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Arq. bras. med. vet. zootec. (Online) ; 73(1): 256-260, Jan.-Feb. 2021. tab, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1153048


As vantagens dos animais transgênicos têm sido demonstradas em diferentes aplicações, entretanto muitas metodologias usadas para gerar animais geneticamente modificados (GM) apresentam baixas taxas de eficiência. O objetivo deste estudo foi avaliar a entrega dos vetores lentivirais (VLs) em zigotos durante a fertilização in vitro (FIV), para gerar embriões GM, com o gene da proteína verde fluorescente (GFP) ou do fator IX de coagulação humana (FIX). Vetores lentivirais com os genes GFP (pLGW-GFP-LV) ou FIX (pLWE2-FIX-LV) foram utilizados na FIV ou na cultura de embriões in vitro (CIV). A coincubação de pLWE2-FIX-LV com espermatozoides e complexos oócitos-células do cumulus (COCs) durante a FIV diminuiu (P<0,05) as taxas de clivagem e de blastocistos, enquanto com pLGW-GFP-LV diminuiu (P<0,05) a taxa de blastocisto quando se comparou ao controle sem VL. A coincubação de pLWE2-FIX-LV e pLGW-GFP-LV com presumíveis zigotos durante a CIV não afetou (P>0,05) o desenvolvimento embrionário. A expressão da proteína GFP não foi detectada em embriões após a coincubação de FIV ou CIV, embora as células do cumulus expressassem a proteína até o dia oito de cultivo in vitro. Reações em cadeia da polimerase (PCR) não detectaram os genes GFP ou FIX em embriões, mas ambos foram detectados em células do cumulus. Assim, a coincubação de VL com espermatozoide bovino e COCs não é eficaz para produzir embriões geneticamente modificados por meio de FIV.(AU)

Animals , Cattle , Zygote , Animals, Genetically Modified/genetics , Transgenes , Embryo, Mammalian , Genetic Vectors/analysis , Fertilization in Vitro/veterinary , Gene Transfer Techniques/veterinary
Chinese Journal of Biotechnology ; (12): 2171-2180, 2020.
Article in Chinese | WPRIM | ID: wpr-878476


In most insects, polyunsaturated fatty acids (PUFAs) are mainly polyunsaturated fatty acids with a carbon-chain length less than 18 carbon atoms, hardly any long-chain polyunsaturated fatty acids such as C20 and C22 that are more valuable and bioactive. This study, by using Drosophila melanogaster (Fruit fly) as a model organism, optimized the Δ6-fatty acid elongase enzyme Elovl5 gene from mice and transferred it to fruit flies for expression. Vectors containing Elovl5 gene were successfully injected into drosophila embryo through the microscopic injection. There were enhanced green fluorescent proteins expressed in the whole developmental stage of Drosophila be means of fluorescence microscope. At the same time, expression of Elovl5 gene significantly contributed to the transformation of fruit flies C18-polyunsaturated fatty acids in the body towards the biosynthesis of longer-chain polyunsaturated fatty acids. The transgenic fruit fly model rich in long-chain polyunsaturated fatty acids such as C20 and C22 were obtained, providing a basis for further research on biosynthesis of polyunsaturated fatty acids in fruit flies.

Acetyltransferases/genetics , Animals , Drosophila melanogaster/genetics , Fatty Acid Elongases/metabolism , Fatty Acids/genetics , Gene Transfer Techniques , Mice
Article in Chinese | WPRIM | ID: wpr-828535


Mesenchymal stem cells (MSCs) have the inherent tumor-homing ability with the attraction of multiple chemokines released by tumor tissues or tumor microenvironments, which can be utilized as promising cellular carriers for targeted delivery of anti-tumor drugs and genes. In most circumstances, large amount of systemicly administrated MSCs will be firstly trapped by lungs, following with re-distribution and homing to tumor tissues after lung clearance. Several approaches like enhanced interactions between chemokines and receptors on MSCs or reducing the retention of MSCs by changes of administration methods are firstly reviewed for improving the homing of MSCs towards tumor tissues. Additionally, the potentials and gains of utilizing MSCs to carry several chemotherapeutics, such as doxorubicin, paclitaxel and gemcitabine are summarized, showing the advantages of overcoming the short half-life and poor tumor targeting of these chemotherapeutics. Moreover, the applications of MSCs to protect and deliver therapeutic genes to tumor sites for selectively tumor cells eliminating or promoting immune system are highlighted. In addition, the potentials of using MSCs for tumor-targeting delivery of diagnostic and therapeutic agents are addressed. We believed that the continuous improvement and optimization of this stem cells-based cellular delivery system will provide a novel delivery strategy and option for tumor treatment.

