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1.
Chinese Journal of Biotechnology ; (12): 2283-2292, 2021.
Article in Chinese | WPRIM | ID: wpr-887796

ABSTRACT

Immunotherapy is becoming an effective and less invasive strategy that can be applied to the treatment of various malignancies. Lentiviral vectors (LVs) have shown great potential in immunotherapy as they can stably integrate relatively large foreign DNA, and effectively transduce dividing and non-dividing cells. Clinical application needs high quality LVs, and therefore strict quality control of the final products is necessary to ensure their purity, efficacy and safety. The quantitative detection of LVs is among the key parts of product development and quality control. In this paper, the existing methods for quantitative detection of LVs are summarized, including fluorescence activated cell sorter (FACS), P24 enzyme-linked immuno sorbent assay (P24 ELISA), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing(TRPS) and virus counter(VC).Their advantages and disadvantages are listed, and future development and challenges are discussed.


Subject(s)
Genetic Vectors/genetics , Humans , Immunotherapy , Lentivirus/genetics , Neoplasms , Transduction, Genetic
2.
Braz. j. med. biol. res ; 53(3): e8876, 2020. graf
Article in English | LILACS | ID: biblio-1089338

ABSTRACT

The immune stimulatory and anti-neoplastic functions of type I interferon have long been applied for the treatment of melanoma. However, the systemic application of high levels of this recombinant protein is often met with toxicity. An approach that provides localized, yet transient, production of type I interferon may overcome this limitation. We propose that the use of mesenchymal stem cells (MSCs) as delivery vehicles for the production of interferon-β (IFNβ) may be beneficial when applied together with our cancer gene therapy approach. In our previous studies, we have shown that adenovirus-mediated gene therapy with IFNβ was especially effective in combination with p19Arf gene transfer, resulting in immunogenic cell death. Here we showed that MSCs derived from mouse adipose tissue were susceptible to transduction with adenovirus, expressed the transgene reliably, and yet were not especially sensitive to IFNβ production. MSCs used to produce IFNβ inhibited B16 mouse melanoma cells in a co-culture assay. Moreover, the presence of p19Arf in the B16 cells sensitizes them to the IFNβ produced by the MSCs. These data represent a critical demonstration of the use of MSCs as carriers of adenovirus encoding IFNβ and applied as an anti-cancer strategy in combination with p19Arf gene therapy.


Subject(s)
Animals , Male , Rabbits , Melanoma, Experimental/therapy , Interferon-beta/metabolism , Cyclin-Dependent Kinase Inhibitor p16/administration & dosage , Mesenchymal Stem Cells/metabolism , Transduction, Genetic , Melanoma, Experimental/metabolism , Genetic Therapy , Cell Line, Tumor , Cell Proliferation , Disease Models, Animal , Mice, Inbred C57BL
3.
Electron. j. biotechnol ; 41: 9-12, sept. 2019. tab, ilus
Article in English | LILACS | ID: biblio-1053556

ABSTRACT

Background: Horizontal gene transfer (HGT) is the most important mechanism in the evolution of new genetic capabilities in bacteria, including specific degradative pathways, virulence factors, and resistance to antibiotics. Among the processes involved in HGT, transduction is noteworthy. This is a mechanism for gene transmission mediated by a bacteriophage that functions both as a reservoir and as a vector of exogenous genes, which remain protected from environmental effects in the bacteriophage's capsid. Within this context, this investigation aimed to evaluate the ability of the generalized transducing bacteriophage P1 to productively infect and transduce in the bacterial species Salmonella bongori. Results: We could establish that a derivative of bacteriophage P1, P1Cm, infects strains of S. bongori with frequencies of lysogenization in the order of ~10−2 lysogens/UFP. Through thermal induction, infective viral progeny was obtained, and we could show that P1Cm readily formed plaques on S. bongori lawns, a phenomenon thus far not reported for other members of the genus Salmonella. Finally, we showed P1Cm-mediated transduction of the model plasmid RP4 at frequencies of ~10−7 transductants/donor. Conclusion: Therefore, bacteriophage P1 can be used as a tool for the genetic manipulation in the species S. bongori.


