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Sample records for 293t cells transfected

  1. Effect of the ARG1 gene on arsenic resistance of 293T cells.

    PubMed

    Mu, X L; Li, L; Li, H

    2013-01-01

    To study the relationship between arsenic resistance of 293T cells and overexpression of ARG1, the ARG1 gene in a recombinant plasmid was transfected into 293T cells via liposomes, and then ARG1 overexpression was examined by real-time PCR and immunocytochemistry. The survival rate, arsenic accumulation and arsenic efflux, GSH level, and GST activity of 293T cells overexpressing ARG1 were assayed by MTT, atomic absorption spectrophotometry, and DTNB, and expression of MRP-2 was detected by Western blot analysis. Compared to that in the control cells, the survival rate of ARG1 gene-overexpressing cells was much higher following exposure to lower sodium arsenite (≤ 8 µM). When cells were exposed to lower sodium arsenite for 24 h, the arsenite content of ARG1 gene-overexpressing cells decreased and arsenic efflux increased. After 48 h, the GSH level, GST activity, and expression of MRP2 increased in a concentration-dependent manner. We conclude that the ARG1 gene increases arsenic resistance of 293T cells. PMID:24391030

  2. Effect of the ARG1 gene on arsenic resistance of 293T cells.

    PubMed

    Mu, X L; Li, L; Li, H

    2013-12-19

    To study the relationship between arsenic resistance of 293T cells and overexpression of ARG1, the ARG1 gene in a recombinant plasmid was transfected into 293T cells via liposomes, and then ARG1 overexpression was examined by real-time PCR and immunocytochemistry. The survival rate, arsenic accumulation and arsenic efflux, GSH level, and GST activity of 293T cells overexpressing ARG1 were assayed by MTT, atomic absorption spectrophotometry, and DTNB, and expression of MRP-2 was detected by Western blot analysis. Compared to that in the control cells, the survival rate of ARG1 gene-overexpressing cells was much higher following exposure to lower sodium arsenite (≤ 8 µM). When cells were exposed to lower sodium arsenite for 24 h, the arsenite content of ARG1 gene-overexpressing cells decreased and arsenic efflux increased. After 48 h, the GSH level, GST activity, and expression of MRP2 increased in a concentration-dependent manner. We conclude that the ARG1 gene increases arsenic resistance of 293T cells.

  3. Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells

    PubMed Central

    Mao, Yingying; Yan, Renhe; Li, Andrew; Zhang, Yanling; Li, Jinlong; Du, Hongyan; Chen, Baihong; Wei, Wenjin; Zhang, Yi; Sumners, Colin; Zheng, Haifa; Li, Hongwei

    2015-01-01

    Objectives:Lentiviral vectors have been used successfully to rapidly produce decigram quantities of active recombinant proteins in mammalian cell lines. To optimize the protein production platform, the roles of Ubiquitous Chromatin Opening Element (UCOE), an insulator, and selected promoters were evaluated based on efficiency and stability of foreign gene expression mediated by lentiviral vectors. Methods: Five lentiviral vectors, pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, p'HR.cppt.3'1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, pTYF-CMV(β-globin intron)-eGFP containing CMV promoter and β-globin intron, pTYF-CMV-eGFP containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell). The transduced cells were passaged once every three days at a ratio of 1:10. Expression level and stability of the foreign gene, green fluorescence protein (GFP), was evaluated using fluorescent microscopy and flow cytometry. Furthermore, we constructed a hepatitis C virus (HCV) E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was packaged and transduced into 293T cells, and the recombinant cell lines with stable expression of E1 protein were established by limiting dilution. Results:GFP expression in 293T cells transduced with the five lentiviral vectors peaked between passages 3 and 5 and persisted for more than 5 weeks. The expression was prolonged in the cells transduced with TYF-CMV (β-globin intron)-eGFP or TYF-CMV-eGFP, demonstrating less than a 50% decrease even at 9 weeks post transduction (p>0.05). The TYF-CMV-eGFP-transduced cells began with a higher level of GFP expression than other vectors did. The percentage of GFP positive cells for any of the five lentiviral vectors sustained over time. Moreover, the survival rates of all transfected cells exceeded 80% at both 5 and 9 weeks post transduction

  4. Display and selection of scFv antibodies on HEK-293T cells

    PubMed Central

    Ho, Mitchell; Pastan, Ira

    2009-01-01

    We describe a human cell display strategy to isolate high affinity single chain antibody fragments (scFvs) specific for CD22 for the treatment of B-cell malignancies. Our strategy uses flow cytometry and human embryonic kidney 293T (HEK-293T) cells that are widely used for transient protein expression. Flow cytometry enhances the screen's sensitivity thereby allowing us to isolate high affinity scFvs. Using human cell display one could isolate and engineer scFvs, single domains, Fabs or whole IgGs for increased affinity and other biological functions. PMID:19554290

  5. Measuring Ca2+-Dependent Modulation of Voltage-Gated Ca2+ Channels in HEK-293T Cells.

    PubMed

    Thomas, Jessica R; Lee, Amy

    2016-01-01

    Voltage-gated Ca(2+) (Cav) channels regulate a variety of biological processes, such as muscle contraction, gene expression, and neurotransmitter release. Cav channels are subject to diverse forms of regulation, including those involving the Ca(2+) ions that permeate the pore. High voltage-activated Cav channels undergo Ca(2+)-dependent inactivation (CDI) and facilitation (CDF), which can regulate processes such as cardiac rhythm and synaptic plasticity. CDI and CDF differ slightly between Cav1 (L-type) and Cav2 (P/Q-, N-, and R-type) channels. Human embryonic kidney cells transformed with SV40 large T-antigen (HEK-293T) are advantageous for studying CDI and CDF of a particular type of Cav channel. HEK-293T cells do not express endogenous Cav channels, but Cav channels can be expressed exogenously at high levels in these cells by transient transfection. This protocol describes how to characterize and analyze Ca(2+)-dependent modulation of recombinant Cav channels in HEK-293T cells. PMID:27587775

  6. Characterization of crude Echis carinatus venom-induced cytotoxicity in HEK 293T cells.

    PubMed

    Pierce, Rebecca D; Kim, Ethan S; Girton, Lance W; McMurry, Jonathan L; Francis, Joshua W; Albrecht, Eric A

    2011-01-01

    Echis carinatus (saw-scaled viper) produces potent hemorrhagic venom that causes the development of apoptotic and necrotic tissues. In this study, we used polyethyleneimine (PEI) to enhance cellular adherence, and to determine whether the substrate attachment influenced the survival of cells treated with crude E. carinatus venom. Human embryonic kidney (HEK) 293T cells were grown for 18hr in tissue culture plates with or without polyethyleneimine (PEI), and were then stimulated with crude E. carinatus venom for 3 or 12hr. HEK 293T cells grown without PEI displayed a robust oxidative response to corresponding substrate detachment, loss of plasma membrane integrity and decreased cell viability. Cells grown on PEI adsorbed substrates demonstrated prolonged substrate attachment resulting in significantly higher cell viabilities. These observations suggest that the cytotoxicity of crude E. carinatus venom is dependent upon cellular detachment. PMID:22331993

  7. Pharmacological characterization of emerging synthetic cannabinoids in HEK293T cells and hippocampal neurons.

    PubMed

    Costain, Willard J; Tauskela, Joseph S; Rasquinha, Ingrid; Comas, Tanya; Hewitt, Melissa; Marleau, Vincent; Soo, Evelyn C

    2016-09-01

    There has been a worldwide proliferation of synthetic cannabinoids that have become marketed as legal alternatives to cannabis (marijuana). Unfortunately, there is a dearth of information about the pharmacological effects of many of these emerging synthetic cannabinoids (ESCs), which presents a challenge for regulatory authorities that need to take such scientific evidence into consideration in order to regulate ECSs as controlled substances. We aimed to characterize the pharmacological properties of ten ESCs using two cell based assays that enabled the determination of potency and efficacy relative to a panel of well-characterized cannabinoids. Agonist-mediated inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels was monitored in live HEK293T cells transfected with human cannabinoid receptor 1 gene (CNR1) and pGloSensor-22F. Pharmacological analysis of this data indicated that all of the ESCs tested were full agonists, with the following rank order of potency: Win 55212-2≈5F-PB-22≈AB-PINACA≈EAM-2201≈MAM-2201>JWH-250≈ PB-22>AKB48 N-(5FP)>AKB-48≈STS-135>XLR-11. Assessment of agonist-stimulated depression of Ca(2+) transients was also used to confirm the efficacy of five ESCs (XLR-11, JWH-250, AB-PINACA, 5F-PB-22, and MAM-2201) in cultured primary hippocampal neurons. This work aims to help inform decisions made by regulatory agencies concerned with the profusion of these poorly characterized recreational drugs. PMID:27260125

  8. Analysis of Wnt signaling β-catenin spatial dynamics in HEK293T cells

    PubMed Central

    2014-01-01

    Background Wnt/β-catenin signaling is involved in different stages of mammalian development and implicated in various cancers (e.g. colorectal cancer). Recent experimental and computational studies have revealed characteristics of the pathway, however a cell-specific spatial perspective is lacking. In this study, a novel 3D confocal quantitation protocol is developed to acquire spatial (two cellular compartments: nucleus and cytosol-membrane) and temporal quantitative data on target protein (e.g. β-catenin) concentrations in Human Epithelial Kidney cells (HEK293T) during perturbation (with either cycloheximide or Wnt3A). Computational models of the Wnt pathway are constructed and interrogated based on this data. Results A single compartment Wnt pathway model is compared with a simple β-catenin two compartment model to investigate Wnt3A signaling in HEK293T cells. When protein synthesis is inhibited, β-catenin decreases at the same rate in both cellular compartments, suggesting diffusional transport is fast compared to β-catenin degradation in the cytosol. With Wnt3A stimulation, the total amount of β-catenin rises throughout the cell, however the increase is initially (~first hour) faster in the nuclear compartment. While both models were able to reproduce the whole cell changes in β-catenin, only the compartment model reproduced the Wnt3A induced changes in β-catenin distribution and it was also the best fit for the data obtained when active transport was included alongside passive diffusion transport. Conclusions This integrated 3D quantitation imaging protocol and computational modeling approach allowed cell-specific compartment models of the signaling pathways to be constructed and analyzed. The Wnt models constructed in this study are the first for HEK293T and have suggested potential roles of inter-compartment transport to the dynamics of signaling. PMID:24712863

  9. HEK293T Cells Are Heterozygous for CCR5 Delta 32 Mutation

    PubMed Central

    Qi, Chunxia; Jia, Xiaopeng; Lu, Lingling; Ma, Ping; Wei, Min

    2016-01-01

    C-C chemokine receptor 5 (CCR5) is a receptor for chemokines and a co-receptor for HIV-1 entry into the target CD4+ cells. CCR5 delta 32 deletion is a loss-of-function mutation, resistant to HIV-1 infection. We tried to induce the CCR5 delta 32 mutation harnessing the genome editing technique, CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR and CRISPR associated protein 9, Cas9) in the commonly used cell line human embryonic kidney HEK 293T cells. Surprisingly, we found that HEK293T cells are heterozygous for CCR5 delta 32 mutation, in contrast to the wild type CCR5 cells, human acute T cell leukemia cell line Jurkat and human breast adenocarcinoma cell line MDA-MB-231 cells. This finding indicates that at least one human cell line is heterozygous for the CCR5 delta 32 mutation. We also found that in PCR amplification, wild type CCR5 DNA and mutant delta 32 DNA can form mismatched heteroduplex and move slowly in gel electrophoresis. PMID:27042825

  10. HEK293T Cells Are Heterozygous for CCR5 Delta 32 Mutation.

    PubMed

    Qi, Chunxia; Jia, Xiaopeng; Lu, Lingling; Ma, Ping; Wei, Min

    2016-01-01

    C-C chemokine receptor 5 (CCR5) is a receptor for chemokines and a co-receptor for HIV-1 entry into the target CD4+ cells. CCR5 delta 32 deletion is a loss-of-function mutation, resistant to HIV-1 infection. We tried to induce the CCR5 delta 32 mutation harnessing the genome editing technique, CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR and CRISPR associated protein 9, Cas9) in the commonly used cell line human embryonic kidney HEK 293T cells. Surprisingly, we found that HEK293T cells are heterozygous for CCR5 delta 32 mutation, in contrast to the wild type CCR5 cells, human acute T cell leukemia cell line Jurkat and human breast adenocarcinoma cell line MDA-MB-231 cells. This finding indicates that at least one human cell line is heterozygous for the CCR5 delta 32 mutation. We also found that in PCR amplification, wild type CCR5 DNA and mutant delta 32 DNA can form mismatched heteroduplex and move slowly in gel electrophoresis.

  11. Inhibition of NAMPT pathway by FK866 activates the function of p53 in HEK293T cells

    SciTech Connect

    Thakur, Basant Kumar; Dittrich, Tino; Chandra, Prakash; Becker, Annette; Lippka, Yannick; Selvakumar, Divakarvel; Klusmann, Jan-Henning; Reinhardt, Dirk; Welte, Karl

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer In 293T cells, p53 is considered to be inactive due to its interaction with the large T-antigen. Black-Right-Pointing-Pointer Acetylation of p53 at lysine 382 is important for its functional activation. Black-Right-Pointing-Pointer First evidence to document the presence of a functional p53 in 293T cells. Black-Right-Pointing-Pointer Inhibition of NAMPT/SIRT pathway by FK866 in 293T cells increases the functional activity of p53. Black-Right-Pointing-Pointer This activation of p53 involves reversible acetylation of p53 at lysine 382. -- Abstract: Inactivation of p53 protein by endogenous and exogenous carcinogens is involved in the pathogenesis of different human malignancies. In cancer associated with SV-40 DNA tumor virus, p53 is considered to be non-functional mainly due to its interaction with the large T-antigen. Using the 293T cell line (HEK293 cells transformed with large T antigen) as a model, we provide evidence that p53 is one of the critical downstream targets involved in FK866-mediated killing of 293T cells. A reduced rate of apoptosis and an increased number of cells in S-phase was accompanied after knockdown of p53 in these cells. Inhibition of NAMPT by FK866, or inhibition of SIRT by nicotinamide decreased proliferation and triggered death of 293T cells involving the p53 acetylation pathway. Additionally, knockdown of p53 attenuated the effect of FK866 on cell proliferation, apoptosis, and cell cycle arrest. The data presented here shed light on two important facts: (1) that p53 in 293T cells is active in the presence of FK866, an inhibitor of NAMPT pathway; (2) the apoptosis induced by FK866 in 293T cells is associated with increased acetylation of p53 at Lys382, which is required for the functional activity of p53.

  12. Biotransport and intracellular ice formation phenomena in freezing human embryonic kidney cells (HEK293T).

    PubMed

    Xu, Yunpeng; Zhao, Gang; Zhou, Xiaoming; Ding, Weiping; Shu, Zhiquan; Gao, Dayong

    2014-04-01

    The objective of this study is to determine the cryobiological characteristics of human embryonic kidney (HEK293T) cells. The cell membrane hydraulic conductivity (L(pg)) and the activation energy of water transport (E(Lp)) were determined in the absence/presence of cryoprotectant agent (CPA), while the nucleation rate kinetic and thermodynamic parameters (Ωo(SCN) and κo(SCN)) were determined in the absence of CPA. Since dehydration and intracellular ice formation (IIF) are two factors that may cause damage to cells during the freezing process, systematical freezing experiments were carried out at different cooling rates (5, 10, 15, 20, 30, and 60°C/min) under the commercial available cryomicroscopy (FDCS 196, Linkham, Waterfield, UK) to further explore the cryoinjury mechanism for HEK293T cells. By simultaneously fitting the water transport equation to the experimentally measured volumetric shrinkage data at 5, 10, and 15°C/min, the "combined best fit" membrane permeability parameters for HEK293T cells in both phosphate buffer saline (PBS) and CPA media (0.75M Me2SO in PBS) are determined. They are L(pg)=2.85×10(-14)m/s/Pa (0.17μm/min/atm), E(Lp)=142.91kJ/mol (34.13kcal/mol) (R(2)=0.990), and L(pg)[cpa]=2.73±0.44×10(-14)m/s/Pa (0.16±0.03μm/min/atm), E(Lp)[cpa]=152.52±27.69kJ/mol (36.42±6.61kcal/mol) (R(2)=0.993), respectively. An optimal cooling rate B(opt) (the highest cooling rate without IIF) was determined to be 14.24°C/min in the absence of CPA. Additionally, the ice nucleation parameters (Ωo(SCN) and κo(SCN)) were averaged to be 1.31±0.11×10(8)m(-2)s(-1) and 7.67±2.55×10(9)K(5) for the cooling rates 20, 30, and 60°C/min.

  13. Biotransport and intracellular ice formation phenomena in freezing human embryonic kidney cells (HEK293T).

    PubMed

    Xu, Yunpeng; Zhao, Gang; Zhou, Xiaoming; Ding, Weiping; Shu, Zhiquan; Gao, Dayong

    2014-04-01

    The objective of this study is to determine the cryobiological characteristics of human embryonic kidney (HEK293T) cells. The cell membrane hydraulic conductivity (L(pg)) and the activation energy of water transport (E(Lp)) were determined in the absence/presence of cryoprotectant agent (CPA), while the nucleation rate kinetic and thermodynamic parameters (Ωo(SCN) and κo(SCN)) were determined in the absence of CPA. Since dehydration and intracellular ice formation (IIF) are two factors that may cause damage to cells during the freezing process, systematical freezing experiments were carried out at different cooling rates (5, 10, 15, 20, 30, and 60°C/min) under the commercial available cryomicroscopy (FDCS 196, Linkham, Waterfield, UK) to further explore the cryoinjury mechanism for HEK293T cells. By simultaneously fitting the water transport equation to the experimentally measured volumetric shrinkage data at 5, 10, and 15°C/min, the "combined best fit" membrane permeability parameters for HEK293T cells in both phosphate buffer saline (PBS) and CPA media (0.75M Me2SO in PBS) are determined. They are L(pg)=2.85×10(-14)m/s/Pa (0.17μm/min/atm), E(Lp)=142.91kJ/mol (34.13kcal/mol) (R(2)=0.990), and L(pg)[cpa]=2.73±0.44×10(-14)m/s/Pa (0.16±0.03μm/min/atm), E(Lp)[cpa]=152.52±27.69kJ/mol (36.42±6.61kcal/mol) (R(2)=0.993), respectively. An optimal cooling rate B(opt) (the highest cooling rate without IIF) was determined to be 14.24°C/min in the absence of CPA. Additionally, the ice nucleation parameters (Ωo(SCN) and κo(SCN)) were averaged to be 1.31±0.11×10(8)m(-2)s(-1) and 7.67±2.55×10(9)K(5) for the cooling rates 20, 30, and 60°C/min. PMID:24582893

  14. DAPIT Over-Expression Modulates Glucose Metabolism and Cell Behaviour in HEK293T Cells

    PubMed Central

    Kontro, Heidi; Cannino, Giuseppe; Rustin, Pierre; Dufour, Eric; Kainulainen, Heikki

    2015-01-01

    Introduction Diabetes Associated Protein in Insulin-sensitive Tissues (DAPIT) is a subunit of mitochondrial ATP synthase and has also been found to associate with the vacuolar H+-ATPase. Its expression is particularly high in cells with elevated aerobic metabolism and in epithelial cells that actively transport nutrients and ions. Deletion of DAPIT is known to induce loss of mitochondrial ATP synthase but the effects of its over-expression are obscure. Results In order to study the consequences of high expression of DAPIT, we constructed a transgenic cell line that constitutively expressed DAPIT in human embryonal kidney cells, HEK293T. Enhanced DAPIT expression decreased mtDNA content and mitochondrial mass, and saturated respiratory chain by decreasing H+-ATP synthase activity. DAPIT over-expression also increased mitochondrial membrane potential and superoxide level, and translocated the transcription factors hypoxia inducible factor 1α (Hif1α) and β-catenin to the nucleus. Accordingly, cells over-expressing DAPIT used more glucose and generated a larger amount of lactate compared to control cells. Interestingly, these changes were associated with an epithelial to mesenchymal (EMT)-like transition by changing E-cadherin to N-cadherin and up-regulating several key junction/adhesion proteins. At physiological level, DAPIT over-expression slowed down cell growth by G1 arrest and migration, and enhanced cell detachment. Several cancers also showed an increase in genomic copy number of Usmg5 (gene encoding DAPIT), thereby providing strong correlative evidence for DAPIT possibly having oncogenic function in cancers. Conclusions DAPIT over-expression thus appears to modulate mitochondrial functions and alter cellular regulations, promote anaerobic metabolism and induce EMT-like transition. We propose that DAPIT over-expression couples the changes in mitochondrial metabolism to physiological and pathophysiological regulations, and suggest it could play a critical

  15. The role of sensitivity of ALA (PpIX)-based PDT on Human embryonic kidney cell line (HEK293T)

    NASA Astrophysics Data System (ADS)

    Fakhar-e-Alam, M.; Atif, M.; Rehman, T.; Sadia, H.; Firdous, S.

    2011-08-01

    Present study evaluates the effects of photodynamic therapy (PDT) with aminolevulinic acid (5-ALA) as photo sensitizer using Human embryonic kidney (HEK293T) cell line as an experimental model. Porphyrins derivatives are used as active cytotoxic antitumor agents in PDT. Above mentioned cell line were irradiated with red light (a diode laser, λ = 635 nm) at different doses (0-160 J/cm2) of light. The influence/effectiveness of incubation time, various concentrations of aminolevulinic acid (5-ALA) and light doses on the cellular viability was studied. HEK293T cells were deliberated by exposing the ALA-PpIX (0-1000 μg/ml) of concentrations. The optimal uptakes of photosensitizer (PS) in cell lines were investigated by means of spectro photo metric measurements. Cells viability was determined by means of neutral red assay (NRA). It was observed that alone, neither photosensitizer nor light dose have significant effect on cells viability, but optimal concentration of PS along with suitable dose of light exhibit effective impact on the viability of cell. Our results showed that light doses of 40 J/cm2 demonstrates effective PDT outcome for HEK293T cell line when incubated with 400 μg/ml, with wrapping up view that HEK293T cell line is very sensitive to ALA-mediated PDT as compared to cell line published in our data. At the end results has been verified by using reactive oxygen species (ROS) measure test.

  16. Increasing cellular uptake of mesoporous silica nanoparticles in human embryonic kidney cell line 293T cells by using Lipofectamine 2000.

    PubMed

    Wang, Jiandong; Teng, Zhaogang; Tian, Ying; Fang, Tian; Ma, Jie; Sun, Jin; Zhu, Feipeng; Wu, Jinrong; Wang, Xin; Yang, Nannan; Zhou, Xiaojun; Yun, Shifeng; Lu, Guangming

    2013-11-01

    Mesoporous silica nanoparticles (MSNs) are ideal nanocarriers that have recently gained attention in important bioapplications such as drug, gene, and protein delivery. The efficacy of endocytosis greatly affects the biological functions of MSNs. In the present study, we investigated the effect of cationic liposomes of Lipofectamine 2000 on cellular uptake of MSNs and the cytotoxicity of cationic liposomes combining with MSNs both in vitro and in vivo. Therefore, mesoporous silica nanoparticles with an average diameter of 130 nm and negative surface charge were synthesized and characterized. The possible role of Lipofectamine 2000 in cellular uptake of MSNs was evaluated in human embryonic kidney cell line 293T cells by transmission electron microscopy (TEM) and with inductively coupled plasma (ICP) analysis. The toxicities of liposomes combining with MSNs were tested in vitro via cell apoptosis assay and MTT cell viability assay, and in vivo by histological examination of six organs of mice after intravenous injection. The endocytosis efficiency of MSNs in human embryonic kidney 293T cells was greatly increased using Lipofectamine 2000 compared with controls (P < 0.001). No apparent in vitro or in vivo cytotoxicity was found for Lipofectamine 2000 combining with MSNs. Our data indicate that cationic liposomes of Lipofectamine 2000 has the potential to greatly increase cellular uptake of MSNs with negative surface charge in human renal 293T cells without apparent toxicity. PMID:24059087

  17. Development of a Model System for Tick-Borne Flavivirus Persistence in HEK 293T Cells

    PubMed Central

    Mlera, Luwanika; Offerdahl, Danielle K.; Martens, Craig; Porcella, Stephen F.; Melik, Wessam

    2015-01-01

    ABSTRACT We devised a model system to study persistent infection by the tick-borne flavivirus Langat virus (LGTV) in 293T cells. Infection with a molecularly cloned LGTV strain produced an acute lytic crisis that left few surviving cells. The culture was repopulated by cells that were ~90% positive for LGTV E protein, thus initiating a persistent infection that was maintained for at least 35 weeks without additional lytic crises. Staining of cells for viral proteins and ultrastructural analysis revealed only minor differences from the acute phase of infection. Infectious LGTV decreased markedly over the study period, but the number of viral genomes remained relatively constant, suggesting the development of defective interfering particles (DIPs). Viral genome changes were investigated by RNA deep sequencing. At the initiation of persistent infection, levels of DIPs were below the limit of detection at a coverage depth of 11,288-fold, implying that DIPs are not required for initiation of persistence. However, after 15 passages, DIPs constituted approximately 34% of the total LGTV population (coverage of 1,293-fold). Furthermore, at this point, one specific DIP population predominated in which nucleotides 1058 to 2881 had been deleted. This defective genome specified an intact polyprotein that coded for a truncated fusion protein containing 28 N-terminal residues of E and 134 C-terminal residues of NS1. Such a fusion protein has not previously been described, and a possible function in persistent infection is uncertain. DIPs are not required for the initiation of persistent LGTV infection but may play a role in the maintenance of viral persistence. PMID:26045539

  18. Identification and Characterization of 293T Cell-Derived Exosomes by Profiling the Protein, mRNA and MicroRNA Components.