Antineoplastic Agents , Doxorubicin , Drug Delivery Systems , Gene Transfer Techniques , Humans , Mesenchymal Stem Cells , Neoplasms , Therapeutics , Paclitaxel , Research
Electron. j. biotechnol ; 41: 22-29, sept. 2019. ilus
Article in English | LILACS | ID: biblio-1087255


We highlight the importance of the mixed genetic approaches (classical and currents) to improve the social perception related to the GMOs acceptance. We pointed out that CRISPR/Cas9 events could carry DNA variability/rearrangements related to somaclonal variations or epigenetic changes that are independent from the editing per se. The transformation of single cells, followed by plant regeneration, is used to generate modified plants, transgenic or genome editing (CRISPR/Cas9). The incidence of undesirable somaclonal variations and/or epigenetic changes that might have occurred during in vitro multiplication and regeneration processes, must be carefully analyzed in replicates in field trials. One remarkable challenge is related to the time lapse that selects the modified elite genotypes, because these strategies may spend a variable amount of time before the results are commercialized, where in all the cases it should be take into account the genotype × environment interactions. Furthermore, this combination of techniques can create an encouraging bridge between the public opinion and the community of geneticists who are concerned with plant genetic improvement. In this context, either transgenesis or genomic editing strategies become complementary modern tools to facing the challenges of plant genetic improvement. Their applications will depend on case-by-case analysis, and when possible will necessary associate them to the schemes and bases of classic plant genetic improvement.

Plants, Genetically Modified , Gene Transfer Techniques , CRISPR-Cas Systems , Gene Editing , Transformation, Genetic , Mutagenesis , DNA Methylation , Genetic Enhancement , Epigenesis, Genetic
Braz. j. med. biol. res ; 52(5): e8108, 2019. tab
Article in English | LILACS | ID: biblio-1001521


Animal models of diseases are invaluable tools of modern medicine. More than forty years have passed since the first successful experiments and the spectrum of available models, as well as the list of methods for creating them, have expanded dramatically. The major step forward in creating specific disease models was the development of gene editing techniques, which allowed for targeted modification of the animal's genome. In this review, we discuss the available tools for creating transgenic animal models, such as transgenesis methods, recombinases, and nucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9 systems. We then focus specifically on the models of atherosclerosis, especially mouse models that greatly contributed to improving our understanding of the disease pathogenesis and we outline their characteristics and limitations.

Humans , Animals , Male , Female , Rabbits , Animals, Genetically Modified , Genetic Engineering/methods , Disease Models, Animal , Atherosclerosis/physiopathology , Transcription Activator-Like Effector Nucleases/metabolism , Gene Transfer Techniques , Biomedical Research/methods , Atherosclerosis/genetics
Article in Chinese | WPRIM | ID: wpr-812999


Gene therapy plays an important role in Alzheimer's disease (AD). In recent years, the research on gene delivery vector has gradually transferred from adenovirus vector, adeno-associated virus vector and lentivirus vector to liposomes and nanomaterial carrier systems. Graphene, the newest member of nanomaterial carrier system, has attracted extensive attention for its well permeability and biocompatibility. The methods of gene therapy can be divided into direct and indirect method. The stem cell therapy, which is the most-well studied one, belongs to the indirect method. In the gene therapy of AD, the selection of appropriate carrier and method will determine the therapeutic effect.