Subject(s)
Salmonella , Transduction, Genetic , Bacteriophage P1/genetics , Bacteriophage P1/pathogenicity , Capsid , Gene Transfer, Horizontal , Escherichia coli , Lysogeny
4.
Chinese Journal of Biotechnology ; (12): 1307-1316, 2019.
Article in Chinese | WPRIM | ID: wpr-771798

ABSTRACT

Gene therapy is a rapidly developing field. The most widely used technique for foreign gene transfer is lentiviral-mediated gene therapy. Lentiviral vector has been developed from the first generation to the third generation in terms of safety. The preparation of lentiviruses with high titer remains difficult. In this study, a Fibra-Cel sheet carrier was used as an HEK293T cell carrier matrix, and several sterile cell culture spinners were combined and cultured on a roller bottle machine to scale up the adherent cells. The virus titer was maximized by screening the factors to optimize the lentivirus titer in the third-generation lentivirus packaging process one by one. Fibra-Cel sheet vector was successfully used as the matrix of HEK293T cell adhesion to culture adherent cells at large scale. The optimal conditions for large-scale preparation of the third-generation lentivirus by bottle roller were screened and three batches of lentiviruses were produced on pilot scale. The production time of lentivirus was shortened from 120 hours to 54 hours from plasmid transfection to virus collection; in terms of cost, a rolling bottle machine was used instead of a bioreactor, leading to lower cost and no need for repeated sterilization during the whole process. The safe, effective and low-cost operation of successful production will provide a technical base for the large-scale preparation of lentivirus and thus lay a firm foundation for its clinical application.


Subject(s)
Genetic Vectors , HEK293 Cells , Humans , Lentivirus , Transduction, Genetic , Transfection
5.
Neuroscience Bulletin ; (6): 485-496, 2018.
Article in English | WPRIM | ID: wpr-777034

ABSTRACT

The GABAergic neurons in the parafacial zone (PZ) play an important role in sleep-wake regulation and have been identified as part of a sleep-promoting center in the brainstem, but the long-range connections mediating this function remain poorly characterized. Here, we performed whole-brain mapping of both the inputs and outputs of the GABAergic neurons in the PZ of the mouse brain. We used the modified rabies virus EnvA-ΔG-DsRed combined with a Cre/loxP gene-expression strategy to map the direct monosynaptic inputs to the GABAergic neurons in the PZ, and found that they receive inputs mainly from the hypothalamic area, zona incerta, and parasubthalamic nucleus in the hypothalamus; the substantia nigra, pars reticulata and deep mesencephalic nucleus in the midbrain; and the intermediate reticular nucleus and medial vestibular nucleus (parvocellular part) in the pons and medulla. We also mapped the axonal projections of the PZ GABAergic neurons with adeno-associated virus, and defined the reciprocal connections of the PZ GABAergic neurons with their input and output nuclei. The newly-found inputs and outputs of the PZ were also listed compared with the literature. This cell-type-specific neuronal whole-brain mapping of the PZ GABAergic neurons may reveal the circuits underlying various functions such as sleep-wake regulation.


Subject(s)
Animals , Axons , Physiology , Brain , Brain Mapping , Brain Stem , Cell Biology , GABAergic Neurons , Physiology , Green Fluorescent Proteins , Genetics , Metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neural Pathways , Physiology , Peptide Elongation Factor 1 , Genetics , Metabolism , Rabies virus , Genetics , Metabolism , Transduction, Genetic , Vesicular Inhibitory Amino Acid Transport Proteins , Genetics , Metabolism
6.
Electron. j. biotechnol ; 19(4): 75-80, July 2016. ilus
Article in English | LILACS | ID: lil-793956

ABSTRACT

Background: Using recombinant adeno-associated virus 2 (rAAV-2), we attempted to establish a HEK293T cell line that is able to site-specifically integrate and stably express glial cell line-derived neurotrophic factor (GDNF). Results:Recombinant vector with enhanced green fluorescent protein (EGFP) and GDNF (pTR-P5-EGFP-IRES-GDNF), as well as that carrying Rep genes and SV40 promoters (pSVAV2) were constructed and packed. HEK293T cells were co-infected with rAAV-2/EGFP-GDNF and rAAV-2/SVAV2 virus separately at 1 x 10(4),1 x 10(5),and 1x10(6) of multiplicity of infection (MOI). The efficiency of transduction was detected using flow cytometry. Additionally, the infected HEK293T cells were separately validated by touchdown polymerase chain reaction (PCR) and Western-blot. After 72 h of transduction, the rate of EGFP positive cell was 22%, 45% and 49% at the MOIs of 1 x 10(4),1 x 10(5) and 1 x 10(6), respectively. On the 3rd, 6th and 9th day of cell passage, there was no significant difference in the cell viability and proliferation rate between transduction and control groups. Importantly, touchdown PCR showed that there was a specific PCR amplified product band in the lane of infected cells. Furthermore, GDNF expression was detected in the infected cells after 15 and 180 d of cultivation. Conclusions: A HEK293T cell line able to site-specifically integrate and stably express GDNF was established.