    PubMed

    Li, Jing; Chen, Xiulan; Yi, Jiao; Liu, Yuchen; Li, Dameng; Wang, Jifeng; Hou, Dongxia; Jiang, Xiaohong; Zhang, Junfeng; Wang, Jin; Zen, Ke; Yang, Fuquan; Zhang, Chen-Yu; Zhang, Yujing

    2016-01-01

    Cell-derived exosomes are leading candidates for in vivo drug delivery carriers. In particular, exosomes derived from 293T cells are used most frequently, although exosome dosing has varied greatly among studies. Considering their biological origin, it is crucial to characterize the molecular composition of exosomes if large doses are to be administered in clinical settings. In this study, we present the first comprehensive analysis of the protein, messenger RNA and microRNA profiles of 293T cell-derived exosomes; then, we characterized these data using Gene Ontology annotation and Kyoto Encyclopedia for Genes and Genomes pathway analysis. Our study will provide the basis for the selection of 293T cell-derived exosome drug delivery systems. Profiling the exosomal signatures of 293T cells will lead to a better understanding of 293T exosome biology and will aid in the identification of any harmful factors in exosomes that could cause adverse clinical effects. PMID:27649079

  19. Identification and Characterization of 293T Cell-Derived Exosomes by Profiling the Protein, mRNA and MicroRNA Components

    PubMed Central

    Li, Dameng; Wang, Jifeng; Hou, Dongxia; Jiang, Xiaohong; Zhang, Junfeng; Wang, Jin; Zen, Ke; Yang, Fuquan; Zhang, Chen-Yu

    2016-01-01

    Cell-derived exosomes are leading candidates for in vivo drug delivery carriers. In particular, exosomes derived from 293T cells are used most frequently, although exosome dosing has varied greatly among studies. Considering their biological origin, it is crucial to characterize the molecular composition of exosomes if large doses are to be administered in clinical settings. In this study, we present the first comprehensive analysis of the protein, messenger RNA and microRNA profiles of 293T cell-derived exosomes; then, we characterized these data using Gene Ontology annotation and Kyoto Encyclopedia for Genes and Genomes pathway analysis. Our study will provide the basis for the selection of 293T cell-derived exosome drug delivery systems. Profiling the exosomal signatures of 293T cells will lead to a better understanding of 293T exosome biology and will aid in the identification of any harmful factors in exosomes that could cause adverse clinical effects. PMID:27649079

  20. Impact of phosphomimetic and non-phosphorylatable mutations of phospholemman on L-type calcium channels gating in HEK 293T cells

    PubMed Central

    Guo, Kai; Wang, Yue-Peng; Zhou, Zhi-Wen; Jiang, Yi-Bo; Li, Wei; Chen, Xiao-Meng; Li, Yi-Gang

    2015-01-01

    Background: Phospholemman (PLM) is an important phosphorylation substrate for protein kinases A and C in the heart. Until now, the association between PLM phosphorylation status and L-type calcium channels (LTCCs) gating has not been fully understood. We investigated the kinetics of LTCCs in HEK 293T cells expressing phosphomimetic or nonphosphorylatable PLM mutants. Methods: The LTCCs gating was measured in HEK 293T cells transfected with LTCC and wild-type (WT) PLM, phosphomimetic or nonphosphorylatable PLM mutants: 6263AA, 6869AA, AAAA, 6263DD, 6869DD or DDDD. Results: WT PLM significantly slowed LTCCs activation and deactivation while enhanced voltage-dependent inactivation (VDI). PLM mutants 6869DD and DDDD significantly increased the peak of the currents. 6263DD accelerated channel activation, while 6263AA slowed it more than WT PLM. 6869DD significantly enhanced PLM-induced increase of VDI. AAAA slowed the channel activation more than 6263AA, and DDDD accelerated the channel VDI more than 6869DD. Conclusions: Our results demonstrate that phosphomimetic PLM could stimulate LTCCs and alter their dynamics, while PLM nonphosphorylatable mutant produced the opposite effects. PMID:25656605

  1. Transcriptome Analysis Reveals a Signature Profile for Tick-Borne Flavivirus Persistence in HEK 293T Cells

    PubMed Central

    Lam, Jennifer; Offerdahl, Danielle K.; Martens, Craig; Sturdevant, Daniel; Turner, Charles V.; Porcella, Stephen F.; Bloom, Marshall E.

    2016-01-01

    ABSTRACT Tick-borne flaviviruses (TBFVs) cause febrile illnesses, which may progress to severe encephalitis and/or death in humans globally. Most people who recover from severe acute disease suffer from debilitating neurological sequelae, which may be due to viral persistence, infection-induced neurological cell damage, host response, or some combination of these. Acute TBFV infection of human embryonic kidney (HEK) 293T cells in vitro results in the death of >95% of infected cells by day 5. However, replacing cell growth medium allows surviving cells to repopulate and become persistently infected for extended periods of time. The mechanisms responsible for initiation and maintenance of viral persistence remain vague. We subjected the HEK 293T cell transcriptome to deep sequencing to identify genes differentially expressed during acute infection and persistent infection. A total of 451 genes showed unique significant differential expression levels in persistently infected cells relative to the acute phase of infection. Ingenuity Pathway Analysis results suggested that the expression of prosurvival oncogenes AKT2 and ERBB2 was upregulated in persistently infected cells, whereas proapoptotic genes, such as Bad and the beta interferon 1 (IFN-β1) gene, were downregulated. Genes encoding antiviral cytokines such as the CCL5, tumor necrosis factor alpha (TNF-α), and CXCL10 genes were upregulated during the acute phase, but the same genes were relatively quiescent in persistently infected cells. Exogenous induction of apoptosis demonstrated that persistently infected cells were resistant to apoptosis in a dose-dependent manner. In summary, the differential transcriptome profiles of acute-phase compared to persistently infected HEK 293T cells demonstrated an evasion of apoptosis, which may be critical for a chronic TBFV infection state. These results provide a basis for further study of the mechanisms of TBFV persistence. PMID:27222466

  2. Protective effect of reduced glutathione C60 derivative against hydrogen peroxide-induced apoptosis in HEK 293T cells.

    PubMed

    Huang, Jin; Zhou, Chi; He, Jun; Hu, Zheng; Guan, Wen-Chao; Liu, Sheng-Hong

    2016-06-01

    Hydrogen peroxide (H2O2) and free radicals cause oxidative stress, which induces cellular injuries, metabolic dysfunction, and even cell death in various clinical abnormalities. Fullerene (C60) is critical for scavenging oxygen free radicals originated from cell metabolism, and reduced glutathione (GSH) is another important endogenous antioxidant. In this study, a novel water-soluble reduced glutathione fullerene derivative (C60-GSH) was successfully synthesized, and its beneficial roles in protecting against H2O2-induced oxidative stress and apoptosis in cultured HEK 293T cells were investigated. Fourier Transform infrared spectroscopy and (1)H nuclear magnetic resonance were used to confirm the chemical structure of C60-GSH. Our results demonstrated that C60-GSH prevented the reactive oxygen species (ROS)-mediated cell damage. Additionally, C60-GSH pretreatment significantly attenuated H2O2-induced superoxide dismutase (SOD) consumption and malondialdehyde (MDA) elevation. Furthermore, C60-GSH inhibited intracellular calcium mobilization, and subsequent cell apoptosis via bcl-2/bax-caspase-3 signaling pathway induced by H2O2 stimulation in HEK 293T cells. Importantly, these protective effects of C60-GSH were superior to those of GSH. In conclusion, these results suggested that C60-GSH has potential to protect against H2O2-induced cell apoptosis by scavenging free radicals and maintaining intracellular calcium homeostasis without evident toxicity.

  3. Protective effect of reduced glutathione C60 derivative against hydrogen peroxide-induced apoptosis in HEK 293T cells.

    PubMed

    Huang, Jin; Zhou, Chi; He, Jun; Hu, Zheng; Guan, Wen-Chao; Liu, Sheng-Hong

    2016-06-01

    Hydrogen peroxide (H2O2) and free radicals cause oxidative stress, which induces cellular injuries, metabolic dysfunction, and even cell death in various clinical abnormalities. Fullerene (C60) is critical for scavenging oxygen free radicals originated from cell metabolism, and reduced glutathione (GSH) is another important endogenous antioxidant. In this study, a novel water-soluble reduced glutathione fullerene derivative (C60-GSH) was successfully synthesized, and its beneficial roles in protecting against H2O2-induced oxidative stress and apoptosis in cultured HEK 293T cells were investigated. Fourier Transform infrared spectroscopy and (1)H nuclear magnetic resonance were used to confirm the chemical structure of C60-GSH. Our results demonstrated that C60-GSH prevented the reactive oxygen species (ROS)-mediated cell damage. Additionally, C60-GSH pretreatment significantly attenuated H2O2-induced superoxide dismutase (SOD) consumption and malondialdehyde (MDA) elevation. Furthermore, C60-GSH inhibited intracellular calcium mobilization, and subsequent cell apoptosis via bcl-2/bax-caspase-3 signaling pathway induced by H2O2 stimulation in HEK 293T cells. Importantly, these protective effects of C60-GSH were superior to those of GSH. In conclusion, these results suggested that C60-GSH has potential to protect against H2O2-induced cell apoptosis by scavenging free radicals and maintaining intracellular calcium homeostasis without evident toxicity. PMID:27376803

  4. A High Affinity hRpn2-Derived Peptide That Displaces Human Rpn13 from Proteasome in 293T Cells

    PubMed Central

    Durham, Sarah E.; Tarasov, Sergey G.; Walters, Kylie J.

    2015-01-01

    Rpn13 is a proteasome ubiquitin receptor that has emerged as a therapeutic target for human cancers. Its ubiquitin-binding activity is confined to an N-terminal Pru (pleckstrin-like receptor for ubiquitin) domain that also docks it into the proteasome, while its C-terminal DEUBAD (DEUBiquitinase ADaptor) domain recruits deubiquitinating enzyme Uch37 to the proteasome. Bis-benzylidine piperidone derivatives that were found to bind covalently to Rpn13 C88 caused the accumulation of polyubiquitinated proteins as well as ER stress-related apoptosis in various cancer cell lines, including bortezomib-resistant multiple myeloma lines. We find that a 38-amino acid peptide derived from the C-terminus of proteasome PC repeat protein hRpn2/PSMD1 binds to hRpn13 Pru domain with 12 nM affinity. By using NMR, we identify the hRpn13-interacting amino acids in this hRpn2 fragment, some of which are conserved among eukaryotes. Importantly, we find the hRpn2-derived peptide to immunoprecipitate endogenous Rpn13 from 293T cells, and to displace it from the proteasome. These findings indicate that this region of hRpn2 is the primary binding site for hRpn13 in the proteasome. Moreover, the hRpn2-derived peptide was no longer able to interact with endogenous hRpn13 when a strictly conserved phenylalanine (F948 in humans) was replaced with arginine or a stop codon, or when Y950 and I951 were substituted with aspartic acid. Finally, over-expression of the hRpn2-derived peptide leads to an increased presence of ubiquitinated proteins in 293T cells. We propose that this hRpn2-derived peptide could be used to develop peptide-based strategies that specifically target hRpn13 function in the proteasome. PMID:26466095

  5. Plasmonic activation of gold nanorods for remote stimulation of calcium signaling and protein expression in HEK 293T cells.

    PubMed

    Sanchez-Rodriguez, Sandra P; Sauer, Jeremy P; Stanley, Sarah A; Qian, Xi; Gottesdiener, Andrew; Friedman, Jeffrey M; Dordick, Jonathan S

    2016-10-01

    Remote activation of specific cells of a heterogeneous population can provide a useful research tool for clinical and therapeutic applications. Here, we demonstrate that photostimulation of gold nanorods (AuNRs) using a tunable near-infrared (NIR) laser at specific longitudinal surface plasmon resonance wavelengths can induce the selective and temporal internalization of calcium in HEK 293T cells. Biotin-PEG-Au nanorods coated with streptavidin Alexa Fluor-633 and biotinylated anti-His antibodies were used to decorate cells genetically modified with His-tagged TRPV1 temperature-sensitive ion channel and AuNRs conjugated to biotinylated RGD peptide were used to decorate integrins in unmodified cells. Plasmonic activation can be stimulated at weak laser power (0.7-4.0 W/cm(2) ) without causing cell damage. Selective activation of TRPV1 channels could be controlled by laser power between 1.0 and 1.5 W/cm(2) . Integrin targeting robustly stimulated calcium signaling due to a dense cellular distribution of nanoparticles. Such an approach represents a functional tool for combinatorial activation of cell signaling in heterogeneous cell populations. Our results suggest that it is possible to induce cell activation via NIR-induced gold nanorod heating through the selective targeting of membrane proteins in unmodified cells to produce calcium signaling and downstream expression of specific genes with significant relevance for both in vitro and therapeutic applications. Biotechnol. Bioeng. 2016;113: 2228-2240. © 2016 Wiley Periodicals, Inc. PMID:27563853

  6. Plasmonic activation of gold nanorods for remote stimulation of calcium signaling and protein expression in HEK 293T cells.

    PubMed

    Sanchez-Rodriguez, Sandra P; Sauer, Jeremy P; Stanley, Sarah A; Qian, Xi; Gottesdiener, Andrew; Friedman, Jeffrey M; Dordick, Jonathan S

    2016-10-01

    Remote activation of specific cells of a heterogeneous population can provide a useful research tool for clinical and therapeutic applications. Here, we demonstrate that photostimulation of gold nanorods (AuNRs) using a tunable near-infrared (NIR) laser at specific longitudinal surface plasmon resonance wavelengths can induce the selective and temporal internalization of calcium in HEK 293T cells. Biotin-PEG-Au nanorods coated with streptavidin Alexa Fluor-633 and biotinylated anti-His antibodies were used to decorate cells genetically modified with His-tagged TRPV1 temperature-sensitive ion channel and AuNRs conjugated to biotinylated RGD peptide were used to decorate integrins in unmodified cells. Plasmonic activation can be stimulated at weak laser power (0.7-4.0 W/cm(2) ) without causing cell damage. Selective activation of TRPV1 channels could be controlled by laser power between 1.0 and 1.5 W/cm(2) . Integrin targeting robustly stimulated calcium signaling due to a dense cellular distribution of nanoparticles. Such an approach represents a functional tool for combinatorial activation of cell signaling in heterogeneous cell populations. Our results suggest that it is possible to induce cell activation via NIR-induced gold nanorod heating through the selective targeting of membrane proteins in unmodified cells to produce calcium signaling and downstream expression of specific genes with significant relevance for both in vitro and therapeutic applications. Biotechnol. Bioeng. 2016;113: 2228-2240. © 2016 Wiley Periodicals, Inc.

  7. Acute effects of pregabalin on the function and cellular distribution of Ca(V)2.1 in HEK293t cells.

    PubMed

    Weissmann, Carina; Di Guilmi, Mariano N; Urbano, Francisco J; Uchitel, Osvaldo D

    2013-01-01

    We established a cell model to study the acute effects of pregabalin (PGB), a drug widely used in epilepsy and neuropathic pain, on voltage gated Ca(V)2.1 (P/Q-type) calcium channels function and distribution at the membrane level. HEK293t cells were transfected with plasmids coding for all subunits of the Ca(V)2.1 channel. We used a α1 fused to an eGFP tag to follow its distribution in time and at different experimental conditions. The expressed channel was functional as shown by the presence of barium-mediated, calcium currents of transfected cells measured by 'whole-cell voltage-clamp' recordings, showing a maximum current peak in the I-V curve at +20 mV. The GFP fluorescent signal was confined to the periphery of the cells. Incubation with 500 μM PGB, that binds α2δ subunits, for 30 min induced changes in localization of the fluorescent subunits as measured by fluorescent time lapse microscopy. These changes correlated with a reversible reduction of barium currents through Ca(V)2.1 calcium channels under the same conditions. However, no changes in the cellular distribution of the subunits were visualized for cells either expressing another membrane associated protein or after exposure of the Ca(V)2.1 channels to isoleucine, another α2δ ligand. Together these results show strong evidence for an acute effect of PGB on Ca(V)2.1 calcium channels' currents and distribution and suggest that internalization of Ca(V)2.1 channels might be a mechanism of PGB action. PMID:23063705

  8. COG Complex Complexities: Detailed Characterization of a Complete Set of HEK293T Cells Lacking Individual COG Subunits

    PubMed Central

    Bailey Blackburn, Jessica; Pokrovskaya, Irina; Fisher, Peter; Ungar, Daniel; Lupashin, Vladimir V.

    2016-01-01

    The Conserved Oligomeric Golgi complex is an evolutionarily conserved multisubunit tethering complex (MTC) that is crucial for intracellular membrane trafficking and Golgi homeostasis. The COG complex interacts with core vesicle docking and fusion machinery at the Golgi; however, its exact mechanism of action is still an enigma. Previous studies of COG complex were limited to the use of CDGII (Congenital disorders of glycosylation type II)-COG patient fibroblasts, siRNA mediated knockdowns, or protein relocalization approaches. In this study we have used the CRISPR approach to generate HEK293T knock-out (KO) cell lines missing individual COG subunits. These cell lines were characterized for glycosylation and trafficking defects, cell proliferation rates, stability of COG subunits, localization of Golgi markers, changes in Golgi structure, and N-glycan profiling. We found that all KO cell lines were uniformly deficient in cis/medial-Golgi glycosylation and each had nearly abolished binding of Cholera toxin. In addition, all cell lines showed defects in Golgi morphology, retrograde trafficking and sorting, sialylation and fucosylation, but severities varied according to the affected subunit. Lobe A and Cog6 subunit KOs displayed a more severely distorted Golgi structure, while Cog2, 3, 4, 5, and 7 knock outs had the most hypo glycosylated form of Lamp2. These results led us to conclude that every subunit is essential for COG complex function in Golgi trafficking, though to varying extents. We believe that this study and further analyses of these cells will help further elucidate the roles of individual COG subunits and bring a greater understanding to the class of MTCs as a whole. PMID:27066481

  9. Single cell optical transfection.

    PubMed

    Stevenson, David J; Gunn-Moore, Frank J; Campbell, Paul; Dholakia, Kishan

    2010-06-01

    The plasma membrane of a eukaryotic cell is impermeable to most hydrophilic substances, yet the insertion of these materials into cells is an extremely important and universal requirement for the cell biologist. To address this need, many transfection techniques have been developed including viral, lipoplex, polyplex, capillary microinjection, gene gun and electroporation. The current discussion explores a procedure called optical injection, where a laser field transiently increases the membrane permeability to allow species to be internalized. If the internalized substance is a nucleic acid, such as DNA, RNA or small interfering RNA (siRNA), then the process is called optical transfection. This contactless, aseptic, single cell transfection method provides a key nanosurgical tool to the microscopist-the intracellular delivery of reagents and single nanoscopic objects. The experimental possibilities enabled by this technology are only beginning to be realized. A review of optical transfection is presented, along with a forecast of future applications of this rapidly developing and exciting technology. PMID:20064901

  10. [Transfection of HL-60 cells by Venus lentiviral vector].

    PubMed

    Li, Zheng; Hu, Shao-Yan; Cen, Jian-Nong; Chen, Zi-Xing

    2013-06-01

    In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.

  11. A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1.

    PubMed

    Gonçalves, Cristine; Gross, Fabian; Guégan, Philippe; Cheradame, Hervé; Midou, Patrick

    2014-11-01

    Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 × 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 μg/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000™, and gene expression is higher than observed with FreeStyle™ and JetPEI®. Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications. PMID:25215936

  12. Electron tomography of HEK293T cells using scanning electron microscope-based scanning transmission electron microscopy.

    PubMed

    You, Yun-Wen; Chang, Hsun-Yun; Liao, Hua-Yang; Kao, Wei-Lun; Yen, Guo-Ji; Chang, Chi-Jen; Tsai, Meng-Hung; Shyue, Jing-Jong

    2012-10-01

    Based on a scanning electron microscope operated at 30 kV with a homemade specimen holder and a multiangle solid-state detector behind the sample, low-kV scanning transmission electron microscopy (STEM) is presented with subsequent electron tomography for three-dimensional (3D) volume structure. Because of the low acceleration voltage, the stronger electron-atom scattering leads to a stronger contrast in the resulting image than standard TEM, especially for light elements. Furthermore, the low-kV STEM yields less radiation damage to the specimen, hence the structure can be preserved. In this work, two-dimensional STEM images of a 1-μm-thick cell section with projection angles between ±50° were collected, and the 3D volume structure was reconstructed using the simultaneous iterative reconstructive technique algorithm with the TomoJ plugin for ImageJ, which are both public domain software. Furthermore, the cross-sectional structure was obtained with the Volume Viewer plugin in ImageJ. Although the tilting angle is constrained and limits the resulting structural resolution, slicing the reconstructed volume generated the depth profile of the thick specimen with sufficient resolution to examine cellular uptake of Au nanoparticles, and the final position of these nanoparticles inside the cell was imaged.

  13. Integrated Electrowetting Nanoinjector for Single Cell Transfection

    PubMed Central

    Shekaramiz, Elaheh; Varadarajalu, Ganeshkumar; Day, Philip J.; Wickramasinghe, H. Kumar

    2016-01-01

    Single cell transfection techniques are essential to understand the heterogeneity between cells. We have developed an integrated electrowetting nanoinjector (INENI) to transfect single cells. The high transfection efficiency, controlled dosage delivery and ease of INENI fabrication promote the widespread application of the INENI in cell transfection assays. PMID:27374766

  14. Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases.

    PubMed

    Nerys-Junior, Arildo; Costa, Lendel C; Braga-Dias, Luciene P; Oliveira, Márcia; Rossi, Atila D; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S; Tanuri, Amilcar

    2014-03-01

    Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity.

  15. Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

    PubMed Central

    Nerys-Junior, Arildo; Costa, Lendel C.; Braga-Dias, Luciene P.; Oliveira, Márcia; Rossi, Átila D.; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S.; Tanuri, Amilcar

    2014-01-01

    Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity. PMID:24688299

  16. Cell biology: Targeted transfection by femtosecond laser

    NASA Astrophysics Data System (ADS)

    Tirlapur, Uday K.; König, Karsten

    2002-07-01

    The challenge for successful delivery of foreign DNA into cells in vitro, a key technique in cell and molecular biology with important biomedical implications, is to improve transfection efficiency while leaving the cell's architecture intact. Here we show that a variety of mammalian cells can be directly transfected with DNA without perturbing their structure by first creating a tiny, localized perforation in the membrane using ultrashort (femtosecond), high-intensity, near-infrared laser pulses. Not only does this superior optical technique give high transfection efficiency and cell survival, but it also allows simultaneous evaluation of the integration and expression of the introduced gene.

  17. Nephrotoxicity evaluation of a new cembrane diterpene from Euphorbiae pekinensis Radix with HEK 293T cells and the toxicokinetics study in rats using a sensitive and reliable UFLC-MS/MS.

    PubMed

    Zhang, Yuanyuan; Liu, Ziying; Zhang, Ruowen; Hou, Pengyi; Bi, Kaishun; Chen, Xiaohui

    2016-02-01

    (-)-(1S)-15-Hydroxy-18-carboxycembrene, the first cembrane-type diterpenoid found in the family Euphorbiaceae, isolated from Euphorbiae pekinensis Radix, was identified to be nephrotoxic using HEK 293T cells. Tests on cell morphology, cell viability and biochemical markers about oxidation stress were carried out using inverted microscope, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and commercial kits respectively, which proved the diterpene time- and dose-dependently decreased cells proliferation. Besides, a sensitive and robust UFLC-MS/MS method was developed and fully validated to investigate the in vivo behavior in rats of the diterpene after oral administration of Euphorbiae pekinensis Radix extracts at a dosage of 9g/kg. The method showed a good linearity in tested range (3-1500ng/mL) with acceptable accuracy and precision. The recovery of the diterpene was more than 85% and the matrix effect was within ±20%. The toxicokinetics parameters indicate the diterpene reached Cmax quickly and slowly eliminate. The study proved the newly found diterpene was one of the nephrotoxic substances of Euphorbiae pekinensis Radix and revealed its toxicokinetics behavior.

