression of most UPR markers. In conclusion, we confirmed a role for melatonin in the modulation of UPR signal pathways and reducing ER-stress during IVM of porcine oocytes.


Assuntos
Antioxidantes/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Meiose/efeitos dos fármacos , Melatonina/farmacologia , Oogênese/efeitos dos fármacos , Animais , Células do Cúmulo/efeitos dos fármacos , Feminino , Oócitos/efeitos dos fármacos , Suínos , Resposta a Proteínas não Dobradas/efeitos dos fármacos
11.
Cell Reprogram ; 19(4): 245-254, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28650694

RESUMO

A time-lapse monitoring system has predictive value for selecting good-quality embryos with the highest implantation potential. Using this new tool, we investigated the developmental potential and developmental kinetics of bovine parthenogenetic (PA) and two types of somatic cell nuclear transfer (NT) embryos. Bovine non-transgenic ear cells (bECs) or transgenic cells (bTGCs) were used as donor cells. The cleavage and blastocyst development rates did not significantly differ among the PA, NT-bEC, and NT-bTGC groups, and first cleavage occurred an average of 19.3 hours (n = 70), 21.6 hours (n = 60), and 21.3 hours (n = 62) after activation, respectively (20.4 hours [n = 192] for all embryos). When embryos were classified into early cleaving (≤20 hours) and late cleaving (>20 hours) groups, the blastocyst formation rate was much higher in the early cleaving groups (PA, 46%; NT-bEC, 50%; NT-bTGC, 39%) than in the late cleaving groups (PA, 18%; NT-bEC, 23%; NT-bTGC, 28%), while the percentage of embryos whose development was blocked between the two- and eight-cell stages was increased in the late cleaving groups. The percentage of embryos classified as early cleaving with a normal morphology was twofold higher in the PA group (20.0%, n = 14) than in the NT-bTGC group (9.7%, n = 6). The timing of each developmental stage varied widely; the timing of first cleavage varied from 7.6 hours in the PA group to 34.5 hours in the NT-bEC group and the timing of expanded/hatching blastocyst appearance varied from 141.6 hours in the PA group to 196.3 hours in the NT-bTGC group, differences of 26.9 and 54.7 hours, respectively (PA>NT-bEC>NT-bTGC). These results demonstrate that time-lapse monitoring provides novel data regarding individual embryo developmental kinetics and helps to predict developmental potential for improved bovine NT embryo selection based on early cleavage (≤20 hours) and normal morphology.


Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário , Técnicas de Transferência Nuclear , Partenogênese , Animais , Blastocisto/citologia , Bovinos , Feminino
12.
Asian-Australas J Anim Sci ; 30(9): 1245-1252, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28231693

RESUMO

OBJECTIVE: Phellodendron amurense (P. amurense) and Humulus japonicus (H. japonicus) are closely involved in anti-oxidative response and increasing antioxidant enzymes activities. However, the effects of their extracts on development of preimplantation bovine embryos have not been investigated. Therefore, we investigated the effects of P. amurense and H. japonicus extracts on developmental competence and quality of preimplantation bovine embryos. METHODS: After in vitro fertilization, bovine embryos were cultured for 7 days in Charles Rosenkrans amino acid medium supplemented with P. amurense (0.01 µg/mL) and H. japonicus (0.01 µg/mL). The effect of this supplementation during in vitro culture on development competence and antioxidant was investigated. RESULTS: We observed that the blastocysts rate was significantly increased (p<0.05) in P. amurense (28.9%±2.9%), H. japonicus (30.9%±1.5%), and a mixture of P. amurense and H. japonicus (34.8%± 2.1%) treated groups compared with the control group (25.4%±1.6%). We next confirmed that the intracellular levels of reactive oxygen species (ROS) were significantly decreased (p<0.01) in P. amurense and/or H. japonicus extract treated groups when compared with the control group. Our results also showed that expression of cleaved caspase-3 and apoptotic cells of blastocysts were significantly decreased (p<0.05) in bovine blastocysts derived from both P. amurense and H. japonicus extract treated embryos. CONCLUSION: These results suggest that proper treatment with P. amurense and H. japonicus extracts in the development of preimplantation bovine embryos improves the quality of blastocysts, which may be related to the reduction of ROS level and apoptosis.

13.
Asian-Australas J Anim Sci ; 29(4): 564-70, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26949958

RESUMO

The Sal-like 1 gene (Sall1) is essential for kidney development, and mutations in this gene result in abnormalities in the kidneys. Mice lacking Sall1 show agenesis or severe dysgenesis of the kidneys. In a recent study, blastocyst complementation was used to develop mice and pigs with exogenic organs. In the present study, transcription activator-like effector nuclease (TALEN)-mediated homologous recombination was used to produce Sall1-knockout porcine fibroblasts for developing knockout pigs. The vector targeting the Sall1 locus included a 5.5-kb 5' arm, 1.8-kb 3' arm, and a neomycin resistance gene as a positive selection marker. The knockout vector and TALEN were introduced into porcine fibroblasts by electroporation. Antibiotic selection was performed over 11 days by using 300 µg/mL G418. DNA of cells from G418-resistant colonies was amplified using polymerase chain reaction (PCR) to confirm the presence of fragments corresponding to the 3' and 5' arms of Sall1. Further, mono- and bi-allelic knockout cells were isolated and analyzed using PCR-restriction fragment length polymorphism. The results of our study indicated that TALEN-mediated homologous recombination induced bi-allelic knockout of the endogenous gene.