Alzheimer Disease , Therapeutics , Dependovirus , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors , Humans
Clinics ; 73(supl.1): e479s, 2018. graf
Article in English | LILACS | ID: biblio-952830


While cancer immunotherapy has gained much deserved attention in recent years, many areas regarding the optimization of such modalities remain unexplored, including the development of novel approaches and the strategic combination of therapies that target multiple aspects of the cancer-immunity cycle. Our own work involves the use of gene transfer technology to promote cell death and immune stimulation. Such immunogenic cell death, mediated by the combined transfer of the alternate reading frame (p14ARF in humans and p19Arf in mice) and the interferon-β cDNA in our case, was shown to promote an antitumor immune response in mouse models of melanoma and lung carcinoma. With these encouraging results, we are now setting out on the road toward translational and preclinical development of our novel immunotherapeutic approach. Here, we outline the perspectives and challenges that we face, including the use of human tumor and immune cells to verify the response seen in mouse models and the incorporation of clinically relevant models, such as patient-derived xenografts and spontaneous tumors in animals. In addition, we seek to combine our immunotherapeutic approach with other treatments, such as chemotherapy or checkpoint blockade, with the goal of reducing dosage and increasing efficacy. The success of any translational research requires the cooperation of a multidisciplinary team of professionals involved in laboratory and clinical research, a relationship that is fostered at the Cancer Institute of Sao Paulo.

Humans , Genetic Therapy/methods , Reading Frames/genetics , Interferon-beta/therapeutic use , Gene Transfer Techniques , Immunotherapy/methods , Neoplasms/therapy , Cell Death/genetics , Cyclin-Dependent Kinase Inhibitor p16/genetics , Tumor Suppressor Protein p14ARF/genetics , Neoplasms/immunology
Electron. j. biotechnol ; 30: 1-5, nov. 2017. ilus, tab, graf
Article in English | LILACS | ID: biblio-1021034


Background: The enzymes utilized in the process of beer production are generally sensitive to higher temperatures. About 60% of them are deactivated in drying the malt that limits the utilization of starting material in the fermentation process. Gene transfer from thermophilic bacteria is a promising tool for producing barley grains harboring thermotolerant enzymes. Results: Gene for α-amylase from hydrothermal Thermococcus, optimally active at 75­85°C and pH between 5.0 and 5.5, was adapted in silico to barley codon usage. The corresponding sequence was put under control of the endosperm-specific promoter 1Dx5 and after synthesis and cloning transferred into barley by biolistics. In addition to model cultivar Golden Promise we transformed three Slovak barley cultivars Pribina, Levan and Nitran, and transgenic plants were obtained. Expression of the ~50 kDa active recombinant enzyme in grains of cvs. Pribina and Nitran resulted in retaining up to 9.39% of enzyme activity upon heating to 75°C, which is more than 4 times higher compared to non-transgenic controls. In the model cv. Golden Promise the grain α-amylase activity upon heating was above 9% either, however, the effects of the introduced enzyme were less pronounced (only 1.22 fold difference compared with non-transgenic barley). Conclusions: Expression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at high temperatures.

Seeds/enzymology , Hordeum/enzymology , Thermococcus/metabolism , alpha-Amylases/metabolism , Seeds/genetics , Seeds/microbiology , Transformation, Genetic , Hordeum/genetics , Hordeum/microbiology , Beer , Enzyme Stability , Plants, Genetically Modified/enzymology , Cloning, Molecular , Gene Transfer Techniques , alpha-Amylases/genetics , Fermentation , Thermotolerance , Hot Temperature , Hydrogen-Ion Concentration
Clin. biomed. res ; 37(4): 330-333, 2017. ilus, graf
Article in English | LILACS | ID: biblio-876699


Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder caused by deficiency of alpha-L-iduronidase (IDUA). Limitations such as the need for weekly injections, high morbidity and mortality, and high cost of current treatments show that new approaches to treat this disease are required. In this study, we aimed to correct fibroblasts from a patient with MPS I using non-viral gene therapy. Using a plasmid encoding the human IDUA cDNA, we achieved stable high IDUA levels in transfected fibroblasts up to 6 months of treatment. These results serve as proof of concept that a non-viral approach can correct the enzyme deficiency in cells of patients with lysosomal storage disorders, which can be used as a research tool for a series of disease aspects. Future studies should focus on showing if this approach can be useful in small animals and clinical trials (AU)

Humans , Fibroblasts/enzymology , Gene Transfer Techniques , Genetic Vectors , Iduronidase/metabolism , Mucopolysaccharidosis I/therapy , DNA, Complementary , Genetic Therapy/methods , Iduronidase/genetics , Mucopolysaccharidosis I/genetics , Plasmids/genetics , Transfection/methods
Journal of Medicinal Plants. 2017; 16 (Supp. 10): 141-155
in Persian | IMEMR | ID: emr-185703