  18. Optimization of Gene Transfection in Murine Myeloma Cell Lines using Different Transfection Reagents

    PubMed Central

    Shabani, Mahdi; Hemmati, Sheyda; Hadavi, Reza; Amirghofran, Zahra; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Shokri, Fazel

    2010-01-01

    Purification and isolation of cellular target proteins for monoclonal antibody (MAb) production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines (SP2/0, NS0, NS1, Ag8, and P3U1) were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein (EGFP), were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP2/0, 55.7%, 21.1% and 9.3% for NS0, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NS0 and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NS0 cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production. PMID:23408356

  19. Cell penetrating peptide-based polyplexes shelled with polysaccharide to improve stability and gene transfection

    NASA Astrophysics Data System (ADS)

    Li, Wenyu; Liu, Yajie; Du, Jianwei; Ren, Kefeng; Wang, Youxiang

    2015-04-01

    Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid-labile imine bonds (Az-I-Dex). The supramolecular polymer CDR/Az-I-Dex with high a C/A molar ratio (molar ratio of CD on CDR to Az on Az-I-Dex) was unfavorable for DNA condensation. The dextran shell of CDR/Az-I-Dex/DNA polyplexes improved the stability under physiological conditions. However, once treated with acetate buffer (pH 5.4) for 3 h, large aggregates formed rapidly due to the cleavage of the dextran shell. As expected, the vector had cell viability of 80% even when the CDR concentration increased to 100 μg mL-1. Moreover, due to the effective cellular uptake efficiency, CDR/Az-I-Dex/DNA polyplexes had 6-300 times higher transfection efficiency than CDR/DNA polyplexes. It was even higher than high molecular weight PLL-based polyplexes of HEK293 T cells. Importantly, chloroquine as an endosomal escape agent could not improve the transfection of CDR/Az-I-Dex/DNA polyplexes, which indicated that the CDR/Az-I-Dex supramolecular polymer had its own ability for endosomal escape. These results suggested that the CPP-based polyplexes shelled with polysaccharide can be promising non-viral gene delivery carriers.Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid

  20. Cell penetrating peptide-based polyplexes shelled with polysaccharide to improve stability and gene transfection.

    PubMed

    Li, Wenyu; Liu, Yajie; Du, Jianwei; Ren, Kefeng; Wang, Youxiang

    2015-05-14

    Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid-labile imine bonds (Az-I-Dex). The supramolecular polymer CDR/Az-I-Dex with high a C/A molar ratio (molar ratio of CD on CDR to Az on Az-I-Dex) was unfavorable for DNA condensation. The dextran shell of CDR/Az-I-Dex/DNA polyplexes improved the stability under physiological conditions. However, once treated with acetate buffer (pH 5.4) for 3 h, large aggregates formed rapidly due to the cleavage of the dextran shell. As expected, the vector had cell viability of 80% even when the CDR concentration increased to 100 μg mL(-1). Moreover, due to the effective cellular uptake efficiency, CDR/Az-I-Dex/DNA polyplexes had 6-300 times higher transfection efficiency than CDR/DNA polyplexes. It was even higher than high molecular weight PLL-based polyplexes of HEK293 T cells. Importantly, chloroquine as an endosomal escape agent could not improve the transfection of CDR/Az-I-Dex/DNA polyplexes, which indicated that the CDR/Az-I-Dex supramolecular polymer had its own ability for endosomal escape. These results suggested that the CPP-based polyplexes shelled with polysaccharide can be promising non-viral gene delivery carriers. PMID:25893559

  1. Using HEK293T Expression System to Study Photoactive Plant Cryptochromes

    PubMed Central

    Yang, Liang; Wang, Xu; Deng, Weixian; Mo, Weiliang; Gao, Jie; Liu, Qing; Zhang, Chuanyu; Wang, Qin; Lin, Chentao; Zuo, Zecheng

    2016-01-01

    Cryptochromes are photolyase-like blue light receptors that are conserved in plants and animals. Although the light-dependent catalytic mechanism of photolyase is well studied, the photochemical mechanism of cryptochromes remains largely unknown. Lack of an appropriate protein expression system to obtain photochemically active cryptochrome holoproteins is a technical obstacle for the study of plant cryptochromes. We report here an easy-to-use method to express and study Arabidopsis cryptochrome in HEK293T cells. Our results indicate that Arabidopsis cryptochromes expressed in HEK293T are photochemically active. We envision a broad use of this method in the functional investigation of plant proteins, especially in the large-scale analyses of photochemical activities of cryptochromes such as blue light-dependent protein–protein interactions. PMID:27446167

  2. Lipoplexes of dicationic gemini surfactants with DNA: Structural features of DNA compaction and transfection efficiency.

    PubMed

    Faizullin, D A; Zuev, Yu F; Zakharova, L Ya; Pokrovsky, A G; Korobeinikov, V A; Mukhametzyanov, T A; Konovalov, A I

    2015-01-01

    The internal structure of DNA lipoplexes with hydroxyethylated alkylammonium gemini surfactants (GS) with high transfection activity was studied by circular dichroism. It was shown that the efficiency of transfection of HEK293T cells with the pEGFP-N1 circular plasmid was different from zero only in the region of existence of chiral supramolecular DNA-GS complexes and reaches a maximum at concentrations at which the spontaneous aggregation of components is observed.

  3. Mammalian cell transfection: the present and the future

    PubMed Central

    Kim, Tae Kyung

    2010-01-01

    Transfection is a powerful analytical tool enabling study of the function of genes and gene products in cells. The transfection methods are broadly classified into three groups; biological, chemical, and physical. These methods have advanced to make it possible to deliver nucleic acids to specific subcellular regions of cells by use of a precisely controlled laser-microcope system. The combination of point-directed transfection and mRNA transfection is a new way of studying the function of genes and gene products. However, each method has its own advantages and disadvantages so the optimum method depends on experimental design and objective. PMID:20549496

  4. Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

    SciTech Connect

    Amand, Helene L.

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. Black-Right-Pointing-Pointer Dimer formation enhances peptiplex stability, resulting in increased transfection. Black-Right-Pointing-Pointer By dimerisation, the CPP EB1 even gain endosomal escape properties while lowering cytotoxicity. -- Abstract: Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes ('peptiplexes') enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the 'chelate effect' and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide dimer resulting from

  5. Biotinylated disulfide containing PEI/avidin bioconjugate shows specific enhanced transfection efficiency in HepG2 cells.

    PubMed

    Zeng, Xuan; Sun, Yun-Xia; Zhang, Xian-Zheng; Zhuo, Ren-Xi

    2009-10-21

    Targeting of non-viral gene vectors to liver cells could offer the opportunity to cure liver diseases. In this paper, disulfide-containing polyethylenimine (PEI-SS) was synthesized from low molecular weight branched PEI and cystamine bisacrylamide (CBA), and then grafted with biotin. The obtained biotinylated PEI-SS was bioconjugated with avidin via the biotin-avidin interaction to form a novel gene vector, biotinylated PEI-SS/avidin bioconjugate (ABP-SS). Characteristics of ABP-SS and its pDNA complexes were evaluated in terms of acid-base titration, agarose gel electrophoresis, SEM morphology observation, particle size and zeta-potential measurements, and PEI-SS was used as the control. The acid-base titration results showed that ABP-SS exhibited comparable buffer capability to 25 kDa PEI. The results of gel electrophoresis indicated that ABP-SS was able to condense pDNA efficiently at an N/P ratio of 6 and could be degraded by reducing agent DTT. The ABP-SS/pDNA complexes had a mean particle size of 226 +/- 40 nm and surface charges of 25 mV. The SEM images showed that the complexes had compact structures with spherical or quadrate shapes. In vitro cell viability and transfection of ABP-SS and PEI-SS were compared in HepG2, 293T and H446 cells. Among the three different cell lines, compared with PEI-SS, ABP-SS exhibited much lower cytotoxicity and higher transfection efficacy in HepG2 cells due to the biocompatibility of avidin and the specific interactions between avidin and HepG2 cells. Molecular probes were used to reveal the cellular uptake of complexes, and the results demonstrated that ABP-SS contributes to more cellular uptake of complexes in HepG2 cells, which was consistent with the transfection results.

  6. Transfection of insect cell in suspension for efficient baculovirus generation.

    PubMed

    Roest, S; Kapps-Fouthier, S; Klopp, J; Rieffel, S; Gerhartz, B; Shrestha, B

    2016-01-01

    Baculovirus (BV) mediated insect cell expression system utilizes transfection as a first step to introduce recombinant baculovirus DNA into insect cells. Many labs are still relying on the conventional liposome based transfection method in adherent culture. Here we describe a more efficient method that can replace the existing method. This method is economical and does not require any special adjustment in existing labs. •An innovative method of transfecting insect cells in suspension using polyethyleneimine (PEI) is described here.•The beauty of this method is minimal intermediate manipulation of culture during transfection and virus generation.•The method significantly reduces the chances of cross contamination of viruses while handling multiple targets and constructs as well as the other microbial contamination. PMID:27222826

  7. An analytical strategy to investigate Semen Strychni nephrotoxicity based on simultaneous HILIC-ESI-MS/MS detection of Semen Strychni alkaloids, tyrosine and tyramine in HEK 293t cell lysates.

    PubMed

    Gu, Liqiang; Hou, Pengyi; Zhang, Ruowen; Liu, Ziying; Bi, Kaishun; Chen, Xiaohui

    2016-10-15

    A Previous metabolomics study has demonstrated that tyrosine metabolism might be disrupted by treating with Semen Strychni on the cell nephrotoxicity model. To investigate the relationship between Semen Strychni alkaloids (SAs) and endogenous tyrosine, tyramine under the nephrotoxicity condition, an HILIC-ESI-MS/MS based analytical strategy was applied in this study. Based on the established Semen Strychni nephrotoxicity cell model, strychnine and brucine were identified and screened as the main SAs by an HPLC-Q Exactive hybrid quadrupole Orbitrap mass system. Then, a sensitive HILIC-ESI-MS/MS method was developed to simultaneously monitor strychnine, brucine, tyrosine and tyramine in cell lysate. The analytes were separated by a Shiseido CAPCELL CORE PC (150mm×2.1mm, 2.7μm) HILIC column in an acetonitrile/0.1% formic acid gradient system. All the calibration curves were linear with regression coefficients above 0.9924. The absolute recoveries were more than 80.5% and the matrix effects were between 91.6%-107.0%. With the developed method, analytes were successfully determined in cell lysates. Decreased levels of tyrosine and tyramine were observed only in combination with increased levels of SAs, indicating that the disturbance of tyrosine metabolism might be induced by the accumulation of SAs in kidney cell after exposure of Semen Strychni. The HILIC-ESI-MS/MS based analytical strategy is a useful tool to reveal the relationships between the toxic herb components and the endogenous metabolite profiling in the toxicity investigation of herb medicines.

  8. Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12°C.

    PubMed

    Marivin, E; Mourot, B; Loyer, P; Rime, H; Bobe, J; Fostier, A

    2015-09-15

    Over-expression or inhibition of gene expression can be efficiently used to analyse the functions and/or regulation of target genes. Modulation of gene expression can be achieved through transfection of exogenous nucleic acids into target cells. Such techniques require the development of specific protocols to transfect cell cultures with nucleic acids. The aim of this study was to develop a method of transfection suitable for rainbow trout granulosa cells in primary culture. After the isolation of rainbow trout granulosa cells, chemical transfection of cells with a fluorescent morpholino oligonucleotide (MO) was tested using FuGENE HD at 12 °C. Electroporation was also employed to transfect these cells with either a plasmid or MO. Transfection was more efficient using electroporation (with the following settings: 1200 V/40 ms/1p) than chemical transfection, but electroporation by itself was deleterious, resulting in a decrease of the steroidogenic capacity of the cells, measured via estradiol production from its androgenic substrate. The disturbance of cell biology induced by the transfection method per se should be taken into account in data interpretation when investigating the effects of under- or over-expression of candidate genes.

  9. Transient transfection of mammalian cells using a violet diode laser

    NASA Astrophysics Data System (ADS)

    Torres-Mapa, Maria Leilani; Angus, Liselotte; Ploschner, Martin; Dholakia, Kishan; Gunn-Moore, Frank J.

    2010-07-01

    We demonstrate the first use of the violet diode laser for transient mammalian cell transfection. In contrast to previous studies, which showed the generation of stable cell lines over a few weeks, we develop a methodology to transiently transfect cells with an efficiency of up to ~40%. Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293) cells are exposed to a tightly focused 405-nm laser in the presence of plasmid DNA encoding for a mitochondrial targeted red fluorescent protein. We report transfection efficiencies as a function of laser power and exposure time for our system. We also show, for the first time, that a continuous wave laser source can be successfully applied to selective gene silencing experiments using small interfering RNA. This work is a major step towards an inexpensive and portable phototransfection system.

  10. Carbon nanoparticles for gene transfection in eukaryotic cell lines.

    PubMed

    Zanin, H; Hollanda, L M; Ceragioli, H J; Ferreira, M S; Machado, D; Lancellotti, M; Catharino, R R; Baranauskas, V; Lobo, A O

    2014-06-01

    For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

  11. Culture and transfection of axolotl cells.

    PubMed

    Denis, Jean-François; Sader, Fadi; Ferretti, Patrizia; Roy, Stéphane

    2015-01-01

    The use of cells grown in vitro has been instrumental for multiple aspects of biomedical research and especially molecular and cellular biology. The ability to grow cells from multicellular organisms like humans, squids, or salamanders is important to simplify the analyses and experimental designs to help understand the biology of these organisms. The advent of the first cell culture has allowed scientists to tease apart the cellular functions, and in many situations these experiments help understand what is happening in the whole organism. In this chapter, we describe techniques for the culture and genetic manipulation of an established cell line from axolotl, a species widely used for studying epimorphic regeneration.

  12. Culture and transfection of axolotl cells.

    PubMed

    Denis, Jean-François; Sader, Fadi; Ferretti, Patrizia; Roy, Stéphane

    2015-01-01

    The use of cells grown in vitro has been instrumental for multiple aspects of biomedical research and especially molecular and cellular biology. The ability to grow cells from multicellular organisms like humans, squids, or salamanders is important to simplify the analyses and experimental designs to help understand the biology of these organisms. The advent of the first cell culture has allowed scientists to tease apart the cellular functions, and in many situations these experiments help understand what is happening in the whole organism. In this chapter, we describe techniques for the culture and genetic manipulation of an established cell line from axolotl, a species widely used for studying epimorphic regeneration. PMID:25740487

  13. Photoporation and cell transfection using a violet diode laser

    NASA Astrophysics Data System (ADS)

    Paterson, L.; Agate, B.; Comrie, M.; Ferguson, R.; Lake, T. K.; Morris, J. E.; Carruthers, A. E.; Brown, C. T. A.; Sibbett, W.; Bryant, P. E.; Gunn-Moore, F.; Riches, A. C.; Dholakia, Kishan

    2005-01-01

    The introduction and subsequent expression of foreign DNA inside living mammalian cells (transfection) is achieved by photoporation with a violet diode laser. We direct a compact 405 nm laser diode source into an inverted optical microscope configuration and expose cells to 0.3 mW for 40 ms. The localized optical power density of ~1200 MW/m2 is six orders of magnitude lower than that used in femtosecond photoporation (~104 TW/m2). The beam perforates the cell plasma membrane to allow uptake of plasmid DNA containing an antibiotic resistant gene as well as the green fluorescent protein (GFP) gene. Successfully transfected cells then expand into clonal groups which are used to create stable cell lines. The use of the violet diode laser offers a new and simple poration technique compatible with standard microscopes and is the simplest method of laser-assisted cell poration reported to date.

  14. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    PubMed

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.

  15. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    PubMed

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination. PMID:27349114

  16. Characterization of cell lines stably transfected with rubella virus replicons

    SciTech Connect

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  17. Surface modified gold nanowires for mammalian cell transfection

    NASA Astrophysics Data System (ADS)

    Kuo, Chiung-Wen; Lai, Jun-Jung; Wei, Kung Hwa; Chen, Peilin

    2008-01-01

    Aminothiol modified gold nanowires have been used as vectors for the delivery of plasmid DNA into two different types of mammalian cells: 3T3 and HeLa. It was measured that positively charged gold nanowires with a diameter of 200 nm and a length around 5 µm were capable of carrying 1 pg of plasmid DNA per nanowire into cells. Compared with other transfection reagents, the gold nanowires exhibited the highest transfection efficiency while almost no cytotoxicity was observed. In addition, it has been shown that individual nanowires can be visualized with sub-micrometer resolution, which may allow the use of functionalized multi-segment nanowires as local probes for the investigation of the microenvironment inside cells.

  18. In vitro transformation of mouse testis cells by oncogene transfection.

    PubMed

    Morimoto, Hiroko; Lee, Jiyoung; Tanaka, Takashi; Ishii, Kei; Toyokuni, Shinya; Kanatsu-Shinohara, Mito; Shinohara, Takashi

    2012-05-01

    Germ cell tumors (GCTs) are unique in that they exhibit diverse biological characteristics and pathological features. Although several in vivo GCT models are available, studies on GCTs are hampered because in vivo development of GCTs is time consuming and prevents a detailed molecular analysis of the transformation process. Here we developed a novel strategy to transform mouse testis cells in vitro. Lentivirus-mediated transfection of dominant negative Trp53, Myc, and activated Hras1 into a CD9-expressing testis cells caused tumorigenic conversion in vitro. Although these cells resembled embryonic stem (ES) cells, they were aneuploid and lacked Nanog expression, which is involved in the maintenance of the undifferentiated state in ES cells. Euploid ES-like cells were produced by transfecting the Yamanaka factors (Pou5f1, Myc, Klf4, and Sox2) into the same cell population. Although these cells expressed Nanog, they were distinct from ES cells in that they expressed CD44, a cancer stem cell antigen. Both treatments induced similar changes in the DNA methylation patterns in differentially methylated regions of imprinted genes. Moreover, despite the differences in their phenotype and karyotype, both cell types similarly produced mixed GCTs on transplantation, which were composed of teratomas, seminomas, and embryonal carcinomas. Thus, in vitro testis cell transformation facilitates an analysis of the GCT formation process, and our results also suggest the close similarity between GCT formation and reprogramming. PMID:22357549

  19. Cell transfection as a tool to study growth hormone action

    SciTech Connect

    Norstedt, G.; Enberg, B.; Francis, S.

    1994-12-31

    The isolation of growth hormone receptor (GHR) cDNA clones has made possible the transfection of GHRs into cultured cells. Our aim in this minireview is to show how the application of such approaches have benefited GHR research. GH stimulation of cells expressing GHR cDNAs can cause an alteration of cellular function that mimic those of the endogenous GHR. GHR cDNA transfected cells also offer a system where the mechanism of GH action can be studied. Such a system has been used to demonstrate that the GHR itself becomes tyrosine phosphorylated and that further phosphorylation of downstream proteins is important in GH action. The GH signals are transmitted to the nucleus and GH regulated genes have now begun to be characterized. The ability to use cell transfection for mechanistic studies of GH action will be instrumental to define domains within the receptor that are of functional importance and to determined pathways whereby GH signals are conveyed within the cell. 33 refs., 2 tabs.

  20. Immune Monitoring Using mRNA-Transfected Dendritic Cells.

    PubMed

    Borch, Troels Holz; Svane, Inge Marie; Met, Özcan

    2016-01-01

    Dendritic cells are known to be the most potent antigen presenting cell in the immune system and are used as cellular adjuvants in therapeutic anticancer vaccines using various tumor-associated antigens or their derivatives. One way of loading antigen into the dendritic cells is by mRNA electroporation, ensuring presentation of antigen through major histocompatibility complex I and potentially activating T cells, enabling them to kill the tumor cells. Despite extensive research in the field, only one dendritic cell-based vaccine has been approved. There is therefore a great need to elucidate and understand the immunological impact of dendritic cell vaccination in order to improve clinical benefit. In this chapter, we describe a method for performing immune monitoring using peripheral blood mononuclear cells and autologous dendritic cells transfected with tumor-associated antigen-encoding mRNA. PMID:27236804

  1. Stimulation of human Jurkat cells by monoclonal antibody crosslinking of transfected-Ly-6A.2 (TAP) molecules.

    PubMed

    McGrew, J T; Rock, K L

    1991-10-01

    Monoclonal antibody crosslinking of phosphatidylinositol-anchored Ly-6A.2 molecules on the surface of murine T lymphocytes leads to cell activation and secretion of IL-2. To examine the potential activity of these molecules in human T cells we transfected the Ly-6A.2 gene into Jurkat cells. Transfection of Jurkat cells with genomic Ly-6A.2 sequences results in low levels of Ly-6A.2 on the cell surface. However, linking the Ly-6A.2 sequences to the enhancer from the human CD2 gene results in greatly increased expression of Ly-6A.2. These molecules are anchored to the membrane via a phosphatidylinositol linkage. Crosslinking of Ly-6A.2 molecules with soluble mAb stimulates the transfected Jurkat cells to produce IL-2. This stimulation is abrogated by treatment with phosphatidylinositol-specific phospholipase C. The transfected human T cells displayed the same unusual crosslinking requirements for stimulation with anti-Ly-6A.2 mAbs as previously observed for murine T cells. Crosslinking of Ly-6A.2 with soluble antibodies is stimulatory, whereas immobilized antibodies are inactive. The crosslinking requirements for antiCD3 mAb stimulation display a reciprocal pattern. These data demonstrate that the Ly-6A.2 pathway for T cell activation is conserved between human and murine T cells.

  2. Gold Nanoparticles Enhanced Electroporation for Mammalian Cell Transfection

    PubMed Central

    Zu, Yingbo; Huang, Shuyan; Liao, Wei-Ching; Lu, Yang; Wang, Shengnian

    2015-01-01

    Electroporation figured prominently as an effective nonviral gene delivery approach for its balance on the transfection efficiency and cell viability, no restrictions of probe or cell type, and operation simplicity. The commercial electroporation systems have been widely adopted in the past two decades while still carry drawbacks associated with the high applied electric voltage, unsatisfied delivery efficiency, and/or low cell viability. By adding highly conductive gold nanoparticles (AuNPs) in electroporation solution, we demonstrated enhanced electroporation performance (i.e. better DNA delivery efficiency and higher cell viability) on mammalian cells from two different aspects: the free, naked AuNPs reduce the resistance of the electroporation solution so that the local pulse strength on cells was enhanced; targeting AuNPs (e.g., Tf-AuNPs) were brought to the cell membrane to work as virtual microelectrodes to porate cells with limited area from many different sites. The enhancement was confirmed with leukemia cells in both a commercial batch electroporation system and a home-made flow-through system using pWizGFP plasmid DNA probes. Such enhancement depends on the size, concentration, and the mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increased 2-3 folds at minimum sacrifice of cell viability. This new delivery concept, the combination of nanoparticles and electroporation technologies, may stimulate various in vitro and in vivo biomedical applications which rely on the efficient delivery of nucleic acids, anticancer drugs, or other therapeutic materials. PMID:24749393

  3. Gold nanoparticles enhanced electroporation for mammalian cell transfection.

    PubMed

    Zu, Yingbo; Huang, Shuyan; Liao, Wei-Ching; Lu, Yang; Wang, Shengnian

    2014-06-01

    Electroporation figured prominently as an effective nonviral gene delivery approach for its balance on the transfection efficiency and cell viability, no restrictions of probe or cell type, and operation simplicity. The commercial electroporation systems have been widely adopted in the past two decades while still carry drawbacks associated with the high applied electric voltage, unsatisfied delivery efficiency, and/or low cell viability. By adding highly conductive gold nanoparticles (AuNPs) in electroporation solution, we demonstrated enhanced electroporation performance (i.e., better DNA delivery efficiency and higher cell viability) on mammalian cells from two different aspects: the free, naked AuNPs reduce the resistance of the electroporation solution so that the local pulse strength on cells was enhanced; targeting AuNPs (e.g., Tf-AuNPs) were brought to the cell membrane to work as virtual microelectrodes to porate cells with limited area from many different sites. The enhancement was confirmed with leukemia cells in both a commercial batch electroporation system and a home-made flow-through system using pWizGFP plasmid DNA probes. Such enhancement depends on the size, concentration, and the mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increased 2-3 folds at minimum sacrifice of cell viability. This new delivery concept, the combination of nanoparticles and electroporation technologies, may stimulate various in vitro and in vivo biomedical applications which rely on the efficient delivery of nucleic acids, anticancer drugs, or other therapeutic materials.

  4. Gold nanoparticle-enhanced electroporation for leukemia cell transfection.

    PubMed

    Huang, Shuyan; Zu, Yingbo; Wang, Shengnian

    2014-01-01

    Electroporation serves as an attractive nonviral gene delivery approach for its effectiveness, operational simplicity, and no restrictions of probe or cell type. The commercial electroporation systems have been widely adopted in research and clinics with protocols usually compromising appropriate transfection efficiency and cell viability. By introducing gold nanoparticles (AuNPs), we demonstrated greatly enhanced performance of electroporation from two aspects: the highly conductive, naked AuNPs help reduce the potential drop consumed by the electroporation solution so that the majority of the applied voltage of an electric pulse is truly imposed on cells with enhanced field strength; AuNPs with targeting ligands (e.g., transferrin-AuNPs or Tf-AuNPs) are bound to the cell membrane, working as virtual microelectrodes to create pores on cells with limited opening area while from many different sites. The addition of AuNPs during electroporation therefore benefits not only quicker recovery and better survival of cells but also more efficient uptake of the subjected probes. Such enhancement was successfully confirmed on a chronic myeloid leukemia cell line (i.e., K562 cells) in both a commercial batch electroporation system and a homemade flow system with pWizGFP plasmid DNA probes. The efficiency was found to be dependent on the size, concentration, and mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increase of two to threefold at a minimum sacrifice of the cell viability.

  5. Delivery of episomal vectors into primary cells by means of commercial transfection reagents.

    PubMed

    Han, Na Rae; Lee, Hyun; Baek, Song; Yun, Jung Im; Park, Kyu Hyun; Lee, Seung Tae

    2015-05-29

    Although episomal vectors are commonly transported into cells by electroporation, a number of electroporation-derived problems have led to the search for alternative transfection protocols, such as the use of transfection reagents, which are inexpensive and easy to handle. Polyplex-mediated transport of episomal vectors into the cytoplasm has been conducted successfully in immortalized cell lines, but no report exists of successful transfection of primary cells using this method. Accordingly, we sought to optimize the conditions for polyplex-mediated transfection for effective delivery of episomal vectors into the cytoplasm of primary mouse embryonic fibroblasts. Episomal vectors were complexed with the commercially available transfection reagents Lipofectamine 2000, FuGEND HD and jetPEI. The ratio of transfection reagent to episomal vectors was varied, and the subsequent transfection efficiency and cytotoxicity of the complexes were analyzed using flow cytometry and trypan blue exclusion assay, respectively. No cytotoxicity and the highest transfection yield were observed when the ratio of transfection reagent to episomal vector was 4 (v/wt) in the cases of Lipofectamine 2000 and FuGENE HD, and 2 in the case of jetPEI. Of the three transfection reagents tested, jetPEI showed the highest transfection efficiency without any cytotoxicity. Thus, we confirmed that the transfection reagent jetPEI could be used to effectively deliver episomal vectors into primary cells without electroporation.