Subject(s)
Dependovirus , Glial Cell Line-Derived Neurotrophic Factor , HEK293 Cells , Recombination, Genetic , Transduction, Genetic , Cell Line , Polymerase Chain Reaction , Green Fluorescent Proteins , Genetic Vectors , Microscopy, Fluorescence
7.
Article in Chinese | WPRIM | ID: wpr-353117

ABSTRACT

<p><b>OBJECTIVE</b>To determine the effect of AQP1 gene on facial nerve edema following injury through investigation of the relationship between the expression of AQP1 gene and Schwann cells swelling.</p><p><b>METHODS</b>The AQP1 expression in Schwann cells of mouse facial nerve tissues was detected by immunofluorescent staining. The transgenic protocol by lentivirus transduction was used to specifically upregulate AQP1 expression in Schwann cells. Lenti-AQP1 and CTRL (empty vector) transduced cells were observed during gene overexpression every 24 h for 6 days by using phase contrast microscopy. Cell volume of CTRL and Lenti-AQP1 treated cells was measured daily from the day of treatment, through day 6.</p><p><b>RESULTS</b>Schwann cell primary cultures maintained a high level of AQP1 water channels, representing an ideal cell model to study the role of AQP1 in the facial nerve. The expression of AQP1 mRNA and protein in Schwann cells infected with the Lenti-AQP1 was increased significantly compared with CTRL lentivirus (P < 0.05). Lenti-AQP1 caused cell swelling in cultured Schwann cells, as validated by cell volume determinations (P < 0.01).</p><p><b>CONCLUSIONS</b>AQP1 is an important factor responsible for the fast water transport of cultured Schwann cells. It plays an important role in facial nerve edema.</p>


Subject(s)
Animals , Aquaporin 1 , Genetics , Metabolism , Cell Size , Edema , Facial Nerve , Metabolism , Facial Nerve Diseases , Lentivirus , Mice , RNA, Messenger , Metabolism , Schwann Cells , Cell Biology , Metabolism , Virology , Time Factors , Transduction, Genetic , Methods , Up-Regulation
8.
Chinese Journal of Virology ; (6): 348-356, 2015.
Article in Chinese | WPRIM | ID: wpr-296277

ABSTRACT

Pigs are increasingly recognized as "natural" hosts of infection by human respiratory viruses because of their similarities to humans in terms of lung physiology, airway morphology, cell types, and distribution of cell receptors in the respiratory tract. We wished to explore the mechanisms of infection by respiratory viruses and screening of drug that could be used to treat respiratory-system diseases. Hence, we developed a model of well-differentiated porcine airway epithelial cells (PAECs) derived from pig-lung tissue and cultured them with serum-free medium under an air-liquid interface condition in vitro. We identified the PAEC model using scanning electron microscopy, electrophysiology, and immunohistology. To evaluate application of gene therapy of adeno-associated virus (AAV)6 on the PAEC model, we generated recombinant adeno-associated virus 6-green fluorescent protein (rAAV6-GFP) using the three-plasmid transfection method and infected PAECs from the apical surface with rAAV6-GFP. Results demonstrated that the PAEC model comprised a multilayer epithelial structure containing ciliated mucous secretory cells, with basal cells located directly beneath the multilayer. rAAV6-GFP could infect PAECs from the apical surface and efficiently transduce PAECs to mediate the long-term expression of the exogenous gene. Establishment of a model of well-differentiated PAECs in vitro could lay a solid foundation for the study of infection by respiratory pathogens, as well as the screening and gene therapy of agents used to treat diseases of the respiratory system.


Subject(s)
Animals , Cell Differentiation , Dependovirus , Genetics , Epithelial Cells , Cell Biology , Metabolism , Green Fluorescent Proteins , Genetics , HEK293 Cells , Humans , Lung , Cell Biology , Membrane Potentials , Mucins , Metabolism , Swine , Transduction, Genetic , Tubulin , Metabolism
9.
Article in Chinese | WPRIM | ID: wpr-312580

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the cytotoxicity of normal CD8(+) T lymphocytes retrovirally transduced with WT1 peptide-specific T-cell receptor (TCR) genes against human lung cancer cells.</p><p><b>METHODS</b>HLA-A*2402-restricted and WT1 peptide-specific TCR-α/β genes were cloned from a cytotoxic T lymphocyte clone and inserted into a retroviral TCR expression vector. The cytotoxicity of normal peripheral CD8⁺ T cells transduced with the WT1-TCR genes against human lung cancer cells was evaluated using a standard ⁵¹Cr release assay.</p><p><b>RESULTS</b>The WT1-TCR gene-modified T cells recognized the peptide-pulsed target cells but not the non-pulsed cells. TCR-redirected CD8⁺ T cells lysed WT1-overexpressing human lung cancer cells in an HLA-A*2402-restricted manner, but did not kill normal cells positively expressing HLA-A*2402.</p><p><b>CONCLUSION</b>These data demonstrate the feasibility of adoptive immunotherapy with TCR-redirected T cell for the treatment of lung cancer.</p>