Background: Using Agrobacterium rhizogenes due to create hairy roots, is a useful method to increase secondary metabolites many plants

Objective: Purpose of this research is to transgenic hairy roots culture, in order to produce secondary metabolites in Datura innoxia

Methods: Explants leaf and cotyledon of Datura innoxia were inoculated for two months with A7, A4 and 15834 strains of Agrobacterium rhizogenes; Furthermore injection and Immersion methods were used in this scrutiny. The presence of T-DNA in transgenic hairy roots were confirmed by PCR. Transgenic hairy roots in liquid medium of 1/2MS were cultured. In order to induct elicitors, methyl jasmonate in tow densities of 50 micro M and 100micro M, and salicylic acid in three densities of 1mM, 0.1mM and 0.01 mM were used randomly three times. Atropine and scopolamine content of transgenic hairy roots were examined by HPLC

Results: The highest and lowest rate of transgenic hairy roots production was respectively related to the strains of A4 and 15834. Best explants for inoculation, leaf with A4 strain and cotyledon with A7 strain, were reported. With highest production rate of hairy roots, Simple deposit using a syringes method was recognized as the best method of inoculation. The effect of salicylic acid at a density of 0.1 mM increases the content of atropine concentrations. Also the results showed that usage of Methyl jasmonate at higher doses [100 micro M] reduces the content of atropine and scopolamine

Conclusion: A. rhizogenes as an appropriate method to produce hairy roots and elicitors the best treatment for increase alkaloids

Gene Transfer Techniques/trends , Agrobacterium/genetics , Transformation, Genetic , Salicylates , Oils, Volatile , Scopolamine Derivatives
Article in English | WPRIM | ID: wpr-148744


Animal models, particularly pigs, have come to play an important role in translational biomedical research. There have been many pig models with genetically modifications via somatic cell nuclear transfer (SCNT). However, because most transgenic pigs have been produced by random integration to date, the necessity for more exact gene-mutated models using recombinase based conditional gene expression like mice has been raised. Currently, advanced genome-editing technologies enable us to generate specific gene-deleted and -inserted pig models. In the future, the development of pig models with gene editing technologies could be a valuable resource for biomedical research.

Animals , Gene Expression , Gene Transfer Techniques , Mice , Models, Animal , Recombinases , Swine
Rev. chil. pediatr ; 86(4): 251-256, ago. 2015. tab
Article in Spanish | LILACS | ID: lil-764081


Introducción: Autoconcepto es el conjunto de ideas y actitudes que se tiene acerca de sí mismo. Nuestro objetivo fue evaluar si existen diferencias en el nivel de autoconcepto de niños de 8-12 años con y sin secuelas de quemaduras, e identificar variables predictoras del autoconcepto en los niños con secuelas. Pacientes y método: Estudio comparativo, transversal de 109 niños con secuelas de quemaduras de 8 a 12 años de edad, con 109 niños sin secuelas de quemaduras, del mismo grupo de edad y nivel socioeconómico. Se utilizó la escala de autoconcepto de Piers-Harris, que entrega medida de autoconcepto general y dimensiones: conductual, estatus intelectual y escolar, apariencia y atributos físicos, ansiedad, popularidad, felicidad y satisfacción. Resultados: No hubo diferencias significativas en el nivel de autoconcepto general ni en sus dimensiones al comparar ambos grupos (p > 0,05). Dentro del grupo con secuelas de quemaduras la variable número de secuelas resultó ser un factor protector para las dimensiones ansiedad, popularidad, felicidad-satisfacción y autoconcepto general. La variable localización surgió como factor de riesgo para la dimensión conductual. Discusión: La ausencia de diferencias en autoconcepto entre niños con secuelas de quemaduras y sin ellas es similar a lo reportado por la literatura. El hallazgo en factores de riesgo y protectores motiva a continuar investigando, incorporando antecedentes premórbidos y familiares.