  6. Asymmetric partitioning of transfected DNA during mammalian cell division

    PubMed Central

    Wang, Xuan; Le, Nhung; Denoth-Lippuner, Annina; Barral, Yves; Kroschewski, Ruth

    2016-01-01

    Foreign DNA molecules and chromosomal fragments are generally eliminated from proliferating cells, but we know little about how mammalian cells prevent their propagation. Here, we show that dividing human and canine cells partition transfected plasmid DNA asymmetrically, preferentially into the daughter cell harboring the young centrosome. Independently of how they entered the cell, most plasmids clustered in the cytoplasm. Unlike polystyrene beads of similar size, these clusters remained relatively immobile and physically associated to endoplasmic reticulum-derived membranes, as revealed by live cell and electron microscopy imaging. At entry of mitosis, most clusters localized near the centrosomes. As the two centrosomes split to assemble the bipolar spindle, predominantly the old centrosome migrated away, biasing the partition of the plasmid cluster toward the young centrosome. Down-regulation of the centrosomal proteins Ninein and adenomatous polyposis coli abolished this bias. Thus, we suggest that DNA clustering, cluster immobilization through association to the endoplasmic reticulum membrane, initial proximity between the cluster and centrosomes, and subsequent differential behavior of the two centrosomes together bias the partition of plasmid DNA during mitosis. This process leads to their progressive elimination from the proliferating population and might apply to any kind of foreign DNA molecule in mammalian cells. Furthermore, the functional difference of the centrosomes might also promote the asymmetric partitioning of other cellular components in other mammalian and possibly stem cells. PMID:27298340

  7. B35 neuroblastoma cells: an easily transfected, cultured cell model of central nervous system neurons.

    PubMed

    Otey, Carol A; Boukhelifa, Malika; Maness, Patricia

    2003-01-01

    A panel of neuronal cell lines was derived from tumors of the neonatal rat central nervous system (CNS) in 1974, and two of these lines are in wide use today. Both the B35 and B50 lines offer a number of advantages to researchers who study CNS neurons in culture: they are simple to grow, to differentiate, and to transfect. B50 cells have been used extensively in the study of neuronal cell death, toxicology, and differentiation, whereas B35 cells have proven useful in the molecular analysis of endocytosis and of signaling pathways, in particular those that guide axonal outgrowth and cell motility. This chapter provides protocols for growing and transfecting B35 cells, selecting stable transfectants, exploring protein function using an antisense approach, and assaying cell motility in a Transwell chamber. All of these protocols have been written for researchers who have some skill in basic cell culture techniques, but previous experience with cultured neurons is not required.

  8. In vivo transfection of melanoma cells by lithotripter shock waves.

    PubMed

    Bao, S; Thrall, B D; Gies, R A; Miller, D L

    1998-01-15

    The potential for gene transfection during shock wave tumor therapy was evaluated by searching for shock wave-induced DNA transfer in mouse tumor cells. B16 mouse melanoma cells were cultured by standard methods and implanted s.c. in female C57BL/6 mice 10-14 days before treatment. A luciferase reporter vector was used as the DNA plasmid for intratumoral injection at 0.2 mg/ml tumor. Air at 10% of tumor volume was injected after the DNA in some tumors to enhance acoustic cavitation activity. The shock wave generation system was similar to a Dornier HM-3 lithotripter with pressure amplitudes of 24.4 MPa peak positive and 5.2 MPa peak negative. Luciferase production in isolated tumor cells was measured with a luminometer 1 day after treatment to assess gene transfer and expression. Exposure to 800 shock waves, followed by immediate isolation and culture of tumor cells for 1 day, yielded 1.1 (0.43 SE) pg/10(6) cells for plasmid injection only and 7.5 (2.5 SE) pg/10(6) cells for plasmid plus air injection. Significantly increased luciferase production, relative to shams, occurred for 200-, 400-, 800-, and 1200-shock wave treatments with plasmid and air injection. Exposure with the isolation of tumor cells delayed for a day to allow gene expression within the growing tumors gave increased luciferase production for 100- and 400-shock wave exposures without and with air injection. Gene transfer therefore can be induced during lithotripter shock wave treatment in vivo, particularly with enhanced acoustic cavitation, which supports the concept that gene and shock wave therapy might be advantageously merged. PMID:9443395

  9. Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture.

    PubMed

    Majumdar, M; Ratho, R; Chawla, Y; Singh, M P

    2014-01-01

    The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs) were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA) and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001), even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET) technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures. PMID:24713904

  10. Transfection-mediated cell synchronization: acceleration of G1-S phase transition by gamma irradiation.

    PubMed

    Jung, E J; Flemington, E K

    2001-11-01

    We have previously provided evidence that the uptake of DNA into cells is cell cycle specific following transfection. We show here that, immediately after transfection, successfully transfected cells are greatly enriched for cells in early G1 or G0 phase and that, upon removal of the DNA precipitates, cells progress through G1 and enter S phase in a synchronous fashion. We also demonstrate that this approach can be utilized in meaningful cell-cycle experiments, and we show that gamma irradiation accelerates the G1-S phase transition in a cell line with a functionally inactive p53 protein. PMID:11730009

  11. Transplantation of human telomerase reverse transcriptase gene-transfected Schwann cells for repairing spinal cord injury

    PubMed Central

    Zhang, Shu-quan; Wu, Min-fei; Liu, Jia-bei; Li, Ye; Zhu, Qing-san; Gu, Rui

    2015-01-01

    Transfection of the human telomerase reverse transcriptase (hTERT) gene has been shown to increase cell proliferation and enhance tissue repair. In the present study, hTERT was transfected into rat Schwann cells. A rat model of acute spinal cord injury was established by the modified free-falling method. Retrovirus PLXSN was injected at the site of spinal cord injury as a vector to mediate hTERT gene-transfected Schwann cells (1 × 1010/L; 10 μL) or Schwann cells (1 × 1010/L; 10 μL) without hTERT gene transfection. Between 1 and 4 weeks after model establishment, motor function of the lower limb improved in the hTERT-transfected group compared with the group with non-transfected Schwann cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and reverse transcription-polymerase chain reaction results revealed that the number of apoptotic cells, and gene expression of aquaporin 4/9 and matrix metalloproteinase 9/2 decreased at the site of injury in both groups; however, the effect improved in the hTERT-transfected group compared with the Schwann cells without hTERT transfection group. Hematoxylin and eosin staining, PKH26 fluorescent labeling, and electrophysiological testing demonstrated that compared with the non-transfected group, spinal cord cavity and motor and sensory evoked potential latencies were reduced, while the number of PKH26-positive cells and the motor and sensory evoked potential amplitude increased at the site of injury in the hTERT-transfected group. These findings suggest that transplantation of hTERT gene-transfected Schwann cells repairs the structure and function of the injured spinal cord. PMID:26889196

  12. Non-Viral Transfection Methods Optimized for Gene Delivery to a Lung Cancer Cell Line

    PubMed Central

    Salimzadeh, Loghman; Jaberipour, Mansooreh; Hosseini, Ahmad; Ghaderi, Abbas

    2013-01-01

    Background Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. Methods In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Results Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. Conclusion This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods. PMID:23799175

  13. Dielectrophoresis-assisted 3D nanoelectroporation for non-viral cell transfection in adoptive immunotherapy.

    PubMed

    Chang, Lingqian; Gallego-Perez, Daniel; Zhao, Xi; Bertani, Paul; Yang, Zhaogang; Chiang, Chi-Ling; Malkoc, Veysi; Shi, Junfeng; Sen, Chandan K; Odonnell, Lynn; Yu, Jianhua; Lu, Wu; Lee, L James

    2015-08-01

    Current transfection technologies lead to significant inter-clonal variations. Previously we introduced a unique electrotransfection technology, Nanochannel-Electroporation (NEP), which can precisely and benignly transfect small cell populations (~100-200 cells) with single-cell resolution. Here we report on the development of a novel 3D NEP system for large scale transfection. A properly-engineered array of nanochannels, capable of handling/transfecting ~60 000 cells cm(-2), was fabricated using cleanroom technologies. Positive dielectrophoresis was used to selectively position cells on the nanochannels, thus allowing highly efficient transfection. Single-cell dosage control was demonstrated using both small and large molecules, and different cell types. The potential clinical relevance of this system was tested with difficult-to-transfect natural killer cell suspensions, and plasmids encoding for the chimeric antigen receptor (CAR), a model of high relevance for adoptive immunotherapy. Our results show significantly higher CAR transfection efficiencies for the DEP-NEP system (>70% vs. <30%), as well as enhanced cell viabilities.

  14. Characterization of insulin-producing cells derived from PDX-1-transfected neural stem cells.

    PubMed

    Wang, Hailan; Jiang, Zesheng; Li, Aihui; Gao, Yi

    2012-12-01

    Islet cell transplantation is a promising treatment strategy for type-1 diabetes. However, functional islet cells are hard to obtain for transplantation and are in short supply. Directing the differentiation of stem cells into insulin‑producing cells, which serve as islet cells, would overcome this shortage. Bone marrow contains hematopoietic stem cells and mesenchymal stem cells. The present study used bone marrow cells isolated from rats and neural stem cells (NSCs) that were derived from bone marrow cells in culture. Strong nestin staining was detected in NSCs, but not in bone marrow stromal cells (BMSCs). In vitro transfection of the pancreatic duodenal homeobox-1 (PDX-1) gene into NSCs generated insulin‑producing cells. Reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analysis confirmed that PDX-1-transfected NSCs expressed insulin mRNA and released insulin protein. However, insulin release from PDX-1-transfected NSCs did not respond to the challenge of glucose and glucagon-like peptide-1. These results support the use of bone marrow-derived NSCs as a renewable source of insulin-producing cells for autologous transplantation to treat type-1 diabetes.

  15. Optimizing electroporation conditions in primary and other difficult-to-transfect cells.

    PubMed

    Jordan, Elizabeth T; Collins, Michelle; Terefe, Joseph; Ugozzoli, Luis; Rubio, Teresa

    2008-12-01

    Electroporation is a valuable tool for nucleic acid delivery because it can be used for a wide variety of cell types. Many scientists are shifting toward the use of cell types that are more relevant to in vivo applications, including primary cells, which are considered difficult to transfect. The ability to electroporate these cell types with nucleic acid molecules of interest at a relatively high efficiency while maintaining cell viability is essential for elucidating the pathway(s) in which a gene product is involved. We present data demonstrating that by optimizing electroporation parameters, nucleic acid molecules can be delivered in a highly efficient manner. We display transfection results for primary and difficult-to-transfect cell types including human primary fibroblasts, human umbilical vein endothelial cells, Jurkat cells, and two neuroblastoma cell lines [SK-N-SH (human) and Neuro-2A (mouse)] with plasmid DNAs and siRNAs. Our data demonstrate that by determining proper electroporation conditions, glyceraldehyde phosphate dehydrogenase mRNA was silenced in Jurkat cells when compared with negative control siRNA electroporations as early as 4 h post-transfection. Other experiments demonstrated that optimized electroporation conditions using a fluorescently labeled transfection control siRNA resulted in 75% transfection efficiency for Neuro-2A, 93% for human primary fibroblasts, and 94% for HUVEC cells, as analyzed by flow cytometry. PMID:19183796

  16. Femtosecond optical transfection as a tool for genetic manipulation of human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Torres-Mapa, M. L.; Gardner, J.; Bradburn, H.; King, J.; Dholakia, K.; Gunn-Moore, F.

    2013-03-01

    We demonstrate the use of femtosecond optical transfection for the genetic manipulation of human embryonic stem cells. Using a system with an SLM combined with a scanning mirror allows poration of both single-cell and colony-formed human embryonic stem cells in a rapid and targeted manner. In this work, we show successful transfection of plasmid DNA tagged with fluorescent reporters into human embryonic stem cells using three doses of focused femtosecond laser. A significant number of transfected cells retained their undifferentiated morphological feature of large nucleus with high nucleus to cytoplasmic ratio, 48h after photoporation. Furthermore, DNA constructs driven by different types of promoters were also successfully transfected into human embryonic stem cells using this technique.

  17. A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing

    PubMed Central

    Woodruff, Kristina; Maerkl, Sebastian J.

    2016-01-01

    Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration and synthetic gene circuits can be optimized on chip. We co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies. PMID:27030663

  18. Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency

    PubMed Central

    Nii, Takenobu; Kohara, Hiroshi; Marumoto, Tomotoshi; Sakuma, Tetsushi; Yamamoto, Takashi; Tani, Kenzaburo

    2016-01-01

    Abstract Efficient gene transfer into human pluripotent stem cells (hPSCs) holds great promise for regenerative medicine and pharmaceutical development. In the past decade, various methods were developed for gene transfer into hPSCs; however, hPSCs form tightly packed colonies, making gene transfer difficult. In this study, we established a stable culture method of hPSCs at a single-cell state to reduce cell density and investigated gene transfection efficiency followed by gene editing efficiency. hPSCs cultured in a single-cell state were transfected using nonliposomal transfection reagents with plasmid DNA or mRNA encoding enhanced green fluorescent protein. We found that most cells (DNA > 90%; mRNA > 99%) were transfected without the loss of undifferentiated PSC marker expression or pluripotency. Moreover, we demonstrated an efficient gene editing method using transcription activator-like effector nucleases (TALENs) targeting the adenomatous polyposis coli (APC) gene. Our new method may improve hPSC gene transfer techniques, thus facilitating their use for human regenerative medicine. PMID:27257519

  19. Ultrasound-mediated gene transfection: A comparison between cells irradiated in suspension and attachment status

    NASA Astrophysics Data System (ADS)

    Zhang, Yiwei; Azuma, Takashi; Sasaki, Akira; Yoshinaka, Kiyoshi; Takagi, Shu; Matsumoto, Yoichiro

    2012-10-01

    Sonoporation, in the presence of microbubbles, is a promising nonviral gene transfection method. Although the mechanism is not yet fully understood, shock waves emitted by cavitation bubbles have been known to play an important role in creating pores on cell membranes. This work investigates the gene transfection efficiency and influencing parameters of cells in two different statuses: attachment and suspension based on the fact that cells in suspension have more bubbles surrounding them and that shock wave has distinct effects on hit objects whether the object is attached to a rigid wall or not. Fibroblast cells (NIH3T3), both in attachment and suspension, and green fluorescent protein (GFP) plasmid were exposed to variations in acoustic pressure (0.6-1.2 MPa) and 10% duty cycle at fixed settings of 2 MHz central frequency, 5 kHz pulse repetition frequency and 1 minute insonation time, in the presence of 10% v/v microbubbles (Sonazoid, a commercialized product of ultrasound contrast agent). The transfection efficiency and cell viability are compared for two statuses and a distribution map of GFP transfected cells as well as viable cells over the well bottom is given for attachment status. The results show that cells irradiated in suspension status has higher transfection ratio as well as viability than those irradiated in attachment status with the same intensity and that the transfected cells of attachment status experiment are highly concentrated near the center of the well.

  20. Bacterial IMPDH gene used for the selection of mammalian cell transfectants.

    SciTech Connect

    Baccam, M.; Huberman, E.; Energy Systems

    2003-06-01

    Stable cell transfection is used for the expression of exogenous genes or cDNAs in eukaryotic cells. Selection of these transfectants requires a dominant selectable marker. A variety of such markers has been identified and is currently in use. However, many of these are not suitable for all cell types or require unique conditions. Here we describe a simple and versatile dominant selectable marker that involves bacterial IMP dehydrogenase (IMPDH), an enzyme essential for the replication of mammalian and bacterial cells. Although IMPDH is evolutionarily conserved, the bacterial enzyme is orders of magnitude more resistant to the toxic effect of the drug mycophenolic acid, which is an IMPDH inhibitor. We have demonstrated that transfection of human, monkey or Chinese hamster cell lines with an expression vector containing bacterial IMPDH and mycophenolic acid treatment resulted in the selection of colonies with a strikingly increased resistance to mycophenolic acid toxicity. Analysis of cells derived from these colonies indicated that the acquisition of this resistance was associated with bacterial IMPDH protein expression. As a proof of principle, we showed that mammalian cell transfection with a hicistronic IMPDH/GFP expression vector and mycophenolic acid treatment can he used to successfully select transfectants that express the fluorescent protein. These results indicate that bacterial IMPDH is a practical dominant selectable marker that can be used for the selection of transfectants that express exogenous genes or cDNAs in mammalian cells.

  1. DEVELOPMENT OF AN ENVIRONMENTAL ESTROGEN SCREEN USING TRANSIENTLY TRANSFECTED RAINBOW TROUT CELL LINES

    EPA Science Inventory

    Rainbow troutp hepatoma (RTH-149) and gonad cells (RTG-2) were used to develop a screening protocol for estrogen disrupting chemicals. Transfection of an estrogen-responsive luciferase reporter plasmid into...

  2. Targeted microbubbles for ultrasound mediated gene transfection and apoptosis induction in ovarian cancer cells

    PubMed Central

    Zhu, Shenyin; Yan, Yu; Zhu, Yi; Li, Min; Wang, Zhigang; Xu, Ronald X.

    2015-01-01

    Ultrasound-targeted microbubble destruction (UTMD) technique can be potentially used for non-viral delivery of gene therapy. Targeting wild-type p53 (wtp53) tumor suppressor gene may provide a clinically promising treatment for patients with ovarian cancer. However, UTMD mediated gene therapy typically uses non-targeted microbubbles with suboptimal gene transfection efficiency. We synthesized a targeted microbubble agent for UTMD mediated wtp53 gene therapy in ovarian cancer cells. Lipid micro-bubbles were conjugated with a Luteinizing Hormone–Releasing Hormone analog (LHRHa) via an avidin– biotin linkage to target the ovarian cancer A2780/DDP cells that express LHRH receptors. The microbubbles were mixed with the pEGFP-N1-wtp53 plasmid. Upon exposure to 1 MHz pulsed ultrasound beam (0.5 W/cm2) for 30 s, the wtp53 gene was transfected to the ovarian cancer cells. The transfection efficiency was (43.90 ± 6.19)%. The expression of wtp53 mRNA after transfection was (97.08 ± 12.18)%. The cell apoptosis rate after gene therapy was (39.67 ± 5.95)%. In comparison with the other treatment groups, ultrasound mediation of targeted microbubbles yielded higher transfection efficiency and higher cell apoptosis rate (p < 0.05). Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles will enhance the gene transfection efficiency in ovarian cancer cells. PMID:22841613

  3. [Transfection efficiency of adenoviral vector AD5/F35 to malignant hematopoietic cells of different origins].

    PubMed

    Wabg, Kai; Peng, Jian-Qinag; Yuan, Zhen-Hua; Wu, Xiao-Bin

    2006-06-01

    This study was aimed to investigate the transfection efficiency of adenoviral vector AD5/F35 to hematopoietic malignant cells lines of various origins and AD5/F35 cytotoxicity. The hematologic malignant cell lines of various origins were transfected by AD5/F35-EGFP at different multiple of infection (MOI) and AD5-EGFP was used as control; the proportion of fluorescence positive cells was detected by flow cytometry; the killing effect of virus on infective target cells was assayed by MTT and observed by fluorescence microscopy. The results showed that the transfection efficiency of AD5/F35 vector to cell line of myeloid origin was > 99% at MOI = 30, the transfective efficiency of AD5 vector was 26.4% at MOI = 1,000; the transfection efficiency of AD5/F35 vector and AD5 vector to cell line of B cell origin were 11.7% and 5.7%, respectively, at MOI = 1,000. AD5/F35 and AD5 vectors could not effectively transfect cells of T cell origin, no fluorescence positive cells were detected at MOI = 1,000; no significant killing effect of AD5/F35 vector on infective target cells was observed at MOI = 1,000. It is concluded that AD5/F35 vector infection has definite selectivity to hematologic malignant cells of various origin, the infection ability of AD5/F35 vector to cells of myeloid origin is stronger than that to cells of B cell origin, the cytotoxicity of AD5/F35 vector to infective target cells is small. The AD5/F35 vector is preferable to AD5 vector in respect of infection ability and offers good prospects of application in gene therapy for myeloid leukemia cells as target cells.

  4. Fast, Efficient, and Gentle Transfection of Human Adherent Cells in Suspension.

    PubMed

    Agrawal, Pranav; Ingle, Nilesh P; Boyle, William S; Ward, Emily; Tolar, Jakub; Dorfman, Kevin D; Reineke, Theresa M

    2016-04-13

    We demonstrate a highly efficient method for gene delivery into clinically relevant human cell types, such as induced pluripotent stem cells (iPSCs) and fibroblasts, reducing the protocol time by one full day. To preserve cell physiology during gene transfer, we designed a microfluidic strategy, which facilitates significant gene delivery in a short transfection time (<1 min) for several human cell types. This fast, optimized and generally applicable cell transfection method can be used for rapid screening of different delivery systems and has significant potential for high-throughput cell therapy applications. PMID:27035392

  5. Efficient transfection of DNA into primarily cultured rat sertoli cells by electroporation.

    PubMed

    Li, Fuping; Yamaguchi, Kohei; Okada, Keisuke; Matsushita, Kei; Enatsu, Noritoshi; Chiba, Koji; Yue, Huanxun; Fujisawa, Masato

    2013-03-01

    The expression of exogenous DNA in Sertoli cells is essential for studying its functional genomics, pathway analysis, and medical applications. Electroporation is a valuable tool for nucleic acid delivery, even in primarily cultured cells, which are considered difficult to transfect. In this study, we developed an optimized protocol for electroporation-based transfection of Sertoli cells and compared its efficiency with conventional lipofection. Sertoli cells were transfected with pCMV-GFP plasmid by square-wave electroporation under different conditions. After transfection of plasmid into Sertoli cells, enhanced green fluorescent protein (EGFP) expression could be easily detected by fluorescent microscopy, and cell survival was evaluated by dye exclusion assay using Trypan blue. In terms of both cell survival and the percentage expressing EGFP, 250 V was determined to produce the greatest number of transiently transfected cells. Keeping the voltage constant (250 V), relatively high cell survival (76.5% ± 3.4%) and transfection efficiency (30.6% ± 5.6%) were observed with a pulse length of 20 μm. The number of pulses significantly affected cell survival and EGFP expression (P < 0.001). Cell survival clearly decreased following one to three pulses, from 83.9% ± 6.1% to 3.2% ± 1.1%, with EGFP expression increasing from 41.8% ± 9.4% to 66.7% ± 5.2%. The yield of positive cells increased with increasing concentration of plasmid DNA (range, 10-50 μg/ml), from 14.0% ± 2.8% to 35.0% ± 6.3%, but cell viability steadily decreased following 20 μg/ml plasmid DNA, from 73.1% ± 4.9% to 57.0% ± 6.6%. Compared with two popular cationic lipid transfection methods, the transfection efficiency of electroporation (21.5% ± 5.7%) was significantly higher than those of Lipofectamine 2000 (2.9% ± 1.0%) and Effectene (1.9% ± 0.8%) in this experiment (P < 0.001). We describe the process of optimizing electroporation conditions, and the successful electroporation of plasmid

  6. Generation of Partially Reprogrammed Cells and Fully Reprogrammed iPS Cells by Plasmid Transfection.

    PubMed

    Kim, Jong Soo; Choi, Hyun Woo; Hong, Yean Ju; Do, Jeong Tae

    2016-01-01

    Induced pluripotent stem (iPS) cells can be directly generated from somatic cells by overexpression of defined transcription factors. iPS cells can perpetually self-renew and differentiate into all cell types of an organism. iPS cells were first generated through infection with retroviruses that contain reprogramming factors. However, development of an exogene-free iPS cell generation method is crucial for future therapeutic applications, because integrated exogenes result in the formation of tumors in chimeras and regain pluripotency after differentiation in vitro. Here, we describe a method to generate iPS cells by transfection of plasmid vectors and to convert partially reprogrammed cells into fully reprogrammed iPS cells by switching from mouse ESC culture conditions to KOSR-based media with bFGF. We also describe basic methods used to characterize fully reprogrammed iPS cells.

  7. Safety and Efficacy of Activated Transfected Killer Cells for Neutropenic Fungal Infections

    PubMed Central

    Lin, Lin; Ibrahim, Ashraf S.; Baquir, Beverlie; Fu, Yue; Applebaum, David; Schwartz, Julie; Wang, Amy; Avanesian, Valentina; Spellberg, Brad

    2010-01-01

    Background Invasive fungal infections cause considerable morbidity and mortality in neutropenic patients. White blood cell transfusions are a promising treatment for such infections, but technical barriers have prevented their widespread use. Methods To recapitulate white blood cell transfusions, we are developing a cell-based immunotherapy using a phagocytic cell line, HL-60. We sought to stably transfect HL-60 cells with a suicide trap (herpes simplex virus thymidine kinase), to enable purging of the cells when desired, and a bioluminescence marker, to track the cells in vivo in mice. Results Transfection was stable despite 20 months of continuous culture or storage in liquid nitrogen. Activation of these transfected cells with retinoic acid and dimethyl sulfamethoxazole enhanced their microbicidal effects. Activated transfected killer (ATAK) cells were completely eliminated after exposure to ganciclovir, confirming function of the suicide trap. ATAK cells improved the survival of neutropenic mice with lethal disseminated candidiasis and inhalational aspergillosis. Bioluminescence and histopathologic analysis confirmed that the cells were purged from surviving mice after ganciclovir treatment. Comprehensive necropsy, histopathology, and metabolomic analysis revealed no toxicity of the cells. Conclusions These results lay the groundwork for continued translational development of this promising, novel technology for the treatment of refractory infections in neutropenic hosts. PMID:20397927

  8. Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes.