Subject(s)
CD8-Positive T-Lymphocytes , Cell Biology , Cell Line, Tumor , Genes, T-Cell Receptor , Humans , Immunotherapy, Adoptive , Lung Neoplasms , Pathology , Peptides , Receptors, Antigen, T-Cell, alpha-beta , Genetics , Retroviridae , T-Lymphocytes, Cytotoxic , Cell Biology , Transduction, Genetic , WT1 Proteins , Genetics
10.
Article in English | WPRIM | ID: wpr-331129

ABSTRACT

This study investigated the effects of miRNA-155 on malignant biological characteristics of NK/T-cell lymphoma cell lines and the possible mechanism. The expression of miRNA-155 was detected in lymphoma cell lines from different sources (SNK-6, YTS, Jurkat and DOHH2) by real-time PCR. Lentiviral vectors (pLL3.7) that could overexpress or downexpress miRNA-155 were constructed. Recombinant lentiviral particles were prepared and purified, and their titers determined. The expression of miRNA-155 in the infected SNK-6 cells and the cell proliferation were detected by PCR and CCK-8, respectively. Flow cytometry was used to determine the apoptosis of infected SNK-6 cells. The target of miRNA155 was predicted from Targetscan website. The effect of miRNA155 on FOXO3a expression was examined by Western blotting. The results showed that among the human NK/T-cell lymphoma cell lines SNK-6, YTS, Jurkat and DOHH2, the expression of miRNA-155 was highest in SNK-6. The infection efficiency of the recombinant lentivirus in SNK-6 was more than 70% at multiplicity of infection (MOI) of 100. The expression of miRNA-155 was significantly increased in SNK-6 cells infected by lentivirus vectors with high expression of miRNA-155 (4 times higher than the control group), and profoundly decreased in those infected with lentiviruses with low expression of miRNA-155. The proliferation of letivirus-infected SNK-6 cells was decreased as the expression of miRNA-155 reduced. The apoptosis rate was increased with the reduction in the expression of miRNA-155. FOXO3a was found to be a possible target of miRNA155, as suggested by Targetscan website. Western blotting showed that the expression of FOXO3a was significantly elevated in SNK-6 cells with miRNA-155 inhibition. It was concluded that reduction in miRNA-155 expression can inhibit the proliferation of SNK-6 lymphoma cells and promote their apoptosis, which may be associated with regulation of FOXO3a gene.


Subject(s)
Apoptosis , Genetics , Forkhead Box Protein O3 , Forkhead Transcription Factors , Genetics , Metabolism , Gene Expression Regulation, Neoplastic , Humans , Jurkat Cells , Lymphoma, T-Cell , Genetics , Metabolism , Pathology , MicroRNAs , Genetics , Natural Killer T-Cells , Metabolism , Pathology , Neoplasm Proteins , Genetics , Metabolism , RNA, Neoplasm , Genetics , Transduction, Genetic
11.
Chinese Journal of Hepatology ; (12): 260-265, 2014.
Article in Chinese | WPRIM | ID: wpr-252236

ABSTRACT

<p><b>OBJECTIVE</b>To generate a mouse model of chronic hepatitis B (CHB) infection by performing in vivo transduction of hepatitis B virus (HBV) covalently closed circular (ccc)DNA.</p><p><b>METHODS</b>Nude mice were injected with HBV cccDNA at doses of 1.5, 1.0 or 0.5 mug/ml. A control group was generated by giving equal injection volumes of physiological saline. The serum levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) on post-injection days 1 and 3, weeks 1-6, 8 and 10 were assayed by reflection immunoassay. At post-injection week 10, all animals were sacrificed and liver tissues were collected. Copies of HBV DNA in serum and liver tissue were detected by real-time PCR. HBV antigens in liver tissue were detected of by immunohistochemistry. Pathological analysis of liver tissue carried out with hematoxylin-eosin staining. Linear correlation of data was determined by statistical analysis.</p><p><b>RESULTS</b>HBsAg and HBeAg were detected in sera from all three groups of cccDNA-injected mice staring at post-injection day 1 and lasting through week 10. The levels of HBsAg over the 10-week period showed two patterns of increase-decrease;the lowest level was detected at week 4 and the highest level was detected at week 8. In contrast, the levels of HBeAg over the 10-week period showed three patterns of increase-decrease; the lower levels were detected at weeks 2 and 4 and the higher levels at weeks 3 and 6. HBV DNA copies in liver tissues showed a cccDNA dose-dependent descending trend over the 10-week study period (1.5 mug/ml:1.14E+07 ± 6.51E+06 copies/g, 1.0 mug/ml:9.81E+06 ± 9.32E+06 copies/g, and 0.5 mug/ml:3.72E+06 ± 2.35E+06 copies/g; Pearson's r =0.979). HBV DNA copies in sera showed the pattern of 1.0 mug/ml cccDNA more than 1.5 mug/ml cccDNA more than 0.5 mug/ml cccDNA, and in general were higher than those detected in the liver tissues. Liver tissues from all cccDNA-injected mice showed positive immunohistochemistry staining for both HBsAg and HBeAg. HE staining showed that the liver tissues of all cccDNA-injected mice had severe fatty and vacuolar degeneration and less obvious structure of liver lobules (compared to the liver tissues from control mice).</p><p><b>CONCLUSION</b>The CHB mouse model successfully established in this study by in vivo transduction of HBV cccDNA may represent a useful tool to study the pathogenic mechanisms and potential antiviral treatments of human CHB.</p>