Introduction: Self-concept is the set of ideas and attitudes that a person has about him/herself. Objective: To evaluate whether there are differences in the level of self-concept in children 8-12 years old with and without burns sequelae. To identify predictive variables of self-concept in children with sequelae. Patients and method: A comparative cross-sectional study of self-concept in 109 children with burns sequelae, from 8 to 12 years old, with 109 children without burns sequelae, and of the same age and socioeconomic status. The Piers-Harris self-concept scale is used, which provides a general measurement of self-concept and behavioural, intellectual and school status, appearance, and physical attributes, anxiety, popularity, happiness and satisfaction dimensions. Results: There were no significant differences in the level of general self-concept or their dimensions (P > .05). In the group with burns sequelae, the protective factor was the variable number of sequels was associated with the dimensions of anxiety, popularity, happiness-satisfaction and general self-concept. The location variable emerged as a risk factor for the behavioural dimension. Discussion: The absence of differences in self-concept between children with burns sequelae and children without them is similar to that reported in the literature. The finding in the risk and protective factors encourages to further research, and perhaps incorporating pre-morbidity and family background.

Humans , DNA , Quaternary Ammonium Compounds/chemistry , Serine/chemistry , Surface-Active Agents/chemistry , Amides/chemistry , Amines/chemistry , Cell Line, Tumor , Chemistry, Pharmaceutical/methods , Esters/chemistry , Gene Transfer Techniques , Genetic Therapy/methods , HeLa Cells , Lipids/chemistry , Transfection/methods
Rev. chil. pediatr ; 86(4): 287-290, ago. 2015. ilus, graf
Article in Spanish | LILACS | ID: lil-764087


Introducción: La telorragia es un síntoma poco frecuente en pacientes pediátricos, la causa más frecuente en esta población es la ectasia ductal mamaria (EDM), que es una afección benigna y autolimitada, caracterizada por la dilatación del conducto mamario, fibrosis e inflamación periductal. Objetivo: Presentar un caso de EDM, para facilitar el rápido reconocimiento por parte de los médicos, y evitar estudios y tratamientos agresivos. Caso clínico: Lactante de sexo masculino de 6 meses de edad, sano, alimentado por lactancia materna exclusiva; consultó por un nódulo retroareolar derecho y telorragia unilateral. Se realizó una ecografía Doppler que mostró una lesión multiquística, sugerente de una EDM. Se planteó tratamiento expectante y acudió a control a los 6 meses con excelente evolución. Conclusiones: La EDM es la principal causa de telorragia en niños, corresponde a una afección benigna, y la resolución generalmente es espontánea, antes de los 9 meses. Por lo que su conocimiento es de gran relevancia para el adecuado diagnóstico y manejo de estos pacientes.

Introduction: Bloody nipple discharge is an infrequent symptom during childhood. The most common cause in this population is mammary duct ectasia (MDE), which is a benign and self-limiting condition, that is characterized by dilatation of the mammary ducts, fibrosis and periductal inflammation. Objective: Report of a case of MDE in order to improve physicians’ diagnosis accuracy and avoid aggressive studies and treatments. Case report: Six-months old male healthy infant, exclusively breastfeeded, that visited our clinic with a lump beneath his right nipple and bloody discharge from the same nipple. An ultrasound was performed which showed a multicystic lesion suggestive of MDE. Watchful waiting was decided as treatment, with good evolution after six months of follow up. Conclusions: The MDE is the leading cause of bloody discharge in pediatric population, being a benign condition that resolves spontaneously before nine months. The knowledge of this condition is essential so as to accurately diagnose and treat it.

Humans , Cations/chemistry , Indicators and Reagents/chemistry , Lipids/chemistry , Polyenes/chemistry , RNA, Small Interfering/chemistry , Cell Line, Tumor , Chemistry, Pharmaceutical/methods , Gene Transfer Techniques , Genetic Vectors/genetics , HeLa Cells , Liposomes/chemistry , Luciferases/chemistry , Phospholipids/chemistry , RNA, Small Interfering/genetics , Transfection/methods
Einstein (Säo Paulo) ; 13(1): 89-95, Jan-Mar/2015. tab, graf
Article in English | LILACS | ID: lil-745883


Objective Evaluate the effects of VEGF165 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Methods Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF165 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. Results There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF165. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF165, suggesting greater tissue differentiation. Conclusion The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF165 seems to provide a protective effect in the treatment of acute myocardial infarct. .