    PubMed

    Chow, Yu Ting; Chen, Shuxun; Wang, Ran; Liu, Chichi; Kong, Chi-Wing; Li, Ronald A; Cheng, Shuk Han; Sun, Dong

    2016-01-01

    Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired. PMID:27067121

  9. Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes

    NASA Astrophysics Data System (ADS)

    Chow, Yu Ting; Chen, Shuxun; Wang, Ran; Liu, Chichi; Kong, Chi-Wing; Li, Ronald A.; Cheng, Shuk Han; Sun, Dong

    2016-04-01

    Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired.

  10. Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes

    PubMed Central

    Chow, Yu Ting; Chen, Shuxun; Wang, Ran; Liu, Chichi; Kong, Chi-wing; Li, Ronald A.; Cheng, Shuk Han; Sun, Dong

    2016-01-01

    Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired. PMID:27067121

  11. Cadherin transfection of Xenopus XTC cells downregulates expression of substrate adhesion molecules.

    PubMed

    Finnemann, S; Kühl, M; Otto, G; Wedlich, D

    1995-09-01

    Cadherins are discussed not in terms of their adhesive function but rather as morphoregulatory proteins. Changes in gene expression following cadherin transfection of cells in culture or by overexpression in embryos have, until now, not been reported. We established a protocol for stable transfection of Xenopus XTC cells and generated cells bearing high levels of membrane-integrated mouse uvomorulin (E-cadherin) or Xenopus XB-cadherin. These cell lines showed drastically impaired substrate adhesion on fibronectin and laminin. In immunoblot and radioimmunoprecipitation experiments, we found that fibronectin and alpha 3/beta 1 integrin are downregulated. The reduced amounts of proteins result from a decrease of the respective mRNAs as proven by RNase protection assays. Coprecipitations revealed that transfected cadherin molecules are complexed with alpha-catenin and beta-catenin at plasma membranes. However, the alpha-catenin present in the XB-cadherin complex differs immunologically from that found in the uvomorulin complex. When a truncated form of XB-cadherin lacking 38 of the most C-terminal amino acids was expressed in XTC cells, complex formation with endogenous catenins was abolished. In these transfectants, substrate adhesion was not affected. These results prove that complex formation of transfected cadherins in XTC cells with endogenous beta-catenin correlates with altered synthesis of certain substrate adhesion molecules.

  12. Cyclic Amphipathic Peptide-DNA Complexes Mediate High-Efficiency Transfection of Adherent Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Legendre, Jean-Yves; Szoka, Francis C., Jr.

    1993-02-01

    A DNA transfection protocol has been developed that makes use of the cyclic cationic amphipathic peptide gramicidin S and dioleoyl phosphatidylethanolamine. The DNA complex is formed by mixing gramicidin S with DNA at a 1:1 charge ratio and then adding phosphatidylethanolamine at a lipid/peptide molar ratio of 5:1. The complex mediates rapid association of DNA with cells and leads to transient expression levels of β-galactosidase ranging from 1 to 30% of the transfected cells, with long-term expression being about an order of magnitude lower. The respective roles of peptide and phospholipid are not yet resolved but optimal transfection requires both the cyclic peptide and the hexagonal phase-competent phospholipid PtdEtn. Transfection in CV-1 cells is not affected by lysomotrophic agents, which suggests that DNA entry into the cell is via the plasma membrane. This technique that is simple, economical, and reproducible mediates transfection levels up to 20-fold higher than cationic liposomes in adherent mammalian cells.

  13. Comparation of enhanced green fluorescent protein gene transfected and wild-type porcine neural stem cells.

    PubMed

    Zheng, Yue-Mao; An, Zhi-Xing; Zhao, Xiao-E; Quan, Fu-Sheng; Zhao, Hui-Ying; Zhang, Ya-Rong; Liu, Jun; He, Xiao-Ying; He, Xiao-Ning

    2010-02-01

    The aim of this study was to transfect and express the enhanced green fluorescence protein (EGFP) gene into porcine neural stem cells (NSCs) to determine whether EGFP can be used as a marker to monitor NSCs. NSCs were isolated from embryonic day 30 fetal pig brain and transfected with EGFP gene using lipofection. Transfected and wild-type NSCs were induced to differentiate into cells of neuronal and myogenic lineages. Markers of passage three NSCs and their differentiated cells were tested by reverse transcription polymerase chain reaction. The results showed that EGFP could be expressed in NSCs and the differentiated cells. NSCs expressed Nestin, NogoA, DCX, Hes1, Oct4, CD-90 and Sox2. NSCs could differentiated into astrocyte (GFAP(+)), oligodendrocyte (GalC(+)), neuron (NF(+), NSE(+) and MAP2(+)) and myocyte (myf-6(+) and myoD(+)). We concluded that EGFP can be used as a marker in monitoring NSCs. PMID:19580981

  14. Comparation of enhanced green fluorescent protein gene transfected and wild-type porcine neural stem cells.

    PubMed

    Zheng, Yue-Mao; An, Zhi-Xing; Zhao, Xiao-E; Quan, Fu-Sheng; Zhao, Hui-Ying; Zhang, Ya-Rong; Liu, Jun; He, Xiao-Ying; He, Xiao-Ning

    2010-02-01

    The aim of this study was to transfect and express the enhanced green fluorescence protein (EGFP) gene into porcine neural stem cells (NSCs) to determine whether EGFP can be used as a marker to monitor NSCs. NSCs were isolated from embryonic day 30 fetal pig brain and transfected with EGFP gene using lipofection. Transfected and wild-type NSCs were induced to differentiate into cells of neuronal and myogenic lineages. Markers of passage three NSCs and their differentiated cells were tested by reverse transcription polymerase chain reaction. The results showed that EGFP could be expressed in NSCs and the differentiated cells. NSCs expressed Nestin, NogoA, DCX, Hes1, Oct4, CD-90 and Sox2. NSCs could differentiated into astrocyte (GFAP(+)), oligodendrocyte (GalC(+)), neuron (NF(+), NSE(+) and MAP2(+)) and myocyte (myf-6(+) and myoD(+)). We concluded that EGFP can be used as a marker in monitoring NSCs.

  15. Single-Tailed Lipidoids Enhance the Transfection Activity of Their Double-Tailed Counterparts.

    PubMed

    Wu, Yihang; Li, Linxian; Chen, Qing; Su, Yi; Levkin, Pavel A; Davidson, Gary

    2016-01-11

    Cationic lipid-like molecules (lipidoids) are widely used for in vitro and in vivo gene delivery. Nearly all lipidoids developed to date employ double-tail or multiple-tail structures for transfection. Single-tail lipidoids are seldom considered for transfection as they have low efficiency in gene delivery. So far, there is no detailed study on the contribution to transfection efficiency of single-tail lipidoids when combined with standard double-tail lipidoids. Here, we use combinatorial chemistry to synthesize 17 double-tail and 17 single-tail lipidoids using thiol-yne and thiol-ene click chemistry, respectively. HEK 293T cells were used to analyze transfection efficiency by fluorescence microscopy and calculated based on the percentage of cells transfected. The size and zeta potential of liposomes and lipoplexes were characterized by dynamic light scattering (DLS). Intracellular DNA delivery and trafficking was further examined using confocal microscopy. Our study shows that combining single with double-tail lipidoids increases uptake of lipoplexes, as well as cellular transfection efficiency.

  16. Optical reprogramming of human cells in an ultrashort femtosecond laser microfluidic transfection platform.

    PubMed

    Uchugonova, Aisada; Breunig, Hans Georg; Batista, Ana; König, Karsten

    2016-09-01

    Induced pluripotent stem cell (iPS cell) technology can be used to produce unlimited numbers of functional cells for both research and therapeutic purposes without ethical controversy. Typically, viruses are applied for efficient intracellular delivery of genes/transcription factors to generate iPS cells. However, the viral genomic integration may cause a risk of mutation as well as tumor formation therefore limits its clinical application. Here we demonstrate that spatially shaped extreme ultrashort laser pulses of sub-20 femtoseconds induce transient membrane permeabilisation which enables contamination-free transfection of cells in a microfluidic tube with multiple genes at the individual cell level in order to achieve optical reprogramming of large cell populations. We found that the ultrashort femtosecond laser-microfluidic cell transfection platform enhanced the efficacy of iPS-like colony-forming following merely a single transfection. Illustration of the spatially shaped femtosecond laser-assisted microfluidic cell transfection platform for production of iPS cell colonies. PMID:26530487

  17. Delivery of proteins to mammalian cells via gold nanoparticle mediated laser transfection

    NASA Astrophysics Data System (ADS)

    Heinemann, D.; Kalies, S.; Schomaker, M.; Ertmer, W.; Murua Escobar, H.; Meyer, H.; Ripken, T.

    2014-06-01

    Nanoparticle laser interactions are in widespread use in cell manipulation. In particular, molecular medicine needs techniques for the directed delivery of molecules into mammalian cells. Proteins are the final mediator of most cellular cascades. However, despite several methodical approaches, the efficient delivery of proteins to cells remains challenging. This paper presents a new protein transfection technique via laser scanning of cells previously incubated with gold nanoparticles. The laser-induced plasmonic effects on the gold nanoparticles cause a transient permeabilization of the cellular membrane, allowing proteins to enter the cell. Applying this technique, it was possible to deliver green fluorescent protein into mammalian cells with an efficiency of 43%, maintaining a high level of cell viability. Furthermore, a functional delivery of Caspase 3, an apoptosis mediating protein, was demonstrated and evaluated in several cellular assays. Compared to conventional protein transfection techniques such as microinjection, the methodical approach presented here enables high-throughput transfection of about 10 000 cells per second. Moreover, a well-defined point in time of delivery is guaranteed by gold nanoparticle mediated laser transfection, allowing the detailed temporal analysis of cellular pathways and protein trafficking.

  18. GAP JUNCTION COMMUNICATON IN A TRANSFECTED HUMAN CELL LINE: ACTION OF MELATONIN AND MAGNETIC FIELDS

    EPA Science Inventory

    GAP JUNCTION COMMUNICTION IN TRANSFECTED HUMAN CELL LINE: ACTION OF MELATONIN AND MAGNETIC FIELDS.

    OBJECTIVE: We previously showed that functional gap junction communication (GJC), as monitored by dye transfer (DT), could be enhanced in mouse C3H 10T112 cells and in mouse...

  19. Delivery of proteins to mammalian cells via gold nanoparticle mediated laser transfection.

    PubMed

    Heinemann, D; Kalies, S; Schomaker, M; Ertmer, W; Murua Escobar, H; Meyer, H; Ripken, T

    2014-06-20

    Nanoparticle laser interactions are in widespread use in cell manipulation. In particular, molecular medicine needs techniques for the directed delivery of molecules into mammalian cells. Proteins are the final mediator of most cellular cascades. However, despite several methodical approaches, the efficient delivery of proteins to cells remains challenging. This paper presents a new protein transfection technique via laser scanning of cells previously incubated with gold nanoparticles. The laser-induced plasmonic effects on the gold nanoparticles cause a transient permeabilization of the cellular membrane, allowing proteins to enter the cell. Applying this technique, it was possible to deliver green fluorescent protein into mammalian cells with an efficiency of 43%, maintaining a high level of cell viability. Furthermore, a functional delivery of Caspase 3, an apoptosis mediating protein, was demonstrated and evaluated in several cellular assays. Compared to conventional protein transfection techniques such as microinjection, the methodical approach presented here enables high-throughput transfection of about 10 000 cells per second. Moreover, a well-defined point in time of delivery is guaranteed by gold nanoparticle mediated laser transfection, allowing the detailed temporal analysis of cellular pathways and protein trafficking.

  20. Electroporation for the efficient transfection of mammalian cells with DNA.

    PubMed Central

    Chu, G; Hayakawa, H; Berg, P

    1987-01-01

    A simple and reproducible procedure for the introduction of DNA into mammalian cells by electroporation is described. The parameters involving the cells, the DNA, and the electric field are investigated. The procedure has been applied to a broad range of animal cells. It is capable of transforming more than 1% of the viable cells to the stable expression of a selectable marker. Images PMID:3029703

  1. Single-cell Transfection by Electroporation Using an Electrolyte/Plasmid-Filled Capillary

    PubMed Central

    Wang, Manyan; Orwar, Owe; Weber, Stephen G.

    2009-01-01

    Single-cell transfection of adherent cells has been accomplished using single-cell electroporation (SCEP) with a pulled capillary. HEPES-buffered physiological saline solution containing pEGFP plasmid at a low concentration (0.16 ∼ 0.78 µg/µL) filled a 15 cm long capillary with a tip opening of 2 µm. The electric field is applied to individual cells by bringing the tip close to the cell and subsequently applying one or two brief electric pulses. Many individual cells can thus be transfected with a small volume of plasmid-containing solution (∼ 1 µL). The extent of electroporation is determined by measuring the percentage loss of freely diffusing thiols (chiefly reduced glutathione) that have been derivatized with the fluorogenic ThioGlo 1. A mass transport model is used to fit the time-dependent fluorescence intensity decay in the target cells. The fits, which are excellent, yield the electroporation-induced fluorescence loss at steady state and the mass transfer rate through the electroporated cell membrane. Steady-state fluorescence loss ranged approximately from 0 to about 80% (based on the fluorescence intensity before electroporation). For the cells having a loss of thiol-ThioGlo 1 fluorescence intensity greater than 10%, and mass transfer rate greater than 0.03 s−1, EGFP fluorescence is observed after 24 hours. The EGFP fluorescence is increased at 48 hours. With a loss smaller than 10% and a mass transfer rate smaller than 0.03 s−1, no EGFP fluorescence is detected. Thus, transfection success is closely related to the small molecule mass transport dynamics as indicated by the loss of fluorescence from thiol-ThioGlo 1 conjugates. The EGFP expression is weaker than bulk lipid-mediated transfection, as indicated by the EGFP fluorescence intensities. However, the success with the single-cell approach is considerably greater than lipid-mediated transfection. PMID:19351139

  2. Functionalized nanomaterials: are they effective to perform gene delivery to difficult-to-transfect cells with no cytotoxicity?

    NASA Astrophysics Data System (ADS)

    Tonelli, F. M. P.; Lacerda, S. M. S. N.; Paiva, N. C. O.; Pacheco, F. G.; Scalzo Junior, S. R. A.; de Macedo, F. H. P.; Cruz, J. S.; Pinto, M. C. X.; Junior, J. D. Corrêa; Ladeira, L. O.; França, L. R.; Guatimosim, S.; Resende, R. R.

    2015-10-01

    Nanodiamonds (NDs), multiwalled carbon nanotubes (MWCNTs) and gold nanorods (NRs) can be functionalized to promote gene delivery to hard-to-transfect cells with higher transfection efficiency than cationic lipids, and inducing less cell death.Nanodiamonds (NDs), multiwalled carbon nanotubes (MWCNTs) and gold nanorods (NRs) can be functionalized to promote gene delivery to hard-to-transfect cells with higher transfection efficiency than cationic lipids, and inducing less cell death. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr04173b

  3. Transfection of cells mediated by biodegradable polymer materials with surface-bound polyethyleneimine.

    PubMed

    Zheng, J; Manuel, W S; Hornsby, P J

    2000-01-01

    Poly(epsilon-CBZ-L-lysine) can be mixed with biodegradable polymers such as poly(D,L-lactic-co-glycolic acid) or poly(L-lactic acid) and formed into films, foams, or microspheres. Surface amino groups may then be deprotected with acid or lithium/liquid ammonia. The amino groups serve as a method to modify the surface by attachment of other molecules. In the present experiments, we show that these polymer materials, as films or foams, may be surface modified by the attachment of polyethyleneimine (PEI). Plasmid DNA attached to the PEI can transfect cells plated on the surface over several days. Covalent atachment of PEI was required for transfection to be efficient. PEI was also attached to surface-bound collagen on cell culture plates and was shown to mediate transfection.

  4. The effect of transfected MHC class I genes on sensitivity to natural killer cells.

    PubMed Central

    Holscher, M; Givan, A L; Brooks, C G

    1991-01-01

    To test the hypothesis that major histocompatibility complex (MHC) molecules protect target cells from lysis by natural killer cells (NKC), we transfected the MHC- B16 melanoma line F10 with the class I genes encoding Dd, Kb, and Kk. Only low levels of Dd expression could be obtained and there was no protection against NKC. By contrast, Kb and Kk transfectants were obtained which displayed significant resistance to NKC, and with the latter transfectants resistance was clearly related to the level of transgene expression. Various mutants of the F10 line with altered patterns of MHC expression were also obtained. These mutant lines provided evidence that (i) the Db molecule is also capable of inducing resistance to NKC and (ii) high MHC class I expression does not by itself guarantee lowered susceptibility to NKC. PMID:1904402

  5. Single-cell optoporation and transfection using femtosecond laser and optical tweezers

    PubMed Central

    Waleed, Muhammad; Hwang, Sun-Uk; Kim, Jung-Dae; Shabbir, Irfan; Shin, Sang-Mo; Lee, Yong-Gu

    2013-01-01

    In this paper, we demonstrate a new single-cell optoporation and transfection technique using a femtosecond Gaussian laser beam and optical tweezers. Tightly focused near-infrared (NIR) femtosecond laser pulse was employed to transiently perforate the cellular membrane at a single point in MCF-7 cancer cells. A distinct technique was developed by trapping the microparticle using optical tweezers to focus the femtosecond laser precisely on the cell membrane to puncture it. Subsequently, an external gene was introduced in the cell by trapping and inserting the same plasmid-coated microparticle into the optoporated cell using optical tweezers. Various experimental parameters such as femtosecond laser exposure power, exposure time, puncture hole size, exact focusing of the femtosecond laser on the cell membrane, and cell healing time were closely analyzed to create the optimal conditions for cell viability. Following the insertion of plasmid-coated microparticles in the cell, the targeted cells exhibited green fluorescent protein (GFP) under the fluorescent microscope, hence confirming successful transfection into the cell. This new optoporation and transfection technique maximizes the level of selectivity and control over the targeted cell, and this may be a breakthrough method through which to induce controllable genetic changes in the cell. PMID:24049675

  6. Micro-optical coherence tomography tracking of magnetic gene transfection via Au-Fe3O4 dumbbell nanoparticles

    NASA Astrophysics Data System (ADS)

    Shi, Wei; Liu, Xinyu; Wei, Chao; Xu, Zhichuan J.; Sim, Stanley Siong Wei; Liu, Linbo; Xu, Chenjie

    2015-10-01

    Heterogeneous Au-Fe3O4 dumbbell nanoparticles (NPs) are composed of Au NPs and Fe3O4 NPs that bring in optical and magnetic properties respectively. This article reports the engineering of Au-Fe3O4 NPs as gene carriers for magnetic gene transfection as well as contrast agents for micro-optical coherence tomography (μOCT). As a proof-of-concept, Au-Fe3O4 NPs are used to deliver the green fluorescent protein to HEK 293T cells and their entrance into the cells is monitored through μOCT.Heterogeneous Au-Fe3O4 dumbbell nanoparticles (NPs) are composed of Au NPs and Fe3O4 NPs that bring in optical and magnetic properties respectively. This article reports the engineering of Au-Fe3O4 NPs as gene carriers for magnetic gene transfection as well as contrast agents for micro-optical coherence tomography (μOCT). As a proof-of-concept, Au-Fe3O4 NPs are used to deliver the green fluorescent protein to HEK 293T cells and their entrance into the cells is monitored through μOCT. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05459a

  7. Alteration of glycolipids in ras-transfected NIH 3T3 cells

    SciTech Connect

    Matyas, G.R.; Aaronson, S.A.; Brady, R.O.; Fishman, P.H.

    1987-09-01

    Glycosphingolipid alterations upon viral transformation are well documented. Transformation of mouse 3T3 cells with murine sarcoma viruses results in marked decreases in the levels of gangliosides GM1 and GD1a and an increase in gangliotriaosylceramide. The transforming oncogenes of these viruses have been identified as members of the ras gene family. The authors analyzed NIH 3T3 cells transfected with human H-, K- and N-ras oncogenes for their glycolipid composition and expression of cell surface gangliosides. Using conventional thin-layer chromatographic analysis, they found that the level of GM3 was increased and that of GD1a was slightly decreased or unchanged, and GM1 was present but not in quantifiable levels. Cell surface levels of GM1 were determined by /sup 125/I-labeled cholera toxin binding to intact cells. GD1a was determined by cholera toxin binding to cells treated with sialidase prior to toxin binding. All ras-transfected cells had decreased levels of surface GM1 and GD1 as compared to logarithmically growing normal NIH 3T3 cells. Levels of GM1 and, to a lesser extent, GD1a increased as the latter cells became confluent. Using a monoclonal antibody assay, they found that gangliotriaosylceramide was present in all ras-transfected cells studied but not in logarithmically growing untransfected cells. These results indicated that ras oncogenes derived form human tumors are capable of inducing alterations in glycolipid composition.

  8. Expression of active human factor IX in transfected cells.

    PubMed

    Busby, S; Kumar, A; Joseph, M; Halfpap, L; Insley, M; Berkner, K; Kurachi, K; Woodbury, R

    Factor IX is the precursor of a serine protease that functions in the intrinsic blood clotting pathway. Deficiencies in this plasma glycoprotein result in haemophilia B (or Christmas disease) and occur in about 1 in 30,000 males. Patients are currently treated with fresh frozen plasma or prothrombin complex concentrates prepared from pooled plasma from normal individuals. There are several problems with this method of treatment, including the probable exposure of the patients to contaminants such as the viral agents responsible for hepatitis and AIDS (acquired immune deficiency syndrome). As a first step towards an alternative source of pure human factor IX, we report here on the use of recombinant DNA techniques to produce biologically active factor IX in cultured mammalian cells. Stable cell lines were produced by cotransfecting a baby hamster kidney (BHK) cell line with a plasmid containing a gene for factor IX and a plasmid containing a selectable marker. Protein secreted by these cell lines reduces the clotting time of plasma from factor IX-deficient patients. We present additional evidence that this protein is authentic human factor IX.

  9. Enhanced photo-transfection efficiency of mammalian cells on graphene coated substrates

    NASA Astrophysics Data System (ADS)

    Mthunzi, Patience; He, Kuang; Ngcobo, Sandile; Warner, Jamie W.

    2014-03-01

    Literature reports graphene, an atomic-thick sheet of carbon atoms as one of the promising biocompatible scaffolds that promotes cellular proliferation in human mesenchymal stem cells. On the other hand, different mammalian cell lines including the induced pluripotent stem cells exhibited an accelerated proliferation rate when cultured on graphene or graphene oxide coated substrates. These findings provide strong motivation to explore the full capability of graphene in further pluripotent stem cell research activities as there exists an urgent requirement to preserve their therapeutic potential. This therefore calls for non-invasive procedures for handling stem cells in-vitro. For example, resent literature has shown successful laser light driven transfection in both multipotent and pluripotent stem cells. In order to explore the non-invasive nature of optical transfection alongside biocompatible qualities of graphene, in this work we investigated the impact of optically transfecting mouse embryonic stem (mES) cells plated on graphene coated sample chambers. Using Chinese Hamster Ovary cells (CHO-K1), we further studied the influence of graphene on cell viability as well as cell cytotoxicity through assessing changes in levels of mitochondrial adenosine triphosphate (ATP) activity and the release of cytosolic lactate dehydrogenase (LHD) respectively. Our results showed that compared to those treated on plain glass, CHO-K1 cells optically treated while plated on graphene coated substrates exhibited a higher production of ATP and a milder release of LDH. In addition there was enhanced photo-transfection efficiency in both CHO-K1 and mES cells irradiated on graphene sample chambers.

  10. Immunity to squamous carcinoma in mice immunized with dendritic cells transfected with genomic DNA from squamous carcinoma cells.

    PubMed

    O-Sullivan, InSug; Ng, Lauren K; Martinez, Don M; Kim, Tae S; Chopra, Amla; Cohen, Edward P

    2005-10-01

    Immunotherapy of squamous cell carcinoma (SCC) at an early stage of the disease increases the likelihood of success. We report a new vaccination strategy designed to prepare SCC vaccines from microgram amounts of tumor tissue, enabling the treatment of patients with minimal residual disease. The vaccine was prepared by transfer of sheared genomic DNA-fragments (25 kb) from KLN205 cells, an SCC cell line of DBA/2 mouse origin, into syngeneic bone marrow-derived mature dendritic cells (DCs). More than 90% of the transfected DCs took up DNA from the neoplasm and transferred genes were expressed as protein. The DCs expressed CD11c, CD11b, and the costimulatory molecules CD40, CD80 and CD86, characteristic of mature DCs. Syngeneic DBA/2J mice, highly susceptible to the growth of KLN205 cells, were injected intravenously (i.v.) with the transfected DCs, followed by a subcutaneous (s.c.) injection of the tumor cells. The strong immunogenic properties of the transfected cells were indicated by the finding that the survival of the tumor-bearing mice was prolonged (P<.001), relative to that of mice in various control groups. Enzyme-linked immuno spot (ELISPOT IFN-gamma) assays revealed the activation of cell-mediated immunity directed toward the SCC in mice immunized with the transfected DCs. Two independent in vitro cytotoxicity assays indicated the presence of robust cell-mediated immunity directed toward the SCC in mice immunized with the transfected cells.