14.
Zygote ; 24(3): 442-56, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26197710

RESUMO

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine ß-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the ß-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine ß-casein gene.


Assuntos
Blastocisto/metabolismo , Sistemas CRISPR-Cas , Caseínas/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Engenharia Genética/métodos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Blastocisto/citologia , Western Blotting , Caseínas/genética , Bovinos , Linhagem Celular , Células Cultivadas , Endonucleases/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Recombinação Homóloga , Humanos , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Asian-Australas J Anim Sci ; 27(11): 1644-51, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25358326

RESUMO

Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine ß-casein gene locus using a knock-in vector for the ß-casein gene locus. We developed the knock-in vector on the porcine ß-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5' arm and 3' arm from the porcine ß-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using ß-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous ß-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.

16.
Acta Histochem ; 116(8): 1490-500, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25265880

RESUMO

Trimethyltin (TMT), a potent neurotoxic chemical, causes dysfunction and neuroinflammation in the brain, particularly in the hippocampus. The present study assessed TMT-induced glial cell activation and inflammatory cytokine alterations in the mouse hippocampus, BV-2 microglia, and primary cultured astrocytes. In the mouse hippocampus, TMT treatment significantly increased the expression of glial cell markers, including the microglial marker ionized calcium-binding adapter molecule 1 and the astroglial marker glial fibrillary acidic protein. The expression of M1 and M2 microglial markers (inducible nitric oxide synthase [iNOS] and CD206, respectively) and pro-inflammatory cytokines (interleukin [IL]-1ß, IL-6 and tumor necrosis factor [TNF]-α) were significantly increased in the mouse hippocampus following TMT treatment. In BV-2 microglia, iNOS, IL-1ß, TNF-α, and IL-6 expression increased significantly, whereas arginase-1 and CD206 expression decreased significantly after TMT treatment in a time- and concentration-dependent manner. In primary cultured astrocytes, iNOS, arginase-1, IL-1ß, TNF-α, and IL-6 expression increased significantly, whereas IL-10 expression decreased significantly after TMT treatment in a time- and concentration-dependent manner. These results indicate that significant up-regulation of pro-inflammatory signals in TMT-induced neurotoxicity may be associated with pathological processing of TMT-induced neurodegeneration.


Assuntos
Astrócitos/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Microglia/efeitos dos fármacos , Compostos de Trimetilestanho/farmacologia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microglia/citologia , Microglia/metabolismo
17.
Brain Behav Immun ; 36: 147-55, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24513875

RESUMO

Individuals with cancer are particularly susceptible to depression and cognitive impairment. However, the precise mechanisms underlying cancer-induced hippocampal dysfunction are poorly understood. We investigated the effects of a peripheral tumor on emotional behavior, hippocampus-dependent memory and associated molecular and cellular features using an experimental animal model. Behavioral alterations were examined; stress-related parameters measured; hippocampal neurogenesis evaluated; and the levels of pro-inflammatory cytokines, brain-derived neurotrophic factor (BDNF) and cyclooxygenase-2 (COX-2) assayed, 2 weeks after inoculation of adult BALB/c mice with cells of a colon carcinoma cell line (CT26). As the tumors developed, CT26-inoculated mice showed significant increases in the depression-like behavior (measured using the tail suspension test) and memory impairment (in terms of object recognition) compared with vehicle-inoculated controls. The presence of a peripheral tumor significantly elevated the hippocampal levels of mRNAs encoding interleukin-6 (IL-6) and tumor necrosis factor-α, as well as plasma IL-6 and corticosterone levels. Additionally, the adrenal glands became enlarged, and the numbers of Ki-67-positive proliferating hippocampal cells and doublecortin-positive immature progenitor neurons, as well as the constitutive levels of mRNAs encoding BDNF and COX-2 were significantly reduced. Therefore, a peripheral tumor alone may be sufficient to induce hippocampal dysfunction, possibly by reducing the rate of neurogenesis and the levels of BDNF and COX-2 in that tissue and also by increasing stress-related parameters and the circulating levels of pro-inflammatory cytokines.