Objetivo Avaliar os efeitos da transferência gênica do VEGF165 no processo de remodelamento da matriz extracelular após infarto agudo do miocárdio. Métodos Ratos Wistar foram submetidos ao infarto do miocárdio por ligação da artéria coronária descendente esquerda, e a fração de ejeção de ventrículo esquerdo foi utilizada para classificar os infartos em grandes e pequenos. Os animais foram divididos em grupos de dez animais, de acordo com o tamanho do infarto (grande ou pequeno), e receberam ou não tratamento com o VEGF165. A avaliação dos diferentes marcadores foi realizada por imuno-histoquímica e quantificação digital. Os anticorpos primários utilizados foram antifibronectina, antivimentina, anti- CD44, anti-E-caderina, anti-CD24, anti-alfa-1-actina e anti-PCNA. Os resultados foram representados como média e erro padrão, e analisados por ANOVA, sendo considerado estatisticamente significativo se p≤0,05. Resultados Houve aumento significativo da expressão de marcadores de células indiferenciadas, como fibronectina (proteína presente na matriz extracelular) e CD44 (glicoproteína presente nas células endoteliais). Entretanto, houve diminuição da expressão de vimentina e PCNA, indicando possível diminuição do processo de proliferação celular após o tratamento com VEGF165. Os marcadores de células diferenciadas, E-caderina (proteína de adesão entre as células do miocárdio), CD24 (proteína presente nos vasos sanguíneos) e alfa-1-actina (marcador especifico de miócitos) também apresentaram maior expressão nos grupos submetidos à terapia gênica, comparativamente com o grupo não tratado. O valor obtido pela relação entre alfa-1-actina e vimentina foi aproximadamente três vezes maior nos grupos tratados com VEGF165, indicando maior diferenciação tecidual. Conclusão O papel dos miócitos se mostrou importante no processo de remodelamento tecidual, confirmando que o VEGF165 parece conferir um efeito protetor no tratamento do infarto agudo do miocárdio. .

Animals , Female , Extracellular Matrix/physiology , Gene Transfer Techniques , Myocardial Infarction/therapy , Vascular Endothelial Growth Factor A/therapeutic use , Actins/analysis , /analysis , /analysis , Cadherins/analysis , Cell Proliferation/physiology , Disease Models, Animal , Fibronectins/analysis , Genetic Therapy/methods , Immunohistochemistry , Myocytes, Cardiac/physiology , Rats, Wistar , Reproducibility of Results , Treatment Outcome , Vascular Endothelial Growth Factor A/genetics , Vimentin/analysis
Chinese Journal of Hepatology ; (12): 837-843, 2015.
Article in Chinese | WPRIM | ID: wpr-303221


<p><b>OBJECTIVE</b>To observe targeted expression of recombinant lentivirus-mediated (Lv)-hTERTp-TK and Lv-hTERTp-tumstatin in HepG2 cells, and explore the inhibitory effect of their combination on HepG2 cells both in vitro and in vivo.</p><p><b>METHODS</b>Lv-hTERTp-TK and Lv-hTERTptumstatin were used to infect HepG2 and L02 cells at different MOIs. Transfection efficiency was observed by fluorescence microscopy. Expression of TK and tumstatin mRNA was detected by reverse-transcriptase PCR. Proliferation and apoptosis were detected by MTT and flow cytometry, respectively. The HepG2 cells were examined by real time-PCR and western blotting to determine expression level of bcl-2 and VEGF mRNA and protein.A murine hepatocellular carcinoma model was established by injecting 1 * 10(7) HepG2 cells into 30 BALB/c nude mice. The modeled mice were randomly divided into a control group, mock group, Lv-hTERTp-tumstatin group, Lv-hTERTp-TK group, and combination group for four weeks of injections at regular intervals of PBS, Lv-hTERTp-null, Lv-hTERTp-tumstatin, Lv-hTERTp-TK, and Lv-hTERTp-tumstatin plus Lv-hTERTp-TK, respectively.Changes in tumor volume and weight, and cell morphology of tumor and major organs, were assessed by hematoxylin-eosin staining.Microvascular density of tumor tissue and cell apoptosis were assessed by immunohistochemical and TUNEL staining, respectively.</p><p><b>RESULTS</b>The Lv-infected HepG2 cells, and not the Lv-infected L02 cells, expressed TK and tumstatin. Lv-hTERTp-TK and Lv-hTERTp-tumstatin, alone or in combination, inhibited proliferation and increased apoptosis of the HepG2 cells, but the combination was more effective than either alone (P less than 0.05). None of the treatments affected proliferation or apoptosis of the L02 cells (P more than 0.05). The combination also led to a greater reduction of bcl-2 and VEGF than either alone (all, P less than 0.05). Tumor growth was significantly inhibited by the combination (P less than 0.05). In vivo, the combination treatment induced the greatest amount of apoptosis of the HepG2 cells. Cell morphology of major organs such as liver, spleen and kidney were similar to the control group. The combination also produced the most significant effect on tumor microvascular density (P less than 0.05) and the highest apoptosis index (P less than 0.05).</p><p><b>CONCLUSION</b>The HTERT promoter can induce targeted expression of TK and tumstatin in HepG2 cells. Lv-hTERTp-TK combined with Lv-hTERTp-tumstatin can significantly inhibit proliferation and induce apoptosis of human HepG2 cells in vitro and in vivo, which may be related to down-regulation ofbcl-2 and VEGF.</p>