  11. Comparison of Different Electroporation Parameters on Transfection Efficiency of Sheep Testicular Cells

    PubMed Central

    Niakan, Sarah; Heidari, Banafsheh; Akbari, Ghasem; Nikousefat, Zahra

    2016-01-01

    Objective Electroporation can be a highly efficient method for introducing the foreign genetic materials into the targeted cells for transient and/or permanent genetic modification. Considering the application of this technique as a very efficient method for drug, oligonucleotide, antibody and plasmid delivery for clinical applications and production of transgenic animals, the present study aimed to optimize the transfection efficiency of sheep testicular cells including spermatogonial stem cells (SSCs) via electroporation. Materials and Methods This study is an experimental research conducted in Biotechnology Research Center (Avicenna Research Institute, Tehran, Iran) from September 2013 to March 2014. Following isolation and propagation of one-month lamb testicular cells (SSCs and somatic testicular cells including; Sertoli, Leydig, and myoid cells), the effect of different electroporation parameters including total voltages (280, 320, and 350 V), burst durations (10, 8, and 5 milliseconds), burst modes (single or double) and addition of dimethyl sulfoxide (DMSO) were evaluated on transfection efficiency, viability rate and mean fluorescent intensity (MFI) of sheep testicular cells. Results The most transfection efficiency was obtained in 320 V/8 milliseconds/single burst group in transduction medium with and without DMSO. There was a significantly inverse correlation between transfection efficiency with application of both following parameters: addition of DMSO and double burst. After transfection, the highest and lowest viability rates of testicular cells were demonstrated in 320 V/8 milliseconds with transduction medium without DMSO and 350 V/5 milliseconds in medium containing DMSO. Ad- dition of DMSO to transduction medium in all groups significantly decreased the viability rate. The comparison of gene expression indicated that Sertoli and SSCs had the most fluorescence intensity in 320 V/double burst/DMSO positive. However, myoid and Leydig cells showed the

  12. Comparison of Different Electroporation Parameters on Transfection Efficiency of Sheep Testicular Cells

    PubMed Central

    Niakan, Sarah; Heidari, Banafsheh; Akbari, Ghasem; Nikousefat, Zahra

    2016-01-01

    Objective Electroporation can be a highly efficient method for introducing the foreign genetic materials into the targeted cells for transient and/or permanent genetic modification. Considering the application of this technique as a very efficient method for drug, oligonucleotide, antibody and plasmid delivery for clinical applications and production of transgenic animals, the present study aimed to optimize the transfection efficiency of sheep testicular cells including spermatogonial stem cells (SSCs) via electroporation. Materials and Methods This study is an experimental research conducted in Biotechnology Research Center (Avicenna Research Institute, Tehran, Iran) from September 2013 to March 2014. Following isolation and propagation of one-month lamb testicular cells (SSCs and somatic testicular cells including; Sertoli, Leydig, and myoid cells), the effect of different electroporation parameters including total voltages (280, 320, and 350 V), burst durations (10, 8, and 5 milliseconds), burst modes (single or double) and addition of dimethyl sulfoxide (DMSO) were evaluated on transfection efficiency, viability rate and mean fluorescent intensity (MFI) of sheep testicular cells. Results The most transfection efficiency was obtained in 320 V/8 milliseconds/single burst group in transduction medium with and without DMSO. There was a significantly inverse correlation between transfection efficiency with application of both following parameters: addition of DMSO and double burst. After transfection, the highest and lowest viability rates of testicular cells were demonstrated in 320 V/8 milliseconds with transduction medium without DMSO and 350 V/5 milliseconds in medium containing DMSO. Ad- dition of DMSO to transduction medium in all groups significantly decreased the viability rate. The comparison of gene expression indicated that Sertoli and SSCs had the most fluorescence intensity in 320 V/double burst/DMSO positive. However, myoid and Leydig cells showed the

  13. Plasmid transfection in mammalian cells spatiotemporally tracked by a gold nanoparticle.

    PubMed

    Muroski, Megan E; Carnevale, Kate J F; Riskowski, Ryan A; Strouse, Geoffrey F

    2015-01-27

    Recent advances in cell transfection have suggested that delivery of a gene on a gold nanoparticle (AuNP) can enhance transfection efficiency. The mechanism of transfection is poorly understood, particularly when the gene is appended to a AuNP, as expression of the desired exogenous protein is dependent not only on the efficiency of the gene being taken into the cell but also on efficient endosomal escape and cellular processing of the nucleic acid. Design of a multicolor surface energy transfer (McSET) molecular beacon by independently dye labeling a linearized plasmid and short duplex DNA (sdDNA) appended to a AuNP allows spatiotemporal profiling of the transfection events, providing insight into package uptake, disassembly, and final plasmid expression. Delivery of the AuNP construct encapsulated in Lipofectamine2000 is monitored in Chinese hamster ovary cells using live-cell confocal microscopy. The McSET beacon signals the location and timing of the AuNP release and endosomal escape events for the plasmid and the sdDNA discretely, which are correlated with plasmid transcription by fluorescent protein expression within the cell. It is observed that delivery of the construct leads to endosomal release of the plasmid and sdDNA from the AuNP surface at different rates, prior to endosomal escape. Slow cytosolic diffusion of the nucleic acids is believed to be the limiting step for transfection, impacting the time-dependent expression of protein. The overall protein expression yield is enhanced when delivered on a AuNP, possibly due to better endosomal escape or lower degradation prior to endosomal escape.

  14. Cell transfection in vitro and in vivo with nontoxic TAT peptide-liposome-DNA complexes

    NASA Astrophysics Data System (ADS)

    Torchilin, Vladimir P.; Levchenko, Tatyana S.; Rammohan, Ram; Volodina, Natalia; Papahadjopoulos-Sternberg, Brigitte; D'Souza, Gerard G. M.

    2003-02-01

    Liposomes modified with TAT peptide (TATp-liposomes) showed fast and efficient translocation into the cell cytoplasm with subsequent migration into the perinuclear zone. TATp-liposomes containing a small quantity (10 mol %) of a cationic lipid formed firm noncovalent complexes with DNA. Here, we present results demonstrating both in vitro and in vivo transfection with TATp-liposome-DNA complexes. Mouse NIH/3T3 fibroblasts and rat H9C2 cardiomyocytes were transfected with such complexes in vitro. The transfection with the TATp-liposome-associated pEGFP-N1 plasmid encoding for the green fluorescent protein (GFP) was high, whereas the cytotoxicity was lower than that of commonly used cationic lipid-based gene-delivery systems. Intratumoral injection of TATp-liposome-DNA complexes into the Lewis lung carcinoma tumor of mice also resulted in an expression of GFP in tumor cells. This transfection system should be useful for various protocols of cell treatment in vitro or ex vivo as well as for localized in vivo gene therapy.

  15. Connexin32 gap junction channels in stably transfected cells: unitary conductance.

    PubMed Central

    Moreno, A P; Eghbali, B; Spray, D C

    1991-01-01

    Pairs of SKHep1 cells, which are derived from a highly metastatic human hepatoma, were studied using the whole cell voltage clamp technique with patch-type electrodes containing CsCl as the major ionic species. In 12 of 81 cell pairs, current flow through junctional membranes was detectable; in the remaining 69 cell pairs, junctional conductance was less than the noise limit of our recording apparatus (worst case: 10 pS). Macroscopic junctional conductance (gj) in the small percentage of pairs where it was detectable ranged from 100 to 600 pS. Unitary junctional conductance (gamma j) determined in the lowest conductance pairs or after reducing conductance with a short exposure to the uncoupling agent halothane was 25-35 pS. To study properties of gap junction channels formed of connexin32, the parental SKHep1 cell line was stably transfected with a plasmid containing cDNA that encodes connexin32, the major gap junction protein of rat liver cells. In 85 of 98 pairs of voltage clamped connexin32-transfected SKHep1 cells, macroscopic gj was greater than 1 nS; gj increased with time after dissociation (from 1.8 +/- 0.6 [mean +/- SE; n = 7] nS at 2 h after plating to 9.3 +/- 2.2 [n = 9] nS, the maximal value, at 24 h). Unitary conductance of gap junction channels between pairs of transfected SKHep1 cells was measured in low conductance pairs and after reducing gj by exposure to halothane or heptanol. Histograms of gamma j values in transfected cells, in 10 experiments where greater than 100 transitions were measurable, displayed two peaks; 120-130 pS and 25-35 pS. The smaller size corresponded to channels that were occasionally detected in the parental cells. We therefore conclude that connexin32 forms gap junctions channels of the 120-130 pS size class. PMID:1722119

  16. Transfection of C6 Glioma Cells with Connexin 43 cDNA: Analysis of Expression, Intercellular Coupling, and Cell Proliferation

    NASA Astrophysics Data System (ADS)

    Zhu, D.; Caveney, S.; Kidder, G. M.; Naus, C. C. G.

    1991-03-01

    C6 glioma cells express low levels of the gap junction protein connexin 43 and its mRNA and display very weak dye coupling. When implanted into the rat cerebrum, these cells quickly give rise to a large glioma. To investigate the role of gap junctions in the tumor characteristics of these cells, we have used Lipofectin-mediated transfection to introduce a full-length cDNA encoding connexin 43. Several transfected clones were obtained that exhibited various amounts of connexin 43 mRNA transcribed from the inserted cDNA. Immunocytochemical analysis revealed an increase in the amount of connexin 43 immunoreactivity in the transfected cells, being localized at areas of intercellular contact as well as in the cytoplasm. The level of dye coupling was also assessed and found to correlate with the amount of connexin 43 mRNA. When cell proliferation was followed over several days, cells expressing the transfected cDNA grew more slowly than nontransfected cells. These transfected cells will be useful in examining the role of gap junctions in tumorigenesis.

  17. Transfection using DEAE-dextran.

    PubMed

    Selden, R F

    2001-05-01

    Two protocols for DEAE-dextran transfection of cells are provided in this unit. The Basic Protocol describes a procedure used to transfect adherent cells and the first Alternate Protocol presents a method used to transfect suspension cells. If an increase in transfection efficiency is needed, cells can be treated with chloroquine as described in the second Alternate Protocol.

  18. Polarized endocytosis by Madin-Darby canine kidney cells transfected with functional chicken liver glycoprotein receptor

    PubMed Central

    1989-01-01

    We have studied the expression of the chicken hepatic glycoprotein receptor (chicken hepatic lectin [CHL]) in Madin-Darby canine kidney (MDCK) cells, by transfection of its cDNA under the control of a retroviral promotor. Transfected cell lines stably express 87,000 surface receptors/cell with a kd = 13 nM. In confluent monolayers, approximately 40% of CHL is localized at the plasma membrane. 98% of the surface CHL is expressed at the basolateral surface where it performs polarized endocytosis and degradation of glycoproteins carrying terminal N-acetylglucosamine at a rate of 50,000 ligand molecules/h. Studies of the half-life of metabolically labeled receptor and of the stability of biotinylated cell surface receptor after internalization indicate that transfected CHL performs several rounds of uptake and recycling before it gets degraded. The successful expression of a functional basolateral receptor in MDCK cells opens the way for the characterization of the mechanisms that control targeting and recycling of proteins to the basolateral membrane of epithelial cells. PMID:2687287

  19. Transfection microarrays for high-throughput phenotypic screening of genes involved in cell migration.

    PubMed

    Onuki-Nagasaki, Reiko; Nagasaki, Akira; Hakamada, Kazumi; Uyeda, Taro Q P; Fujita, Satoshi; Miyake, Masato; Miyake, Jun

    2010-01-01

    Cell migration is important in several biological phenomena, such as cancer metastasis. Therefore, the identification of genes involved in cell migration might facilitate the discovery of antimetastatic drugs. However, screening of genes by the current methods can be complicated by factors related to cell stimulation, for example, abolition of contact inhibition and the release inflammatory cytokines from wounded cells during examinations of wound healing in vitro. To overcome these problems and identify genes involved in cell migration, in this chapter we describe the use of transfection microarrays for high-throughput phenotypic screening. PMID:20387151

  20. The Characteristics of Murine iPS Cells and siRNA Transfection Under Hypoxia.

    PubMed

    Sugimoto, K; Hayashi, Yoshihiko

    2016-01-01

    iPS cells are attractive for the regenerative medicine. The creation of pluripotent cells from somatic cells has great potential for basic and clinical research and application. Retroviral transduction of four or three transfection factors has been shown to initiate a reprogramming process. Here, we describe the effect of transcription factors regarding the growth and differentiation of mouse iPS cells in normoxia or hypoxia. Furthermore, we introduce the function of hypoxia-inducible factors (HIFs) in mouse iPS cells in hypoxia using RT-PCR and western blotting together with HIFs knockdown techniques.

  1. Development and Optimization of AAV hFIX Particles by Transient Transfection in an iCELLis(®) Fixed-Bed Bioreactor.

    PubMed

    Powers, Alicia D; Piras, Bryan A; Clark, Robert K; Lockey, Timothy D; Meagher, Michael M

    2016-06-01

    Adeno-associated virus (AAV) vectors are increasingly popular in gene therapy because they are unassociated with human disease, replication dependent, and less immunogenic than other viral vectors and can infect a variety of cell types. These vectors have been used in over 130 clinical trials, and one AAV product has been approved for treatment of lipoprotein lipase deficiency in Europe. To meet the demand for the increasing quantities of AAV required for clinical trials and treatment, a scalable high-capacity technology is required. Bioreactors meet these requirements but limited options are available for adherent HEK 293T/17 cells. Here we optimize the transient transfection of HEK293T/17 cells for the production of AAV human factor IX in a disposable fixed-bed bioreactor, the iCELLis(®) Nano (PALL Corporation). A fixed bed in the center of the iCELLis bioreactor is surrounded by culture medium that is pumped through the bed from the bottom of the bioreactor so that a thin film of the medium overflows the bed and is replenished with oxygen and depleted of CO2 as it returns to the surrounding medium reservoir. We show that this fixed-bed bioreactor can support as many as 2.5 × 10(8) cells/ml of fixed bed (1.9 × 10(6) cells/cm(2)). By optimizing culture and transfection parameters such as the concentration of DNA for transfection, day of harvest, size of PEI/DNA particles, and transfection medium, and adding an additional medium change to the process, we increased our yield to as high as 9.0 × 10(14) viral particles per square meter of fixed bed. We also show an average GFP transfection of 97% of cells throughout the fixed bed. These yields make the iCELLis a promising scalable technology for the clinical production of AAV gene therapy products.

  2. Experimental research of RB94 gene transfection into retinoblastoma cells using ultrasound-targeted microbubble destruction.

    PubMed

    Zheng, Min-Ming; Zhou, Xi-Yuan; Wang, Li-Ping; Wang, Zhi-Gang

    2012-06-01

    The purpose of this study was to explore the transfection of the recombinant expression plasmid pEGFP-C1/RB94 into human retinoblastoma cells (HXO-Rb44) using ultrasound-targeted microbubble destruction (UTMD). pEGFP-C1/RB94 was transfected into HXO-Rb44 in vitro by UTMD, with liposome as the positive control. After 24 to 72 h, the expression of the reporter gene enhanced green fluorescent protein (EGFP) was observed using fluorescent microscopy and flow cytometry. The cell viability of HXO-Rb44 was measured by a MTT assay. The mRNA and proteins of RB94, caspase-3 and Bax were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Moreover, the apoptosis rate and cell cycle progression of the cells were detected by flow cytometry. This study demonstrated that UTMD can enhance the transfection efficiency of RB94, which has an obvious impact on the inhibition of the growth process of retinoblastoma cells, suggesting that the combination of UTMD and RB94 compounds might be a useful tool for use in the gene therapy of retinoblastoma.

  3. Chitosan as a non-viral co-transfection system in a cystic fibrosis cell line.

    PubMed

    Fernández Fernández, Elena; Santos-Carballal, Beatriz; Weber, Wolf-Michael; Goycoolea, Francisco M

    2016-04-11

    Successful gene therapy requires the development of suitable vehicles for the selective and efficient delivery of genes to specific target cells at the expense of minimal toxicity. In this work, we investigated a non-viral gene delivery system based on chitosan (CS) to specifically address cystic fibrosis (CF). Thus, electrostatic self-assembled CS-pEGFP and CS-pEGFP-siRNA complexes were prepared from high-pure fully characterized CS (Mw ∼ 20 kDa and degree of acetylation ∼ 30%). The average diameter of positively-charged complexes (i.e. ζ ∼+25 mV) was ∼ 200 nm. The complexes were found relatively stable over 14h in Opti-MEM. Cell viability study did not show any significant cytotoxic effect of the CS-based complexes in a human bronchial cystic fibrosis cell line (CFBE41o-). We evaluated the transfection efficiency of this cell line with both CS-pEGFP and co-transfected with CS-pEGFP-siRNA complexes at (N/P) charge ratio of 12. We reported an increase in the fluorescence intensity of CS-pEGFP and a reduction in the cells co-transfected with CS-pEGFP-siRNA. This study shows proof-of-principle that co-transfection with chitosan might be an effective delivery system in a human CF cell line. It also offers a potential alternative to further develop therapeutic strategies for inherited disease treatments, such as CF. PMID:26875537

  4. Effect of TIMP1 transfection on PTEN expression in human kidney proximal tubular cells.

    PubMed

    Chen, J X; Cai, G Y; Chen, X M; Liu, H; Chen, X; Peng, Y M; Liu, F Y; Li, Z; Shi, S Z

    2015-12-21

    To explore the role of metalloproteinase-1 (TIMP-1) tissue inhibitor in the mechanisms of kidney aging, we observed the effects of sense and antisense transfection of TIMP-1 and of metalloproteinase (MMP) inhibitors on phosphatase and tensin homolog (PTEN), vascular endothelial growth factor (VEGF), and Flk-1 expression in TIMP-1 transgenic human proximal tubular epithelial cells (HKCs). Transfected HKCs were co-incubated with 100 μM MMP-2 and MMP-9 inhibitor III for 24 h to affect enzyme inhibition. TIMP-1, MMP-2, MMP-9, PTEN, VEGF, and Flk-1 mRNA expression was detected by reverse transcription-polymerase chain reaction. PTEN, VEGF, and Flk-1 protein expression in cells of each experimental group was measured by indirect immunofluorescence. We found that PTEN expression was up-regulated (P < 0.05) in the sense TIMP-1-transfected group (P < 0.05) compared with the non-transfected and empty vector groups, and that expression of VEGF and Flk-1 was down-regulated (P < 0.05). In contrast, the antisense TIMP-1 transgenic group showed the opposite results (P < 0.05). No significant differences in expression of PTEN, VEGF, or Flk-1 were observed among the MMP- 2/MMP-9 inhibitor III, non-transfected, and empty vector groups (P > 0.05). These results suggest that in the progression of renal aging, high expression of TIMP-1 up-regulates PTEN expression through an MMP-independent pathway, and subsequently down-regulates the expression of VEGF and Flk-1, indicating that PTEN and TIMP-1 are involved in the aging-associated impairment of renal angiogenesis. Our study provides a theoretical basis for further exploration of the mechanism underlying TIMP- 1 participation in renal aging progression.

  5. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control

    NASA Astrophysics Data System (ADS)

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L. James; Lu, Wu

    2015-12-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk

  6. Characterisation of human thyroid epithelial cells immortalised in vitro by simian virus 40 DNA transfection.

    PubMed Central

    Lemoine, N. R.; Mayall, E. S.; Jones, T.; Sheer, D.; McDermid, S.; Kendall-Taylor, P.; Wynford-Thomas, D.

    1989-01-01

    Human primary thyroid follicular epithelial cells were transfected with a plasmid containing an origin-defective SV40 genome (SVori-) to produce several immortal cell lines. Two of the 10 cell lines analysed expressed specific features of thyroid epithelial function (iodide-trapping and thyroglobulin production). These two lines were characterised in detail and found to be growth factor-independent, capable of anchorage-independent growth at low frequency but non-tumorigenic in nude mice. These differentiated, These differentiated, partially transformed cell lines were shown to be suitable for gene transfer at high frequency using simple coprecipitation techniques. Images Figure 2 Figure 3 Figure 4 PMID:2557880

  7. Glyoxalase I in detoxification: studies using a glyoxalase I transfectant cell line.

    PubMed Central

    Ranganathan, S; Walsh, E S; Tew, K D

    1995-01-01

    The glyoxalase system (glyoxalase I, glyoxalase II and GSH as cofactor) is involved in the detoxification of methylglyoxal (a byproduct of the glycolytic pathway) and other alpha-oxoaldehydes. We have transfected a 622 bp cDNA encoding human glyoxalase I into murine NIH3T3 cells. The recipient cells were shown to express elevated transcript and protein levels and a 10-fold increase in glyoxalase I enzyme activity. This was accompanied by an increased tolerance for exogenous methylglyoxal and enhanced resistance to the cytotoxic effects of two glyoxalase I inhibitors (s-p-bromobenzylglutathione diethyl ester and s-p-bromobenzylglutathione dicyclopentyl ester), a glutathione analogue [gamma-glutamyl-(S)-(benzyl)cysteinyl-(R)-(-)-phenylglycine diethyl ester] and the anti-cancer drugs mitomycin C and adriamycin. Steady-state levels of GSH were significantly lower in the transfected cells, perhaps reflecting increased flux as a consequence of elevated glyoxalase activity. This decrease did not alter the sensitivity to the alkylating agent chlorambucil. Although transfection did not affect the growth or doubling time of the NIH3T3 cells, analysis of glyoxalase I activity showed a consistent increase in tumour tissue when compared with pair-matched controls. Thus increased glyoxalase I is associated with the malignant phenotype and may also contribute to protection against the cytotoxicity of certain anti-cancer drugs. Images Figure 1 PMID:7619046

  8. In vitro transfection of bone marrow-derived dendritic cells with TATp-liposomes

    PubMed Central

    Pappalardo, Juan Sebastián; Langellotti, Cecilia A; Di Giacomo, Sebastián; Olivera, Valeria; Quattrocchi, Valeria; Zamorano, Patricia I; Hartner, William C; Levchenko, Tatyana S; Torchilin, Vladimir P

    2014-01-01

    Dendritic cells (DC) are antigen-presenting cells uniquely capable of priming naïve T cells and cross-presenting antigens, and they determine the type of immune response elicited against an antigen. TAT peptide (TATp), is an amphipathic, arginine-rich, cationic peptide that promotes penetration and translocation of various molecules and nanoparticles into cells. TATp-liposomes (TATp-L) used for DC transfection were prepared using TATp derivatized with a lipid-terminated polymer capable of anchoring in the liposomal membrane. Here, we show that the addition of TATp to DNA-loaded liposomes increased the uptake of DNA in DC. DNA-loaded TATp-L increased the in vitro transfection efficiency in DC cultures as evidenced by a higher expression of the enhanced green fluorescent protein and bovine herpes virus type 1 glycoprotein D (gD). The de novo synthesized gD protein was immunologically stimulating when transfections were performed with TATp-L, as indicated by the secretion of interleukin 6. PMID:24611012

  9. In vitro transfection of bone marrow-derived dendritic cells with TATp-liposomes.

    PubMed

    Pappalardo, Juan Sebastián; Langellotti, Cecilia A; Di Giacomo, Sebastián; Olivera, Valeria; Quattrocchi, Valeria; Zamorano, Patricia I; Hartner, William C; Levchenko, Tatyana S; Torchilin, Vladimir P

    2014-01-01

    Dendritic cells (DC) are antigen-presenting cells uniquely capable of priming naïve T cells and cross-presenting antigens, and they determine the type of immune response elicited against an antigen. TAT peptide (TATp), is an amphipathic, arginine-rich, cationic peptide that promotes penetration and translocation of various molecules and nanoparticles into cells. TATp-liposomes (TATp-L) used for DC transfection were prepared using TATp derivatized with a lipid-terminated polymer capable of anchoring in the liposomal membrane. Here, we show that the addition of TATp to DNA-loaded liposomes increased the uptake of DNA in DC. DNA-loaded TATp-L increased the in vitro transfection efficiency in DC cultures as evidenced by a higher expression of the enhanced green fluorescent protein and bovine herpes virus type 1 glycoprotein D (gD). The de novo synthesized gD protein was immunologically stimulating when transfections were performed with TATp-L, as indicated by the secretion of interleukin 6. PMID:24611012

  10. Antigen mRNA-transfected, allogeneic fibroblasts loaded with NKT-cell ligand confer antitumor immunity.

    PubMed

    Fujii, Shin-ichiro; Goto, Akira; Shimizu, Kanako

    2009-04-30

    The maturation of dendritic cells (DCs) in situ by danger signals plays a central role in linking innate and adaptive immunity. We previously demonstrated that the activation of invariant natural killer T (iNKT) cells by administration of alpha-galactosylceramide (alpha-GalCer)-loaded tumor cells can act as a cellular adjuvant through the DC maturation. In the current study, we used allogeneic fibroblasts loaded with alpha-GalCer and transfected with antigen-encoding mRNA, thus combining the adjuvant effects of iNKT-cell activation with delivery of antigen to DCs in vivo. We found that these cells produce antigen protein and activate NK and iNKT cells. When injected into major histocompatibility complex (MHC)-mismatched mice, they elicited antigen-specific T-cell responses and provided tumor protection, suggesting that these immune responses depend on host DCs. In addition, antigen-expressing fibroblasts loaded with alpha-GalCer lead to a more potent T-cell response than those expressing NK cell ligands. Thus, glycolipid-loaded, mRNA-transfected allogeneic fibroblasts act as cellular vectors to provide iNKT-cell activation, leading to DC maturation and T-cell immunity. By harnessing the innate immune system and generating an adaptive immune response to a variety of antigens, this unique tool could prove clinically beneficial in the development of immunotherapies against malignant and infectious diseases. PMID:19164596

  11. Sodium Kinetics of Na,K-ATPase α Isoforms in Intact Transfected HeLa Cells

    PubMed Central

    Zahler, Raphael; Zhang, Zhong-Ting; Manor, Mira; Boron, Walter F.