Subject(s)
Animals , DNA, Circular , DNA, Viral , Disease Models, Animal , Hepatitis B Surface Antigens , Blood , Hepatitis B e Antigens , Blood , Hepatitis B virus , Genetics , Physiology , Hepatitis B, Chronic , Virology , Male , Mice , Mice, Nude , Transduction, Genetic , Virus Replication
12.
São Paulo; s.n; s.n; 2014. 102 p. tab, graf, ilus.
Thesis in Portuguese | LILACS | ID: biblio-847313

ABSTRACT

Sabe-se há décadas que mutações nos genes ras estão presentes em cerca de 20% dos cânceres humanos, mas o desenvolvimento de terapias eficazes para o tratamento de câncer dependente dos oncogenes ras permanece um desafio científico importante. Nesse contexto, o nosso grupo publicou recentemente resultados interessantes mostrando que FGF2 exógeno ou PMA, contrariamente à expectativa geral, inibem a proliferação de células de camundongo malignas dependentes dos oncogenes H- ou K-Ras. Para dar continuidade a estes estudos o projeto desta tese foi planejado para investigar os mecanismos subjacentes a possíveis efeitos citotóxicos de FGF2 e PMA em células humanas transformadas por ras. Para esse fim, a linhagem humana imortalizada HEK 293 foi condicionalmente transformada pela expressão ectópica da construção quimérica de DNA ER:H-rasV12, que codifica a oncoproteína de fusão ER:H-RasV12, cuja atividade é induzível por 4-hidroxi-tamoxifen (4OHT). Essa abordagem nos permitiu verificar os efeitos de FGF2 e PMA em sublinhagens HEK/ER:HrasV12 fenotipicamente "normais" ou transformadas por níveis crescentes da oncoproteína H-RasV12. Os principais resultados mostraram que tanto FGF2 como PMA tem efeito dual promovendo ou inibindo a proliferação das células transformadas em função da concentração intracelular crescente de H-RasV12. Ensaios de crescimento de colônias em suspensão de agarose mostraram que: a) as células parentais HEK293 não desenvolveram colônias mesmo quando tratadas com FGF2 ou PMA, resultados que estão de acordo com seu fenótipo não tumoral; b) mas, as sublinhagens HEK/ER:HrasV12 deram origem a colônias mesmo quando tratadas com concentrações pequenas de 4OHT, que condicionaram níveis intracelulares baixos de ER:HRasV12; nestas condições experimentais, FGF2 foi um forte promotor do crescimento de colônias, condizente com sua reconhecida atividade promotora do crescimento de células tumorais em suspensão; ainda nestas condições, PMA não teve efeito significante sobre o crescimento de colônias; c) coerentemente, concentrações elevadas de 4-OHT levaram aos níveis intracelulares mais altos de ER:HRasV12 e, por conseguinte, a desenvolvimento máximo de colônias de células HEK/ER:HrasV12, no entanto, nestas condições, ambos FGF2 e PMA inibiram completamente o crescimento de colônias. Por outro lado, transformação de HEK293 com um vetor de expressão constitutiva de HrasV12 levou à seleção e isolamento das sublinhagens tumorais HEK/HrasV12, cujo fenótipo se caracterizou por: a) nenhum efeito de FGF2 sobre a sua proliferação e b) forte inibição de sua proliferação por PMA. A ação citotóxica de PMA exclusivamente observada em células HEK 293 transformadas por H-rasV12 se caracterizou por: a) total dependência de PKC, provavelmente mediada pela ativação proteolítica específica de PKC δ; b) envolvimento de níveis elevados e sustentados de ROS com disparo tardio de apoptose