Animals , Apoptosis , Autoantigens , Genetics , Carcinoma, Hepatocellular , Therapeutics , Cell Line, Tumor , Collagen Type IV , Genetics , Down-Regulation , Flow Cytometry , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors , Hep G2 Cells , Humans , Lentivirus , Liver Neoplasms , Therapeutics , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Telomerase , Genetics , Transfection , Vascular Endothelial Growth Factor A , Metabolism
Article in English | WPRIM | ID: wpr-119877


Advantages of DNA vaccination against infectious diseases over more classical immunization methods include the possibilities for rapid manufacture, fast adaptation to newly emerging pathogens and high stability at ambient temperatures. In addition, upon DNA immunization the antigen is produced by the cells of the vaccinated individual, which leads to activation of both cellular and humoral immune responses due to antigen presentation via MHC I and MHC II molecules. However, so far DNA vaccines have shown most efficient immunogenicity mainly in small rodent models, whereas in larger animals including humans there is still the need to improve effectiveness. This is mostly due to inefficient delivery of the DNA plasmid into cells and nuclei. Here, we discuss technologies used to overcome this problem, including physical means such as in vivo electroporation and co-administration of adjuvants. Several of these methods have already entered clinical testing in humans.

Adjuvants, Immunologic , Animals , Antigen Presentation , Communicable Diseases , DNA , Electroporation , Gene Transfer Techniques , Humans , Immunity, Humoral , Immunization , Plasmids , Rodentia , Vaccination , Vaccines, DNA
Korean Journal of Urology ; : 197-204, 2015.
Article in English | WPRIM | ID: wpr-60934


PURPOSE: Electroporation is known to enhance the efficiency of gene transfer through a transient increase in cell membrane permeability. The aim of this study was to determine the optimal conditions for in vivo electroporation-mediated gene delivery into mouse corpus cavernosum. MATERIALS AND METHODS: Diabetes was induced in C57BL/6 mice by intraperitoneal injections of streptozotocin. After intracavernous injection of pCMV-Luc (100 microg/40 microL), different electroporation settings (5-50 V, 8-16 pulses with a duration of 40-100 ms) were applied to the penis to establish the optimal conditions for electroporation. Gene expression was evaluated by luciferase assay. We also assessed the undesired consequences of electroporation by visual inspection and hematoxylin-eosin staining of penile tissue. RESULTS: Electroporation profoundly induced gene expression in the corpus cavernosum tissue of normal mice in a voltage-dependent manner. We observed electrical burn scars in the penis of normal mice who received electroporation with eight 40-ms pulses at a voltage of 50 V and sixteen 40-ms pulses, eight 100-ms pulses, and sixteen 100-ms pulses at a voltage of 30 V. No detectable burn scars were noted in normal mice stimulated with eight 40-ms pulses at a voltage of 30 V. Electroporation also significantly induced gene expression in diabetic mice stimulated with 40-ms pulse at a voltage of 30 V without injury to the penis. CONCLUSIONS: We have established the optimal electroporation conditions for maximizing gene transfer into the corpus cavernosum of mice while avoiding damage to the erectile tissue. The electroporation-mediated gene delivery technique will be a valuable tool for gene therapy in the field of erectile dysfunction.