Assuntos
Hipocampo/metabolismo , Neoplasias Experimentais/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Linhagem Celular Tumoral , Corticosterona/sangue , Ciclo-Oxigenase 2/metabolismo , Citocinas/sangue , Citocinas/metabolismo , Depressão/metabolismo , Feminino , Hipocampo/patologia , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neurogênese/fisiologia , Reconhecimento Psicológico/fisiologia , Estresse Fisiológico
18.
Anim Biotechnol ; 24(4): 253-67, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23947662

RESUMO

Xenotransplantation of pig organs into primates leads to hyperacute rejection (HAR). Functional ablation of the pig α 1,3-galactosyltransferase (GalT) gene, which abrogates expression of the Gal α 1-3Gal ß 1-4GlcNAc-R (Gal) antigen, which inhibits HAR. However, antigens other than Gal may induce immunological rejection by their cognate antibody responses. Ultimately, overexpression of complement regulatory proteins reduces acute humoral rejection by non-Gal antibodies when GalT is ablated. In this study, we developed a vector-based strategy for ablation of GalT function and concurrent expression of membrane cofactor protein (MCP, CD46). We constructed an MCP expression cassette (designated as MCP-IRESneo) and inserted between the left and the right homologous arms to target exon 9 of the GalT gene. Nucleofection of porcine ear skin fibroblasts using the U-023 and V-013 programs resulted in high transfection efficiency and cell survival. We identified 28 clones in which the MCP-IRESneo vector had been successfully targeted to exon 9 of the GalT gene. Two of those clones, with apparent morphologically mitotic fibroblast features were selected through long-term culture. GalT gene expression was downregulated in these 2 clones. Importantly, MCP was shown to be efficiently expressed at the cell surface and to efficiently protect cell lysis against normal human complement serum attack in vitro.


Assuntos
Galactosiltransferases/genética , Proteína Cofatora de Membrana/genética , Transfecção/métodos , Análise de Variância , Animais , Sobrevivência Celular , Células Cultivadas , Regulação para Baixo , Fibroblastos , Galactosiltransferases/metabolismo , Inativação Gênica , Vetores Genéticos/genética , Proteína Cofatora de Membrana/metabolismo , Reação em Cadeia da Polimerase , Suínos , Porco Miniatura , Transplante Heterólogo
19.
Sci Rep ; 3: 1981, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23760311

RESUMO

After the knock-out (KO) of α1,3 galactosyltransfease (Gal-T), the Hanganutziu-Deicher antigen became a major antigen of the "non-Gal antigen" that is implicated in subsequent xenograft rejection. For deletion of non-Gal antigen, we successfully produced zinc finger nuclease (ZFN)-mediated monoallelic/biallelic male and female CMP-N-acetylneuraminic acid hydroxylase (CMAH) KO miniature pigs: the efficiency of the gene targeting (41.7%) was higher when donor DNA was used with the ZFN than those of ZFN alone (9.1%). Monoallelic KO pigs had no integration of exogenous DNA into their genome, indicating that this technique would provide a new avenue to reduce the risk of antibiotics resistance when organs from genetically modified pigs are transplanted into patients. Until now, both monoallelic and biallelic CMAH KO pigs are healthy and show no sign of abnormality and off-target mutations. Therefore, these CMAH null pigs on the Gal-T KO background could serve as an important model for the xenotransplantation.


Assuntos
Animais Geneticamente Modificados , Técnicas de Inativação de Genes , Homozigoto , Oxigenases de Função Mista/genética , Suínos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Reparo do DNA por Junção de Extremidades , Feminino , Fibroblastos/metabolismo , Expressão Gênica , Ordem dos Genes , Marcação de Genes , Loci Gênicos , Vetores Genéticos , Recombinação Homóloga , Cariótipo , Masculino , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Fenótipo , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Dedos de Zinco
20.
PLoS One ; 7(9): e45812, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23049868

RESUMO

The generation of induced pluripotent stem (iPS) cells is a powerful tool in regenerative medicine, and advances in nanotechnology clearly have great potential to enhance stem cell research. Here, we introduce a liposomal magnetofection (LMF) method for iPS cell generation. Efficient conditions for generating virus-free iPS cells from mouse embryonic fibroblast (MEF) cells were determined through the use of different concentrations of CombiMag nanoparticle-DNA(pCX-OKS-2A and pCX-cMyc)-lipoplexes and either one or two cycles of the LMF procedure. The cells were prepared in a short reprogramming time period (≤ 8 days, 0.032-0.040%). Among the seven LMF-iPS cell lines examined, two were confirmed to be integration-free, and an integration-free LMF-iPS cell line was produced under the least toxic conditions (single LMF cycle with a half-dose of plasmid). This cell line also displayed in vitro/in vivo pluripotency, including teratoma formation and chimeric mouse production. In addition, the safety of CombiMag-DNA lipoplexes for the transfection of MEF cells was confirmed through lactate dehydrogenase activity assay and transmission electron microscopy. These results demonstrated that the LMF method is simple, effective, and safe. LMF may represent a superior technique for the generation of virus-free or integration-free iPS cell lines that could lead to enhanced stem cell therapy in the future.


Assuntos
Técnicas de Cultura de Células , Células-Tronco Pluripotentes Induzidas/citologia , Lipossomos/metabolismo , Animais , Células Cultivadas , DNA/química , DNA/metabolismo , Técnicas Genéticas , L-Lactato Desidrogenase/metabolismo , Magnetismo , Camundongos , Microscopia Eletrônica de Transmissão/métodos , Modelos Genéticos , Plasmídeos/metabolismo , Células-Tronco/citologia , Teratoma/metabolismo , Fatores de Tempo , Transgenes
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