    1997-01-01

    By participating in the regulation of ion and voltage gradients, the Na-K pump (i.e., Na,K-ATPase) influences many aspects of cellular physiology. Of the four α isoforms of the pump, α1 is ubiquitous, α2 is predominant in skeletal muscle, and α3 is found in neurons and the cardiac conduction system. To determine whether the isoforms have different intracellular Na+ affinities, we used the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI) to measure pump-mediated Na+ efflux as a function of [Na+]i in human HeLa cells stably transfected with rat Na-K pump isoforms. We Na+-loaded the cells, and then monitored the time course of the decrease in [Na+]i after removing external Na+. All transfected rat α subunits were highly ouabain resistant: the α1 isoform is naturally resistant, whereas the α2 and α3 isoforms had been mutagenized to render them resistant. Thus, the Na+ efflux mediated by endogenous and transfected pumps could be separated by studying the cells at low (1 μM) and high (4 mM) ouabain concentrations. We found that the apparent Km for Na+ efflux attributable to the native human α1 isoform was 12 mM, which was similar to the Km of rat α1. The α2 and α3 isoforms had apparent Km's of 22 and 33 mM, respectively. The cells expressing α3 had a high resting [Na+]i. The maximal activity of native α1 in the α3-transfected cells was only ∼56% of native α1 activity in untransfected HeLa cells, suggesting that transfection with α3 led to a compensatory decrease in endogenous α1 pumps. We conclude that the apparent Km(Na+) for rat Na-K pump isoforms increases in the sequence α1 < α2 < α3. The α3 isoform may be suited for handling large Na+ loads in electrically active cells. PMID:9236212

  12. Biological effects of eukaryotic recombinant plasmid pReceiver-M61-BAI-1 transfection on T24 cells and HUVECs

    PubMed Central

    Tian, Da-Wei; Hu, Hai-Long; Sun, Yan; Tang, Yang; Lei, Ming-De; Liu, Li-Wei; Han, Rui-Fa; Wu, Chang-Li

    2016-01-01

    The aim of the current study was to investigate the biological effect on T24 cells and human umbilical vein endothelial cells (HUVECs) of transfection with brain-specific angiogenesis inhibitor-1 (BAI-1). The recombinant plasmid pReceiver-M61-BAI-1 was transfected into human superficial bladder tumor cells (T24) and HUVECs, in parallel with the vector control. mRNA and protein expression levels of BAI-1 were then detected by quantitative polymerase chain reaction (qPCR) and western blotting, respectively. Cell apoptosis of T24 cells and HUVECs prior and subsequent to transfection with BAI-1 was analyzed by flow cytometric analysis. Proliferation of T24 cells and HUVECs prior and subsequent to transfection of BAI-1 was assessed by the MTT method. T24 cells and HUVECs transfected with pReceiver-M61-BA1-1 were classed as the experimental group; T24 cells and HUVECs transfected with p-Receiver-M61 were the control group. qPCR and western blotting methods confirmed that there was positive expression of BAI-1 in T24 cells and HUVECs transfected with pReceiver-M61-BAI-1, however BAI-1 was not expressed in T24 cells and HUVECs transfected with pReceiver-M61. The results of the MTT assay demonstrated that absorbance was markedly reduced in HUVECs at 12, 48 and 72 h subsequent to transfection with pReceiver-M61-BAI-1 when compared with that of the control group and in T24 cells transfected with p-Receiver-M61-BAI-1. Furthermore, flow cytometry results also indicated that the apoptotic rate of HUVECs transfected with p-Receiver-M61-BAI-1 was significantly increased compared with that of the control group and T24 cells transfected with p-Receiver-M61-BAI-1. BAI-1 was observed to markedly inhibit the proliferation of vascular endothelial cells in vitro, however, no direct inhibition by BAI-1 was observed in T24 cells. In conclusion, BAI-1 is suggested to be a potential novel therapautic target for the inhibition of tumor neovascularization. PMID:27356780

  13. Effects of microbubble size on ultrasound-mediated gene transfection in auditory cells.

    PubMed

    Liao, Ai-Ho; Hsieh, Yi-Lei; Ho, Hsin-Chiao; Chen, Hang-Kang; Lin, Yi-Chun; Shih, Cheng-Ping; Chen, Hsin-Chien; Kuo, Chao-Yin; Lu, Ying-Jui; Wang, Chih-Hung

    2014-01-01

    Gene therapy for sensorineural hearing loss has recently been used to insert genes encoding functional proteins to preserve, protect, or even regenerate hair cells in the inner ear. Our previous study demonstrated a microbubble- (MB-)facilitated ultrasound (US) technique for delivering therapeutic medication to the inner ear. The present study investigated whether MB-US techniques help to enhance the efficiency of gene transfection by means of cationic liposomes on HEI-OC1 auditory cells and whether MBs of different sizes affect such efficiency. Our results demonstrated that the size of MBs was proportional to the concentration of albumin or dextrose. At a constant US power density, using 0.66, 1.32, and 2.83 μm albumin-shelled MBs increased the transfection rate as compared to the control by 30.6%, 54.1%, and 84.7%, respectively; likewise, using 1.39, 2.12, and 3.47 μm albumin-dextrose-shelled MBs increased the transfection rates by 15.9%, 34.3%, and 82.7%, respectively. The results indicate that MB-US is an effective technique to facilitate gene transfer on auditory cells in vitro. Such size-dependent MB oscillation behavior in the presence of US plays a role in enhancing gene transfer, and by manipulating the concentration of albumin or dextrose, MBs of different sizes can be produced.

  14. Effects of Microbubble Size on Ultrasound-Mediated Gene Transfection in Auditory Cells

    PubMed Central

    Liao, Ai-Ho; Hsieh, Yi-Lei; Ho, Hsin-Chiao; Chen, Hang-Kang; Lin, Yi-Chun; Kuo, Chao-Yin; Lu, Ying-Jui

    2014-01-01

    Gene therapy for sensorineural hearing loss has recently been used to insert genes encoding functional proteins to preserve, protect, or even regenerate hair cells in the inner ear. Our previous study demonstrated a microbubble- (MB-)facilitated ultrasound (US) technique for delivering therapeutic medication to the inner ear. The present study investigated whether MB-US techniques help to enhance the efficiency of gene transfection by means of cationic liposomes on HEI-OC1 auditory cells and whether MBs of different sizes affect such efficiency. Our results demonstrated that the size of MBs was proportional to the concentration of albumin or dextrose. At a constant US power density, using 0.66, 1.32, and 2.83 μm albumin-shelled MBs increased the transfection rate as compared to the control by 30.6%, 54.1%, and 84.7%, respectively; likewise, using 1.39, 2.12, and 3.47 μm albumin-dextrose-shelled MBs increased the transfection rates by 15.9%, 34.3%, and 82.7%, respectively. The results indicate that MB-US is an effective technique to facilitate gene transfer on auditory cells in vitro. Such size-dependent MB oscillation behavior in the presence of US plays a role in enhancing gene transfer, and by manipulating the concentration of albumin or dextrose, MBs of different sizes can be produced. PMID:25254216

  15. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control.

    PubMed

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L James; Lu, Wu

    2016-01-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a "3D nano-channel electroporation (NEP) chip" on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40,000 cells per cm(2) on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.

  16. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control.

    PubMed

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L James; Lu, Wu

    2016-01-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a "3D nano-channel electroporation (NEP) chip" on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40,000 cells per cm(2) on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells. PMID:26309218

  17. Gold nanoparticle mediated cell manipulation using fs and ps laser pulses for cell perforation and transfection

    NASA Astrophysics Data System (ADS)

    Heinemann, D.; Schomaker, M.; Motekaitis, D.; Krawinkel, J.; Killian, D.; Escobar, H. M.; Junghanß, Christian; Heisterkamp, Alexander

    2011-03-01

    Manipulation of cells requires the delivery of membrane-impermeable substances like genetic materials or proteins into the cytoplasm. Thus delivery of molecules over the cell membrane barrier is one of the key technologies in molecular biology. Many techniques concerning especially the delivery foreign DNA have been developed. Notwithstanding there still is a range of applications where these standard techniques fail to raise the desired results due to low efficiencies, high toxicity or other safety issues. Especially the transfection of sensitive cell types like primary and stem cells can be problematic. Here we present an alternative, laser based technique to perforate the cell membrane and thus allowing efficient delivery of extra cellular molecules: Gold nanoparticles (GNP) are brought into close contact with the cell, were the laser-GNP interaction leads to membrane perforation. This allows the utilisation of a weakly focused laser beam leading to fast scanning of the sample and thus to a high throughput. To investigate the GNP-laser interaction in more detail we have compared membrane perforation obtained by different laser pulse lengths. From our results we assume strong light absorption for ps laser pulses and relatively small particles as the initiating perforation mechanism, whereas an enhanced near field scattering occurs at 200 nm GNP when using fs laser pulses. SEM and ESEM imaging were applied to give a deeper insight in the GNP-cell interaction and the effects of laser radiation on the GNP. Additionally dextran- FITC derivatives of varying sizes were used to investigate the impact of molecule size on delivery efficiency.

  18. Single cell transfection by laser-induced breakdown of an optically trapped gold nanoparticle

    NASA Astrophysics Data System (ADS)

    Arita, Yoshihiko; Ploschner, Martin; Antkowiak, Maciej; Gunn-Moore, Frank; Dholakia, Kishan

    2014-03-01

    Cell selective introduction of therapeutic agents remains a challenging problem. Cavitation-based therapies including ultrasound-induced sonoporation and laser-induced optoporation have led the way for novel approaches to provide the potential of sterility and cell selectivity compared with viral or biochemical counterparts. Acoustic streaming, shockwaves and liquid microjets associated with the cavitation dynamics are implicated in gene and drug delivery. These approaches, however, often lead to non-uniform and sporadic molecular uptake that lacks refined spatial control and suffers from a significant loss of cell viability. Here we demonstrate spatially controlled cavitation instigated by laser-induced breakdown of an optically trapped single gold nanoparticle. Our unique approach employs optical tweezers to trap a single nanoparticle, which when irradiated by a nanosecond laser pulse is subject to laser-induced breakdown followed by cavitation. Using this method for laser-induced cavitation, we can gain additional degrees of freedom for the cavitation process - the particle material, its size, and its position relative to cells or tissues. We show the energy breakdown threshold of gold nanoparticles of l00nm with a single nanosecond laser pulse at 532 nm is three orders of magnitude lower than that for water, which leads to gentle nanocavitation enabling single cell transfection. We optimize the shear stress to the cells from the expanding bubble to be in the range of 1-10 kPa for transfection by precisely positioning a trapped gold nanoparticle, and thus nanobubble, relative to a cell of interest. The method shows transfection of plasmid-DNA into individual mammalian cells with an efficiency of 75%.

  19. Bead transfection: rapid and efficient gene transfer into marrow stromal and other adherent mammalian cells.

    PubMed

    Matthews, K E; Mills, G B; Horsfall, W; Hack, N; Skorecki, K; Keating, A

    1993-05-01

    We report a simple, rapid, efficient and cost-effective method of gene transfer into bone marrow stromal and other adherent mammalian cells. Our approach involves brief incubation of cells with glass beads in a solution containing the DNA to be transferred. We optimized the technique using COS cells (SV40 transformed kidney cell line from African green monkey) and a transient expression assay for chloramphenicol acetyl transferase (CAT). Factors affecting gene transfer include size and condition of the beads and DNA concentration, but not DNA conformation. Gene transfer efficiency, assessed in a transient expression assay for beta-galactosidase activity, was 5 and 3% in nontransformed human bone marrow stromal cells and COS cells, respectively. Long-term stable expression with the selectable marker, neomycin phosphotransferase, was demonstrated in clonogenic COS cells at a frequency of 27%. Southern analysis of resistant clones revealed the transferred DNA to be integrated in low copy number at one or two sites in the host cell genome. Comparison with electroporation and DEAE-dextran indicates that bead transfection is more efficient than the latter and less costly than either of these methods. In view of its simplicity and because the use of retroviral sequences can be avoided, bead transfection may be an attractive means of gene insertion for gene therapy.

  20. Gene Transfection of Human Turbinate Mesenchymal Stromal Cells Derived from Human Inferior Turbinate Tissues

    PubMed Central

    Kwon, Jin Seon; Park, Seung Hun; Baek, Ji Hye; Dung, Truong Minh; Kim, Sung Won; Min, Byoung Hyun; Kim, Jae Ho; Kim, Moon Suk

    2016-01-01

    Human turbinate mesenchymal stromal cells (hTMSCs) are novel stem cells derived from nasal inferior turbinate tissues. They are easy to isolate from the donated tissue after turbinectomy or conchotomy. In this study, we applied hTMSCs to a nonviral gene delivery system using polyethyleneimine (PEI) as a gene carrier; furthermore, the cytotoxicity and transfection efficiency of hTMSCs were evaluated to confirm their potential as resources in gene therapy. DNA-PEI nanoparticles (NPs) were generated by adding the PEI solution to DNA and were characterized by a gel electrophoresis and by measuring particle size and surface charge of NPs. The hTMSCs were treated with DNA-PEI NPs for 4 h, and toxicity of NPs to hTMSCs and gene transfection efficiency were monitored using MTT assay, fluorescence images, and flow cytometry after 24 h and 48 h. At a high negative-to-positive charge ratio, DNA-PEI NPs treatment led to cytotoxicity of hTMSCs, but the transfection efficiency of DNA was increased due to the electrostatic effect between the NPs and the membranes of hTMSCs. Importantly, the results of this research verified that PEI could deliver DNA into hTMSCs with high efficiency, suggesting that hTMSCs could be considered as untapped resources for applications in gene therapy. PMID:26783402

  1. Impact of Polyethylenimine Conjugation Mode on the Cell Transfection Efficiency of Silica Nanovectors.

    PubMed

    Wang, Xiaolin; Masse, Sylvie; Laurent, Guillaume; Hélary, Christophe; Coradin, Thibaud

    2015-10-13

    The conjugation of polyethylenimine (PEI) to silica nanoparticles has emerged as a useful strategy in gene delivery. Here we investigate the influence of the PEI conjugation mode on the transfection ability of plain silica nanoparticles. Surface functionalization with sulfonate- and chloride-bearing silanes modulates the amount and conformation of PEI and therefore the particles' affinity for the plasmid, without impacting on cytotoxicity. However, transfection efficiency in both immortalized and primary cells is more directly correlated to the nature and strength of the particle-PEI interactions. It suggests that PEI detachment from the particle surface at the stage of endosomal escape is a key event in the plasmid delivery process. These data should provide fruitful guidelines for the fine tuning of colloidal surfaces intended for intracellular delivery of bioactive molecules.

  2. Class I (H-2Kb) gene transfection reduces susceptibility of YAC-1 lymphoma targets to natural killer cells.

    PubMed

    Carlow, D A; Payne, U; Hozumi, N; Roder, J C; Czitrom, A A

    1990-04-01

    A "hybrid gene" (MTKb) comprised of the human metallothionein IIA promoter ligated to the genomic sequence of the major histocompatibility complex class I (H-2Kb) gene was subcloned into the expression vector pSV2neo and transfected into the natural killer (NK) cell-sensitive YAC-1 lymphoma. The Kb gene product was readily detectable on the cell surface of G418-resistant transfectants using both Kb-specific monoclonal antibodies and H-2b-specific cytolytic T cells. Unlike control pSV2neo transfectants, MTKb-pSV2neo transfectants were relatively resistant to lysis by NK cells from H-2a, H-2b, H-2k or H-2 (a x b)F1 haplotype mice. These data strongly suggest that the effects of MHC expression on susceptibility to NK cells can be mediated by a single and well-defined class I molecule, Kb.

  3. Transfection of normal human bronchial epithelial cells with the bcl-2 oncogene

    SciTech Connect

    Kennedy, C.H.; Kenyon, K.D.; Tesfaigzi, J.

    1995-12-01

    In vitro, studies examining the transformation of virus-immortalized human bronchial epithelial (HBE) cells after exposure to chemical and physical carcinogens have contributed to our understanding of the mechanisms that underlie the development of lung cancer. Virus-immortalized HBE cells have been used because of both the limited life span of normal human bronchial epithelial (NHBE) cells in culture (approximately 30-35 population doublins) and their resistance to in vitro malignant transformation. For example, human papillomavirus (HPV)-immortalized HBE cells have been used to study the genetic changes that occur after exposure to {alpha}-particles in vitro. Although this model may prove to be useful for studying the 18% or less of bronchogenic carcinomas found to contain HPV sequences, it is not an appropriate model for studying the majority of lung epithelial malignancies in which HPV DNA is not detected. This view is supported by the fact that HPV-immortalized cell lines commonly exhibit aneuploidy. This results of this study suggest that: (1) NHBE cells can be transiently transfected with the pCMV{Beta} vector; and (2) the antibiotic hygromycin-resistant transfected cells.

  4. Suppression of tumorigenicity in transformed cells after transfection with vinculin cDNA

    PubMed Central

    1992-01-01

    Transfection of chicken vinculin cDNA into two tumor cell lines expressing diminished levels of the endogenous protein, brought about a drastic suppression of their tumorigenic ability. The SV-40-transformed Balb/c 3T3 line (SVT2) contains four times less vinculin than the parental 3T3 cells, and the rat adenocarcinoma BSp73ASML has no detectable vinculin. Restoration of vinculin in these cells, up to the levels found in 3T3 cells, resulted in an apparent increase in substrate adhesiveness, a decrease in the ability to grow in soft agar, and suppression of their capacity to develop tumors after injection into syngeneic hosts or nude mice. These results suggest that vinculin, a cytoplasmic component of cell-matrix and cell-cell adhesions, may have a major suppressive effect on the transformed phenotype. PMID:1400584

  5. Immobilization of gold nanoparticles on cell culture surfaces for safe and enhanced gold nanoparticle-mediated laser transfection.

    PubMed

    Kalies, Stefan; Heinemann, Dag; Schomaker, Markus; Gentemann, Lara; Meyer, Heiko; Ripken, Tammo

    2014-01-01

    In comparison to standard transfection methods, gold nanoparticle-mediated laser transfection has proven to be a versatile alternative. This is based on its minor influence on cell viability and its high efficiency, especially for the delivery of small molecules like small interfering RNA. However, in order to transfer it to routine usage, a safety aspect is of major concern: The avoidance of nanoparticle uptake by the cells is desired. The immobilization of the gold nanoparticles on cell culture surfaces can address this issue. In this study, we achieved this by silanization of the appropriate surfaces and the binding of gold nanoparticles to them. Comparable perforation efficiencies to the previous approaches of gold nanoparticle-mediated laser transfection with free gold nanoparticles are demonstrated. The uptake of the immobilized particles by the cells is unlikely. Consequently, these investigations offer the possibility of bringing gold nanoparticle-mediated laser transfection closer to routine usage.

  6. Optimized production of HIV-1 virus-like particles by transient transfection in CAP-T cells.

    PubMed

    Gutiérrez-Granados, Sonia; Cervera, Laura; Segura, María de Las Mercedes; Wölfel, Jens; Gòdia, Francesc

    2016-05-01

    HIV-1 virus-like particles (VLPs) have great potential as new-generation vaccines. The novel CAP-T cell line is used for the first time to produce Gag-GFP HIV-1 VLPs by means of polyethylenimine (PEI)-mediated transient transfection. CAP-T cells are adapted to grow to high cell densities in serum-free medium, and are able to express complex recombinant proteins with human post-translational modifications. Furthermore, this cell line is easily transfected with PEI, which offers the flexibility to rapidly generate and screen a number of candidates in preclinical studies. Transient transfection optimization of CAP-T cells has been performed systematically in this work. It is determined that for optimal production, cells need to be growing at mid-exponential phase, Protein Expression Medium (PEM) medium has to be added post-transfection, and cells can be transfected by independent addition of DNA and PEI with no prior complexation. A Box-Behnken experimental design is used to optimize cell density at time of transfection, DNA/cell and PEI/cell ratios. The optimal conditions determined are transfection at a density of 3.3E + 06 cells/mL with 0.5 pg of DNA/cell and 3 pg of PEI/cell. Using the optimized protocol, 6 × 10(10) VLP/mL are obtained, demonstrating that CAP-T is a highly efficient cell line for the production of HIV-1 VLPs and potentially other complex viral-based biotherapeutics. PMID:26685677

  7. Optimized production of HIV-1 virus-like particles by transient transfection in CAP-T cells.

    PubMed

    Gutiérrez-Granados, Sonia; Cervera, Laura; Segura, María de Las Mercedes; Wölfel, Jens; Gòdia, Francesc

    2016-05-01

    HIV-1 virus-like particles (VLPs) have great potential as new-generation vaccines. The novel CAP-T cell line is used for the first time to produce Gag-GFP HIV-1 VLPs by means of polyethylenimine (PEI)-mediated transient transfection. CAP-T cells are adapted to grow to high cell densities in serum-free medium, and are able to express complex recombinant proteins with human post-translational modifications. Furthermore, this cell line is easily transfected with PEI, which offers the flexibility to rapidly generate and screen a number of candidates in preclinical studies. Transient transfection optimization of CAP-T cells has been performed systematically in this work. It is determined that for optimal production, cells need to be growing at mid-exponential phase, Protein Expression Medium (PEM) medium has to be added post-transfection, and cells can be transfected by independent addition of DNA and PEI with no prior complexation. A Box-Behnken experimental design is used to optimize cell density at time of transfection, DNA/cell and PEI/cell ratios. The optimal conditions determined are transfection at a density of 3.3E + 06 cells/mL with 0.5 pg of DNA/cell and 3 pg of PEI/cell. Using the optimized protocol, 6 × 10(10) VLP/mL are obtained, demonstrating that CAP-T is a highly efficient cell line for the production of HIV-1 VLPs and potentially other complex viral-based biotherapeutics.

  8. Immortalization and characterization of pleomorphic adenoma cells by transfection with the hTERT gene.

    PubMed

    Kitagawa, Masae; Ogawa, Ikuko; Shima, Kaori; Hashimoto, Sadamitsu; Kudo, Yasusei; Miyauchi, Mutsumi; Tahara, Hidetoshi; Shimono, Masaki; Takata, Takashi

    2007-08-01

    Pleomorphic adenomas (PAs) of salivary glands are characterized by the mixed appearance of epithelial and mesenchymal-like components such as myxoid and chondroid tissues. Although various studies have examined PAs, thus far it is not clear how PAs make these multiple components. Thus, clarification of the histodifferentiation of this unique salivary gland tumor using not only tissues in vivo but also PA cells cultured in vitro is necessary. However, no in vitro model of PA has been reported, because normal and benign tumor cells tend to grow slowly and senesce quickly in culture. Therefore, we immortalized cells using transfection of the hTERT gene without otherwise altering the nature of those cells. The immortalized PA cells expressed mRNA of the pleomorphic adenoma gene 1 and showed epithelial and neoplastic myoepithelial characteristics by immunohistochemical immunofluorescence analyses and ultrastructural study. Our findings suggest that these cells will be a useful model to study the cellular differentiation of PA.

  9. Selection and optimization of transfection enhancer additives for increased virus-like particle production in HEK293 suspension cell cultures.

    PubMed

    Cervera, Laura; Fuenmayor, Javier; González-Domínguez, Irene; Gutiérrez-Granados, Sonia; Segura, Maria Mercedes; Gòdia, Francesc

    2015-12-01

    The manufacturing of biopharmaceuticals in mammalian cells typically relies on the use of stable producer cell lines. However, in recent years, transient gene expression has emerged as a suitable technology for rapid production of biopharmaceuticals. Transient gene expression is particularly well suited for early developmental phases, where several potential therapeutic targets need to be produced and tested in vivo. As a relatively new bioprocessing modality, a number of opportunities exist for improving cell culture productivity upon transient transfection. For instance, several compounds have shown positive effects on transient gene expression. These transfection enhancers either facilitate entry of PEI/DNA transfection complexes into the cell or nucleus or increase levels of gene expression. In this work, the potential of combining transfection enhancers to increase Gag-based virus-like particle production levels upon transfection of suspension-growing HEK 293 cells is evaluated. Using Plackett-Burman design of experiments, it is first tested the effect of eight transfection enhancers: trichostatin A, valproic acid, sodium butyrate, dimethyl sulfoxide (DMSO), lithium acetate, caffeine, hydroxyurea, and nocodazole. An optimal combination of compounds exhibiting the highest effect on gene expression levels was subsequently identified using a surface response experimental design. The optimal consisted on the addition of 20 mM lithium acetate, 3.36 mM valproic acid, and 5.04 mM caffeine which increased VLP production levels 3.8-fold, while maintaining cell culture viability at 94%. PMID:26278533

  10. Partial processing of the neuropeptide Y precursor in transfected CHO cells.

    PubMed

    Wulff, B S; O'Hare, M M; Boel, E; Theill, L E; Schwartz, T W

    1990-02-12

    The activation of regulatory peptides by post-translational modification of their biosynthetic precursors is generally thought to occur only in neuroendocrine cells. We have selected clones of Chinese hamster ovary cells, a non-neuroendocrine cell line, which were transfected with a eukaryotic expression vector coding for the precursor for neuropeptide Y. Although the majority of the immunoreactive NPY was found in the form of pro-NPY, some degree of intracellular proteolytic processing of the precursor occurred in all clones. Part of the intracellular NPY immunoreactivity was even correctly amidated. Extracellular degradation of pro-NPY in the tissue culture medium generated immunoreactivity which corresponded in size to NPY. It is concluded that precursor processing can occur in non-neuroendocrine cells both as a biological process within the cells and as apparent processing, degradation in the tissue culture medium.