It is known for nearly 20 years that mutated ras oncogenes are found in 20% of human malignancies, however efficacious therapies are not yet available for Ras-driven cancer. Along of these lines, our group recently published provocative results showing, against common belief, that FGF2 and PMA inhibited proliferation of Ras-dependent malignant mouse cells. Aiming to gain insight into this intriguing phenomenon, the present thesis project was planned to investigate the possible cytotoxicity of FGF2 and PMA in human Ras-driven malignant cells. To this end an immortalized non-tumorigenic human cell line (HEK293) was stably transformed with the DNA construction ER:H-rasV12, which encodes the fusion protein ER:H-RasV12, whose activity requires activation by 4-hidroxitamoxifen (4-OHT). This approach allowed us to evaluate FGF2 and PMA effects on HEK/ER:HrasV12 sublines under switching from "normal" to transformed phenotypes upon 4-OHT induction. Our main results have shown that both FGF2 and PMA displayed dual effects promoting or inhibiting proliferation of HEK/ER:HrasV12 cells in function of ER:HRasV12 intracellular levels. Clonogenic assays in agarose suspension have shown: a) parental HEK293 line did not develop colonies under FGF2 and PMA treatment or not, in agreement with its non-tumorigenic nature; b) however, HEK/ER:HrasV12 sublines developed colonies even under low 4-OHT concentrations, which led to low ER:HRasV12 intracellular levels; under these conditions FGF2 strongly promoted colony growth and PMA had no effect; c) furthermore, in HEK/ER:HrasV12 sublines, elevated 4-OHT concentrations led to high ER:HRasV12 intracellular levels and maximal colony growth; but, under these experimental conditions both FGF2 and PMA abolished colony growth. On the other hand, HEK293 transformation with a vector that constitutively express HrasV12 yielded HEK/ER:HrasV12 sublines displaying the following phenotypic traits: a) non FGF2 effects on proliferation and b) severe proliferation inhibition by PMA. PMA toxicity, exclusively observed in HrasV12 -transformed HEK293 cells, was characterized by: a) total dependency on PKC, likely mediated by specific proteolytic activation of PKCδ; b) involvement of high and sustained ROS levels correlated with late apoptosis triggering


Subject(s)
Animals , Male , Female , Mice , Fibroblast Growth Factor 2/analysis , Genes, ras/genetics , HEK293 Cells , Neoplasms/complications , Cell Culture Techniques/methods , Flow Cytometry/methods , Reverse Transcription/genetics , Tamoxifen , Transduction, Genetic/methods
13.
Article in English | WPRIM | ID: wpr-243219

ABSTRACT

Induced pluripotent stem (iPS) cells were originally generated from mouse fibroblasts by enforced expression of Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc). The technique was quickly reproduced with human fibroblasts or mesenchymal stem cells. Although having been showed therapeutic potential in animal models of sickle cell anemia and Parkinson's disease, iPS cells generated by viral methods do not suit all the clinical applications. Various non-viral methods have appeared in recent years for application of iPS cells in cell transplantation therapy. These methods mainly include DNA vector-based approaches, transfection of mRNA, and transduction of reprogramming proteins. This review summarized these non-viral methods and compare the advantages, disadvantages, efficiency, and safety of these methods.


Subject(s)
Animals , Cellular Reprogramming , Humans , Induced Pluripotent Stem Cells , Physiology , Transduction, Genetic , Transfection , Transgenes
14.
Chinese Journal of Biotechnology ; (12): 1696-1700, 2013.
Article in Chinese | WPRIM | ID: wpr-242421

ABSTRACT

Corynebacterium glutamicum SA001 is a mutant with lactate dehydrogenase (ldhA) deletion. In order to increase metabolic flux from isocitrate to succinate, and to improve the production of succinate under anaerobic conditions,we transducted the gene aceA coding isocitrate lyase (ICL) from Escherichia coli K12 into Corynebacterium glutamicum SA001 (SA001/pXMJ19-aceA). After 12 h aerobic induction by adding 0.8 mmol/L of IPTG, the recombinant strain was transferred to anaerobic fermentation for 16 h. Succinate reached 14.84 g/L, with a productivity of 0.83 g/(L x h). Compared to C. glutamicum SA001, the activity of ICL of the recombinant strain was increased 5.8-fold, and the succinate productivity was increased 48%. Overexpression of isocitrate lyase will increase the metabolic flux of glyoxylate bypass flowing to succinate.