Animals , Diabetes Mellitus, Experimental/complications , Electroporation/methods , Erectile Dysfunction/therapy , Gene Expression , Gene Transfer Techniques , Genes, Reporter , Genetic Therapy/methods , Luciferases/metabolism , Male , Mice , Mice, Inbred C57BL , Penile Erection/physiology , Penis/physiopathology , Transfection
Rev. Assoc. Med. Bras. (1992) ; 60(6): 520-524, Nov-Dec/2014.
Article in English | LILACS | ID: lil-736322


Methods and techniques employed in gene therapy are reviewed in parallel with pertinent ethical conflicts. Clinical interventions based on gene therapy techniques preferentially use vectors for the transportation of therapeutic genes, however little is known about the potential risks and damages to the patient. Thus, attending carefully to the clinical complications arising as well as to security is essential. Despite the scientific and technological advances, there are still many uncertainties about the side effects of gene therapy. Moreover, there is a need, above all, to understand the principles of bioethics as both science and ethics, in accordance with its socioecological responsibility, in order to prioritize the health and welfare of man and nature, using properly natural resources and technology. Therefore, it is hard to determine objective results and to which extent the insertion of genes can affect the organism, as well as the ethical implication.

Métodos e técnicas empregadas na terapia gênica são revisados em paralelo a conflitos éticos pertinentes. Intervenções clínicas com base em técnicas de terapia gênica são usadas preferencialmente em vetores para o transporte de genes terapêuticos; porém, pouco se sabe sobre os possíveis riscos e danos para o paciente, sendo necessário atender cuidadosamente às complicações clínicas resultantes, bem como à segurança. Apesar dos avanços científicos e tecnológicos relacionados à terapia gênica, ainda há muitas incertezas sobre os efeitos colaterais do uso dessa terapia. Além disso, é necessário, acima de tudo, compreender os princípios da bioética como uma ética da ciência para com a responsabilidade socioecológica, a fim de priorizar a saúde e o bem-estar do homem e da natureza, utilizando adequadamente recursos naturais e tecnologia. Portanto, é difícil afirmar qual é o rendimento real, bem como os resultados do aumento da genética inserida no organismo e as implicações éticas.

Humans , Bioethical Issues , Gene Transfer Techniques , Genetic Therapy , Attitude of Health Personnel , Patient Safety , Risk Assessment/standards , Social Responsibility
Braz. j. microbiol ; 45(3): 785-789, July-Sept. 2014. tab
Article in English | LILACS | ID: lil-727003


Streptococcus agalactiae (GBS) is a major source of human perinatal diseases and bovine mastitis. Erythromycin (Ery) and tetracycline (Tet) are usually employed for preventing human and bovine infections although resistance to such agents has become common among GBS strains. Ery and Tet resistance genes are usually carried by conjugative transposons (CTns) belonging to the Tn916 family, but their presence and transferability among GBS strains have not been totally explored. Here we evaluated the presence of Tet resistance genes (tetM and tetO) and CTns among Ery-resistant (Ery-R) and Ery-susceptible (Ery-S) GBS strains isolated from human and bovine sources; and analyzed the ability for transferring resistance determinants between strains from both origins. Tet resistance and int-Tn genes were more common among Ery-R when compared to Ery-S isolates. Conjugative transfer of all resistance genes detected among the GBS strains included in this study (ermA, ermB, mef, tetM and tetO), in frequencies between 1.10-7 and 9.10-7, was possible from bovine donor strains to human recipient strain, but not the other way around. This is, to our knowledge, the first report of in vitro conjugation of Ery and Tet resistance genes among GBS strains recovered from different hosts.

Animals , Cattle , Humans , Conjugation, Genetic , Gene Transfer Techniques , Streptococcus agalactiae/genetics , Anti-Bacterial Agents/pharmacology , DNA Transposable Elements , Drug Resistance, Bacterial , Erythromycin/pharmacology , Streptococcal Infections/microbiology , Streptococcal Infections/veterinary , Streptococcus agalactiae/drug effects , Streptococcus agalactiae/isolation & purification , Tetracycline/pharmacology