Subject(s)
Corynebacterium glutamicum , Genetics , Metabolism , Escherichia coli , Genetics , Gene Deletion , Industrial Microbiology , Isocitrate Lyase , Genetics , L-Lactate Dehydrogenase , Genetics , Succinic Acid , Metabolism , Transduction, Genetic
15.
Article in Chinese | WPRIM | ID: wpr-233266

ABSTRACT

We evaluated the anti-HBV effects of nucleotide analogues, Entecavir (ETV) and Lamivudine (LAM) targeting mouse model of HBV persistent infection with recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome (rAAV8-1.3HBV). Ninety percent (27 of 30 mice) of rAAVS-treated mice were chosen as mouse model. Four groups were orally administrated with different doses of ETV (1 mg/(kgd) or 0.1 mg/(kgd)) and LAM (500 mg/(kgd) or 100 mg/(kgd)) once a day for 10 days. The other two groups were set as normal saline treated and untreated control. We detected the levels of HBV DNA, HBeAg and HBsAg in sera at different time. Results indicate that HBV DNA level decreased significantly (P < 0.05) in drug-treated groups compared with normal saline group after drug administration. Fifteen days after the drug withdrawal, HBV DNA level rebounded back obviously (P < 0.05) in groups with low doses of ETV and LAM. However, there was no apparent change of HBeAg and HBsAg in the whole process among all groups. These results showed that our model could reflect the anti-viral effect of nucleotide analogues. This model can be a useful and convenient tool for anti-HBV drug discovery.


Subject(s)
Animals , Antiviral Agents , Pharmacology , Dependovirus , Genetics , Metabolism , Disease Models, Animal , Genetic Vectors , Genome, Viral , Guanine , Pharmacology , Hepatitis B Antibodies , Blood , Hepatitis B virus , Genetics , Physiology , Hepatitis B, Chronic , Virology , Lamivudine , Pharmacology , Mice , Mice, Inbred C57BL , Nucleotides , Pharmacology , Transduction, Genetic , Virus Replication
16.
Chinese Journal of Biotechnology ; (12): 235-242, 2013.
Article in Chinese | WPRIM | ID: wpr-233251

ABSTRACT

Adeno-associated virus (AAV) has many advantages for gene therapy over other vector systems. However, after the production of recombinant AAV (Raav) vectors, the biological titration of rAAV stocks is still cumbersome. Different investigators used laboratory-specific methods or internal reference standards that may limit preclinical and clinical applications. The inverted terminal repeats (ITR) sequences are the only cis-regulated viral elements required for rAAV packaging and remain within viral vector genomes. ITR is the excellent target sequences for qPCR quantification of rAAV titer. In this study, we developed a novel qPCR strategy to quantify rAAVs' vector genome titer via targeting the ITR2 or ITR2-CMV element. In conclusion, the method is fast and accurate for the titration of rAAV2-derived vector genomes. It will promote the standardization of rAAV titration in the future.


Subject(s)
Dependovirus , Classification , Genetics , Genetic Vectors , Genetics , Genome, Viral , Genetics , Polymerase Chain Reaction , Methods , Recombination, Genetic , Serotyping , Terminal Repeat Sequences , Genetics , Transduction, Genetic
17.
Chinese Journal of Biotechnology ; (12): 520-531, 2013.
Article in Chinese | WPRIM | ID: wpr-233224

ABSTRACT

In this study, we expressed and purified supercharged green fluorescent protein (+36GFP) that we used to study its combination with nucleic acid and its cell transduction efficiency as carrier of DNA. We transformed pET+36GFP-HA2 plasmid into Escherichia coli BL21 (DE3), then expressed and purified the target protein. We used the protein to transduce a variety of mammalian cell lines including B16 cells, 293 cells, A549 cells and HepG2 cells at specified protein concentrations. Transduction efficiency of the protein was analyzed by flow cytometry. Under laser scanning confocal microscope, we observed visually transduction efficiency of +36GFP protein (100 nmol/L) to A549 cells. We incubated +36GFP with plasmid DNA and analyzed their binding ability with gel mobility shift assay. Then we transduced cells with the mixture of plasmid DNA/+36GFP protein at various ratio and detected the expression of reporter gene by using laser scanning confocal microscope and flow cytometry. The experimental results demonstrate that +36GFP had high transduction efficiency, and as the concentration increased, the efficiency improved in a dose-dependent manner. Gel mobility shift assay indicates that +36GFP could bind to plasmid DNA, blocking the migration of DNA in the gel in a concentration-dependent manner. After the plasmid wrapped by +36GFP penetrated into cells, the cells could express target protein efficiently, proving that +36GFP had the ability to carry nucleic acids into cells. Sucussful expression and purification of +36GFP protein confirms its high efficiency of cell transduction and its ability as carrier to deliver exogenous nucleic acids into cells.


Subject(s)
Cell Line, Tumor , DNA , Genetics , Metabolism , Escherichia coli , Genetics , Metabolism , Green Fluorescent Proteins , Genetics , Pharmacology , Humans , Recombinant Fusion Proteins , Genetics , Pharmacology , Transduction, Genetic , Methods , Transfection
18.
Article in English | WPRIM | ID: wpr-199830

ABSTRACT

Human mesenchymal stem cells (MSCs) have emerged as attractive cellular vehicles to deliver therapeutic genes for ex-vivo therapy of diverse diseases; this is, in part, because they have the capability to migrate into tumor or lesion sites. Previously, we showed that MSCs could be utilized to deliver a bacterial cytosine deaminase (CD) suicide gene to brain tumors. Here we assessed whether transduction with a retroviral vector encoding CD gene altered the stem cell property of MSCs. MSCs were transduced at passage 1 and cultivated up to passage 11. We found that proliferation and differentiation potentials, chromosomal stability and surface antigenicity of MSCs were not altered by retroviral transduction. The results indicate that retroviral vectors can be safely utilized for delivery of suicide genes to MSCs for ex-vivo therapy. We also found that a single retroviral transduction was sufficient for sustainable expression up to passage 10. The persistent expression of the transduced gene indicates that transduced MSCs provide a tractable and manageable approach for potential use in allogeneic transplantation.


Subject(s)
Adolescent , Animals , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Transformation, Neoplastic/drug effects , Child , Cytosine Deaminase/genetics , Fluorouracil/pharmacology , Genetic Therapy , Genomic Instability/drug effects , Humans , Karyotype , Mesenchymal Stem Cells/cytology , Mice , Multipotent Stem Cells/cytology , Neoplasms/therapy , Retroviridae/metabolism , Time Factors , Transduction, Genetic
19.
Article in Chinese | WPRIM | ID: wpr-353065

ABSTRACT

<p><b>OBJECTIVE</b>To construct one lentiviral vector containing mouse SRY-related high mobility group-box gene 9 (SOX9) and transfect the murine bone mesenchymal stem cells (mBMSCs) in vitro and observe the expression of target gene.</p><p><b>METHODS</b>RNA from the vectors containing mouse SOX9 gene were extracted and SOX9 genes were amplified by reverse transcription-Polymerase Chain Reaction (RT-PCR). The SOX9 genes were connected into lentiviral vectors pGC-FU. Then pGC-FU-SOX9 transduced into 293T cells to produce recombinant lentivirus called as Lenti-SOX9-EGFP. mBMSCs were transfected. The expression of target gene was detected by immunofluorescence, RT-PCR and Western Blot.</p><p><b>RESULTS</b>Lenti-SOX9-EGFP was recombined successfully and transduced efficiently into mBMSCs. The expression of SOX9 gene was confirmed by RT-PCR and Western Blot.</p><p><b>CONCLUSION</b>Lentiviral vector of mouse SOX9 gene can transfect successfully into mBMSCs. Meanwhile, SOX9 gene may be expressed in mBMSCs. This will provide the target cells for the following study about SOX9 gene repairing cartilage injury.</p>


Subject(s)
Animals , Female , Gene Expression , Genetic Therapy , Genetic Vectors , Lentivirus , Genetics , Male , Mesenchymal Stem Cells , Metabolism , Mice , Osteoarthritis , Therapeutics , Reverse Transcriptase Polymerase Chain Reaction , SOX9 Transcription Factor , Genetics , Transduction, Genetic , Transfection
20.
Article in Chinese | WPRIM | ID: wpr-353022

ABSTRACT

<p><b>OBJECTIVE</b>To construct one lentiviral vector containing mouse SRY-related silencing group--box gene 9 (SOX9) and to transfect murine bone mesenehymal stem cells (mBMSCs) in vitro and observe the expression of target gene.</p><p><b>METHODS</b>RNA inteference target sequence was designed in connectin with mice SOX9 gene sequence. The double strands DNAoligo containing interference sequence were synthesized and cloned into lentivirus vector. The siRNA lentiviral vector with SOX9 gene silencing was constructed and identified, which was transfected into rat bone mesenehymal stem cells. The expression of target gene was detected by immunofluorescence, RT-PCR and Western blot.</p><p><b>RESULTS</b>Lenti-SOX9-siRNA-EGFP was recombined successfully and transduced efficiently into mBMSCs. The expression of SOX9 gene silencing was confirmed by RT-PCR and Western blot.</p><p><b>CONCLUSION</b>Mouse SOX9 gene silencing by RNA interference and Lentiviral vector can transfected successfully into mBMSCs. Meanwhile,SOX9 gene may be silenced in SOX9 transduced mBMSCs. This will provide target cells for the following study about SOX9 gene respairing cartilage injury.</p>


Subject(s)
Animals , Female , Gene Expression , Gene Silencing , Genetic Therapy , Genetic Vectors , Lentivirus , Genetics , Male , Mesenchymal Stem Cells , Metabolism , Mice , SOX9 Transcription Factor , Genetics , Transduction, Genetic
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