  11. Using the gene pulser MXcell electroporation system to transfect primary cells with high efficiency.

    PubMed

    McCoy, Adam M; Collins, Michelle L; Ugozzoli, Luis A

    2010-01-01

    It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments. PMID:20057352

  12. Immunological recognition of different forms of the neurotensin receptor in transfected cells and rat brain.

    PubMed Central

    Boudin, H; Grauz-Guyon, A; Faure, M P; Forgez, P; Lhiaubet, A M; Dennis, M; Beaudet, A; Rostene, W; Pelaprat, D

    1995-01-01

    In this work, the molecular forms of the rat neurotensin receptor (NTR) expressed in transfected Chinese hamster ovary (CHO) cells, in infected Sf9 insect cells and in rat cerebral cortex were immunologically detected by means of an anti-peptide antibody raised against a fragment of the third intracellular loop of the receptor. Immunoblot experiments against a fusion protein indicated that the anti-peptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence within the NTR. In immunoblot analysis of membranes from NTR-transfected CHO cells, high levels of immunoreactivity were observed between 60 and 72 kDa, while only a faint labelling was observed at 47 kDa, the molecular mass deduced for the rat NTR cDNA. The bands of high molecular mass were no longer observed after deglycosylation of membrane proteins by peptide N-glycosidase F, indicating that they represented glycosylated forms of the receptor. Extracts of membranes derived from baculovirus-infected Sf9 insect-cells expressing the NTR provided a quite different immunoblot pattern, since the major band detected in that case was at 47 kDa, the molecular size of the non-glycosylated receptor. Taken together, these data show that, while most of the NTR protein was glycosylated in CHO cells, it was unglycosylated in Sf9 insect-cells. In addition, molecular sizes of the receptor proteins observed in these two cell lines differed from those obtained for the NTR endogenously expressed in the rat cerebral cortex of 7 day-old rats, where bands at 56 and 54 kDa were detected. Binding experiments carried out on membrane preparations obtained from baculovirus-infected Sf9 cells demonstrated that the immunogenic sequence was still accessible to the antibody when the receptor was embedded in the cell membrane. Immunohistochemical studies carried out on both transfected CHO cells and infected Sf9 cells confirmed this interpretation and further indicated that the antibody could be applied

  13. A cost-effective approach to microporate mammalian cells with the Neon Transfection System.

    PubMed

    Brees, Chantal; Fransen, Marc

    2014-12-01

    Electroporation is one of the most efficient nonviral methods for transferring exogenous DNA into mammalian cells. However, the relatively high costs of electroporation kits and reagents temper the routine use of this fast and easy to perform technique in many laboratories. Several years ago, a new flexible and easy to operate electroporation device was launched under the name Neon Transfection System. This device uses specialized pipette tips containing gold-plated electrodes as electroporation chamber. Here we report a protocol to regenerate these expensive tips as well as some other Neon kit accessories, thereby reducing the cost of electroporation at least 10-fold. PMID:25172131

  14. A cost-effective approach to microporate mammalian cells with the Neon Transfection System.

    PubMed

    Brees, Chantal; Fransen, Marc

    2014-12-01

    Electroporation is one of the most efficient nonviral methods for transferring exogenous DNA into mammalian cells. However, the relatively high costs of electroporation kits and reagents temper the routine use of this fast and easy to perform technique in many laboratories. Several years ago, a new flexible and easy to operate electroporation device was launched under the name Neon Transfection System. This device uses specialized pipette tips containing gold-plated electrodes as electroporation chamber. Here we report a protocol to regenerate these expensive tips as well as some other Neon kit accessories, thereby reducing the cost of electroporation at least 10-fold.

  15. Differentiation of Odontoblast-Like Cells From Mouse Induced Pluripotent Stem Cells by Pax9 and Bmp4 Transfection

    PubMed Central

    Seki, Daisuke; Takeshita, Nobuo; Oyanagi, Toshihito; Sasaki, Shutaro; Takano, Ikuko; Hasegawa, Masakazu

    2015-01-01

    The field of tooth regeneration has progressed in recent years, and human tooth regeneration could become viable in the future. Because induced pluripotent stem (iPS) cells can differentiate into odontogenic cells given appropriate conditions, iPS cells are a potential cell source for tooth regeneration. However, a definitive method to induce iPS cell-derived odontogenic cells has not been established. We describe a novel method of odontoblast differentiation from iPS cells using gene transfection. We generated mouse iPS cell-derived neural crest-like cells (iNCLCs), which exhibited neural crest markers. Next, we differentiated iNCLCs into odontoblast-like cells by transfection of Pax9 and Bmp4 expression plasmids. Exogenous Pax9 upregulated expression of Msx1 and dentin matrix protein 1 (Dmp1) in iNCLCs but not bone morphogenetic protein 4 (Bmp4) or dentin sialophosphoprotein (Dspp). Exogenous Bmp4 upregulated expression of Msx1, Dmp1, and Dspp in iNCLCs, but not Pax9. Moreover, cotransfection of Pax9 and Bmp4 plasmids in iNCLCs revealed a higher expression of Pax9 than when Pax9 plasmid was used alone. In contrast, exogenous Pax9 downregulated Bmp4 overexpression. Cotransfection of Pax9 and Bmp4 synergistically upregulated Dmp1 expression; however, Pax9 overexpression downregulated exogenous Bmp4-induced Dspp expression. Together, these findings suggest that an interaction between exogenous Pax9- and Bmp4-induced signaling modulated Dmp1 and Dspp expression. In conclusion, transfection of Pax9 and Bmp4 expression plasmids in iNCLCs induced gene expression associated with odontoblast differentiation, suggesting that iNCLCs differentiated into odontoblast-like cells. The iPS cell-derived odontoblast-like cells could be a useful cell source for tooth regeneration. Significance It has been reported that induced pluripotent stem (iPS) cells differentiate into odontogenic cells by administration of recombinant growth factors and coculture with odontogenic cells

  16. Human allospecific cytolytic T lymphocyte lysis of a murine cell transfected with HLA-A2.

    PubMed

    Koller, T D; Clayberger, C; Maryanski, J L; Krensky, A M

    1987-04-01

    A variety of molecules are involved in the interaction of human allospecific cytolytic T lymphocytes (CTL) with target cells. Monoclonal antibodies specific for these molecules inhibit CTL-target conjugate formation and/or lysis. To further study recognition and lysis of targets by human CTL, we used a murine mastocytoma cell line transfected with the histocompatibility leukocyte antigen (HLA)-A2 gene (P815-A2+) as a target for human HLA-A2-specific CTL. We find that only a subset of human HLA-A2-specific CTL can lyse murine P815-A2+ cells, suggesting that the murine cells may lack one or more accessory molecules needed for CTL recognition and lysis. PMID:3549894

  17. A paradigm change: efficient transfection of human leukemia cells by stimuli-responsive multicompartment micelles.

    PubMed

    Rinkenauer, Alexandra C; Schallon, Anja; Günther, Ulrike; Wagner, Michael; Betthausen, Eva; Schubert, Ulrich S; Schacher, Felix H

    2013-11-26

    The controlled nonviral delivery of genetic material using cationic polymers into cells has been of interest during the past three decades, yet the ideal delivery agent featuring utmost transfection efficiency and low cytotoxicity still has to be developed. Here, we demonstrate that multicompartment micelles from stimuli-responsive triblock terpolymers, polybutadiene-block-poly(methacrylic acid)-block-poly(2-(dimethylamino)ethyl methacrylate) (BMAAD), are promising candidates. The structures exhibit a patchy shell, consisting of amphiphilic (interpolyelectrolyte complexes, MAA and D) and cationic patches (excess D), generating a surface reminiscent to those of certain viruses and capable of undergoing pH-dependent changes in charge stoichiometry. After polyplex formation with plasmid DNA, superior transfection efficiencies can be reached for both adherent cells and human leukemia cells. Compared to the gold standard PEI, remarkable improvements and a number of advantages were identified for this system, including increased cellular uptake and an improved release of the genetic material, accompanied by fast and efficient endosomal escape. Furthermore, high sedimentation rates might be beneficial regarding in vitro applications. PMID:24147450

  18. Three-dimensional imaging of nucleolin trafficking in normal cells, transfectants, and heterokaryons

    NASA Astrophysics Data System (ADS)

    Ballou, Byron T.; Fisher, Gregory W.; Deng, Jau-Shyong; Hakala, Thomas R.; Srivastava, Meera; Farkas, Daniel L.

    1996-04-01

    The study of intracellular trafficking using labeled molecules has been aided by the development of the cyanine fluorochromes, which are easily coupled, very soluble, resist photobleaching, and fluoresce at far-red wavelengths where background fluorescence is minimal. We have used Cy3-, Cy5-, and Cy5.5-labeled antibodies, antigen-binding fragments, and specifically binding single-stranded oligonucleotides to follow expression and trafficking of nucleolin, the most abundant protein of the nucleolus. Nucleolin shuttles between the nucleolus and the cytoplasm, and is also expressed on the cell surface, allowing us to test our techniques at all three cellular sites. Differentially cyanine-labeled non-specific antibodies were used to control for non-specific binding. Similarly, the differentially labeled non-binding strand of the cloned oligonucleotide served as a control. The multimode microscope allowed us to follow both rapid and slow redistributions of labeled ligands in the same study. We also performed 3-D reconstructions of nucleolin distribution in cells using rapid acquisition and deconvolution. Microinjection of labeled ligands was used to follow intracellular distribution, while incubation of whole cells with antibody and antigen-binding fragments was used to study uptake. To unambiguously define trafficking, and eliminate the possibility of interference by cross-reactive proteins, we transfected mouse renal cell carcinoma cells that express cell surface nucleolin with human nucleolin. We used microinjection and cell surface staining with Cy3- or Cy5- labeled monoclonal antibody D3 (specific for human nucleolin) to assess the cellular distribution of the human protein. Several clones expressed human nucleolin on their surfaces and showed high levels of transport of the human protein into the mouse nucleus and nucleolus. This distribution roughly parallels that of mouse nucleolin as determined by labeled polyclonal antibody. We have used these engineered

  19. Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection.

    PubMed

    van der Loo, J C M; Swaney, W P; Grassman, E; Terwilliger, A; Higashimoto, T; Schambach, A; Baum, C; Thrasher, A J; Williams, D A; Nordling, D L; Reeves, L; Malik, P

    2012-03-01

    The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10-14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6 × 10(7) infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner.

  20. Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection

    PubMed Central

    van der Loo, JCM; Swaney, WP; Grassman, E; Terwilliger, A; Higashimoto, T; Schambach, A; Baum, C; Thrasher, AJ; Williams, DA; Nordling, DL; Reeves, L; Malik, P

    2014-01-01

    The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10–14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6×107 infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner. PMID:21753795

  1. Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection.

    PubMed

    van der Loo, J C M; Swaney, W P; Grassman, E; Terwilliger, A; Higashimoto, T; Schambach, A; Baum, C; Thrasher, A J; Williams, D A; Nordling, D L; Reeves, L; Malik, P

    2012-03-01

    The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10-14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6 × 10(7) infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner. PMID:21753795

  2. Efficient protection and transfection of small interfering RNA by cationic shell-crosslinked knedel-like nanoparticles.

    PubMed

    Shen, Yuefei; Fang, Huafeng; Zhang, Ke; Shrestha, Ritu; Wooley, Karen L; Taylor, John-Stephen A

    2013-04-01

    Despite the great potential of small interfering RNA (siRNA) as a therapeutic agent, progress in this area has been hampered by a lack of efficient biocompatible transfection agents. Recently, cationic shell-crosslinked knedel-like nanoparticles (cSCKs) were found to possess lower cytotoxicity and better transfection ability for phosphorothioate ODNs and plasmid DNA than the commonly used cationic lipid-based agent Lipofectamine. To determine the usefulness of cSCKs for siRNA transfection, a small library of cSCKs with varying percentage of primary and tertiary amines was assessed for its ability to bind to siRNA, inhibit siRNA degradation in human serum, and to transfect HeLa and mouse macrophage cell lines. The silencing efficiency in HeLa cells was greatest with the cSCK with 100% primary amines (pa100) as determined by their viability following transfection with cytotoxic and non-cytotoxic siRNAs. cSCK-pa100 showed greater silencing efficiency than Lipofectamine 2000 in the HeLa cells, as well in 293T and human bronchial epithelial (HEK) cells, but was comparable in human bronchial epithelial (BEAS-2B) cells and human mammary epithelial (MCF10a) cells. cSCK-pa100 also showed greater silencing of iNOS expression than Lipofectamine 2000 in a mouse macrophage cell line, and provided greater protection from serum degradation, demonstrating its potential usefulness as an siRNA transfection agent. The siRNA silencing of iNOS at lower concentrations of siRNA could be enhanced by complexation with the fusogenic GALA peptide, which was shown to enhance endosomal escape following uptake.

  3. Physicochemical properties of polymers: An important system to overcome the cell barriers in gene transfection.

    PubMed

    Namvar, Ali; Bolhassani, Azam; Khairkhah, Niloofardokht; Motevalli, Fatemeh

    2015-07-01

    Delivery of the macromolecules including DNA, miRNA, and antisense oligonucleotides is typically mediated by carriers due to the large size and negative charge. Different physical (e.g., gene gun or electroporation), and chemical (e.g., cationic polymer or lipid) vectors have been already used to improve the efficiency of gene transfer. Polymer-based DNA delivery systems have attracted special interest, in particular via intravenous injection with many intra- and extracellular barriers. The recent progress has shown that stimuli-responsive polymers entitled as multifunctional nucleic acid vehicles can act to target specific cells. These nonviral carriers are classified by the type of stimulus including reduction potential, pH, and temperature. Generally, the physicochemical characterization of DNA-polymer complexes is critical to enhance the transfection potency via protection of DNA from nuclease digestion, endosomal escape, and nuclear localization. The successful clinical applications will depend on an exact insight of barriers in gene delivery and development of carriers overcoming these barriers. Consequently, improvement of novel cationic polymers with low toxicity and effective for biomedical use has attracted a great attention in gene therapy. This article summarizes the main physicochemical and biological properties of polyplexes describing their gene transfection behavior, in vitro and in vivo. In this line, the relative efficiencies of various cationic polymers are compared.

  4. Suppression of vascular smooth muscle cell responses induced by TNF-α in GM3 synthase gene transfected cells.

    PubMed

    Park, Sung-Suk; Kim, Wun-Jae; Moon, Sung-Kwon

    2011-01-01

    The natural accumulation of ganglioside GM3 (N-glycolylneuraminic acid) on atherosclerotic lesions is a common theory. The present study is the first to examine the effects of the GM3 synthase gene on the responses of vascular smooth muscle cells (VSMC) to tumor necrosis factor-α (TNF-α). We found that overexpression of the GM3 synthase gene inhibited DNA synthesis and ERK1/2 activity induced by TNF-α in VSMC, whereas the basal levels of DNA synthesis and ERK1/2 activity remained unchanged. In addition, GM3 synthase gene transfectants significantly reduced the migration and invasion of VSMC following TNF-α treatment, compared with empty vector transfectants. Furthermore, TNF-α-induced matrix metalloproteinase-9 (MMP-9) expression and promoter activity were also decreased in GM3 synthase gene transfectants. GM3 synthase gene expression markedly suppressed the TNF-α-stimulated transcriptional activity of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB), which are the controlling factors of MMP-9 expression. Consistent with these results, the addition of anti-GM3 antibody into the GM3 synthase gene transfectants blocked inhibition of DNA synthesis, ERK1/2 activity, migration and invasion. Finally, GM3 synthase gene transfectants treated with anti-GM3 antibody reversed the suppression of MMP-9 expression by reducing AP-1 and NF-κB binding activity. These results suggest regulatory roles for the GM3 synthase gene in VSMC proliferation and migration during the formation of atherosclerotic lesions.

  5. Nerve growth factor (NGF) induces neuronal differentiation in neuroblastoma cells transfected with the NGF receptor cDNA

    SciTech Connect

    Matsushima, H.; Bogenmann, E. )

    1990-09-01

    Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment.

  6. Towards high-throughput automated targeted femtosecond laser-based transfection of adherent cells

    NASA Astrophysics Data System (ADS)

    Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Gunn-Moore, Frank; Dholakia, Kishan

    2011-03-01

    Femtosecond laser induced cell membrane poration has proven to be an attractive alternative to the classical methods of drug and gene delivery. It is a selective, sterile, non-contact technique that offers a highly localized operation, low toxicity and consistent performance. However, its broader application still requires the development of robust, high-throughput and user-friendly systems. We present a system capable of unassisted enhanced targeted optoinjection and phototransfection of adherent mammalian cells with a femtosecond laser. We demonstrate the advantages of a dynamic diffractive optical element, namely a spatial light modulator (SLM) for precise three dimensional positioning of the beam. It enables the implementation of a "point-and-shoot" system in which using the software interface a user simply points at the cell and a predefined sequence of precisely positioned doses can be applied. We show that irradiation in three axial positions alleviates the problem of exact beam positioning on the cell membrane and doubles the number of viably optoinjected cells when compared with a single dose. The presented system enables untargeted raster scan irradiation which provides transfection of adherent cells at the throughput of 1 cell per second.

  7. Novel cancer vaccines prepared by anchoring cytokines to tumor cells avoiding gene transfection

    NASA Astrophysics Data System (ADS)

    Nizard, Philippe; Gross, David-Alexandre; Chenal, Alexandre; Beaumelle, Bruno; Kosmatopoulos, Konstadinos; Gillet, Daniel

    2002-06-01

    Cytokines have a strong potential for triggering anticancer immunity if released in the tumor microenvironment. Successful vaccines have been engineered using tumor cells genetically modified to secrete the cytokines. Unfortunately, this approach remains difficult and hazardous to perform in the clinic. We describe a new way of combining cytokines with tumor cells to prepare anticancer vaccines. This consists in anchoring recombinant cytokines to the membrane of killed tumor cells. Attachment is mediated by a fragment of diphtheria toxin (T) genetically connected to the cytokine. It is triggered by an acid pH pulse. The method was applied to IL-2, a potent anti-tumor cytokine. IL-2 anchored to the surface of tumor cells by the T anchor retained its IL-2 activity and remained exposed several days. Interestingly, vaccination of mice with these modified tumor cells induced a protective anti-tumor immunity mediated by tumor-specific cytotoxic T lymphocytes. This procedure presents several advantages as compared to the conventional approaches based on the transfection of tumor cells with cytokine genes. It does not require the culture of tumor cells from the patients and eliminates the safety problems connected with viral vectors while allowing the control of the amount of cytokines delivered with the vaccine.

  8. Upregulation of ULK1 expression in PC-3 cells following tumor protein P53 transfection by sonoporation

    PubMed Central

    WANG, YU; CHEN, YI-NI; ZHANG, WEI; YANG, YU; BAI, WEN-KUN; SHEN, E; HU, BING

    2016-01-01

    The aim of the present study was to investigate whether ultrasound combined with microbubbles was able to enhance liposome-mediated transfection of genes into human prostate cancer cells, and to examine the association between autophagy and tumor protein P53 (P53). An MTT assay was used to evaluate cell viability, while flow cytometry and fluorescence microscopy were used to measure gene transfection efficiency. Autophagy was observed using transmission electron microscopy. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were used to assess the expression of autophagy-associated genes. The results of the present study revealed that cell viability was significantly reduced following successfully enhanced transfection of P53 by ultrasound combined with microbubbles. In addition, serine/threonine-protein kinase ULK1 levels were simultaneously upregulated. Castration-resistant prostate cancer is difficult to treat and is investigated in the present study. P53 has a significant role in a number of key biological functions, including DNA repair, apoptosis, cell cycle, autophagy, senescence and angiogenesis. Prior to the present study, to the best of our knowledge, increased transfection efficiency and reduced side effects have been difficult to achieve. Ultrasound is considered to be a ‘gentle’ technique that may be able to achieve increased transfection efficiency and reduced side effects. The results of the present study highlight a potential novel therapeutic strategy for the treatment of prostate cancer. PMID:26870270

  9. Conceptual design of integrated microfluidic system for magnetic cell separation, electroporation, and transfection.

    PubMed

    Durdík, Š; Krafčík, A; Babincová, M; Babinec, P

    2013-09-01

    For the purposes of a successful ex vivo gene therapy we have proposed and analyzed a new concept of an integrated microfluidic system for combined magnetic cell separation, electroporation, and magnetofection. For the analysis of magnetic and electric field distribution (given by Maxwell equations) as well as dynamics of magnetically labeled cell and transfection complex, we have used finite element method directly interfaced to the Matlab routine solving Newton dynamical equations of motion. Microfluidic chamber has been modeled as a channel with height and length 1 mm and 1 cm, respectively. Bottom electrode consisted of 100 parallel ferromagnetic straps and the upper electrode was plate of diamagnetic copper. From the dynamics of magnetic particle motion we have found that the characteristic time-scales for the motion of cells (mean capture time ∼ 4 s) and gene complexes (mean capture time ∼ 3 min), when permanent magnets are used, are in the range suitable for efficient cell separation and gene delivery. The largest electric field intensity (∼10 kV/m) was observed at the edges of the microelectrodes, in the close proximity of magnetically separated cells, which is optimal for subsequent cell electroporation.

  10. Characterization of the estrogen receptor transfected MCF10A breast cell line 139B6.

    PubMed

    Pilat, M J; Christman, J K; Brooks, S C

    1996-01-01

    There has been increasing evidence which suggests that abnormal expression of the estrogen receptor (ER) protein in nonmalignant breast tissue may be important in the carcinogenic process. To examine the effects of ER expression in immortalized nonmalignant mammary epithelial cells, an expression vector containing human ER cDNA was transfected into the ER negative human breast cells, MCF10A. Characterization of a clone stably expressing ER, 139B6, provided evidence for the regulated synthesis of a functional ER capable of binding estradiol-17 beta (E2) and undergoing processing. Expression of the ER gene did not enable E2 to stimulate endogenous genes [progesterone receptor (PgR), pS2, cathepsin D and TGF alpha] which normally respond to estrogens in breast cancer cells. The ER in 139B6 cells was, however, capable of inducing expression of an ERE-regulated reporter gene, indicating its ability to interact with transcriptional machinery. Furthermore, cultures in log growth displayed a slight increase in doubling time in the presence of E2. These results indicate that ER expression alone is not sufficient to induce a transformed phenotype. Thus, the 139B6 cell line should provide a new model for determining what additional changes lead to increased growth potential in response to E2 and for exploring how E2 itself may help bring about changes leading to progression of preneoplastic breast epithelial cells.

  11. Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells.

    PubMed

    Bettinger, T; Carlisle, R C; Read, M L; Ogris, M; Seymour, L W

    2001-09-15

    Synthetic vectors were evaluated for their ability to mediate efficient mRNA transfection. Initial results indicated that lipoplexes, but not polyplexes based on polyethylenimine (PEI, 25 and 22 kDa), poly(L-lysine) (PLL, 54 kDa) or dendrimers, mediated efficient translation of mRNA in B16-F10 cells. Significant mRNA transfection was achieved by lipoplex delivery in quiescent (passage 0) human umbilical vein endothelial cells (HUVEC), and by passage 4, 10.7% of HUVEC were transfected compared to 0.84% with DNA. Lack of expression with PEI 25 kDa/mRNA or PLL 54 kDa/mRNA in a cell-free translation assay and following cytoplasmic injection into Rat1 cells indicated that these polyplexes were too stable to release mRNA. In contrast, polyplexes formed using smaller PEI 2 kDa and PLL 3.4 kDa gave 5-fold greater expression in B16-F10 cells compared to DOTAP, but were dependent on chloroquine for transfection activity. Endosomolytic activity was incorporated by conjugating PEI 2 kDa to melittin and resulting PEI 2 kDa-melittin/mRNA polyplexes mediated high transfection levels in HeLa cells (31.1 +/- 4.1%) and HUVEC (58.5 +/- 2.9%) in the absence of chloroquine, that was potentiated to 52.2 +/- 2.7 and 71.6 +/- 1.7%, respectively, in the presence of chloroquine. These results demonstrate that mRNA polyplexes based on peptide-modified low molecular weight polycations can possess versatile properties including endosomolysis that should enable efficient non-viral mRNA transfection of quiescent and post-mitotic cells. PMID:11557821

  12. G-patch domain containing 2, a gene highly expressed in testes, inhibits nuclear factor-κB and cell proliferation.

    PubMed

    Hu, Fen; Gou, Lixia; Liu, Qing; Zhang, Wendian; Luo, Mengmeng; Zhang, Xiujun

    2015-02-01

    G-patch domain containing 2 (GPATC2), a human gene that is highly expressed in the testes, was implicated as a novel cancer/testis antigen. The present study investigated GPATC2 expression in a number of human cell lines and rat tissues, and its potential biological function in 293T cells. Semi‑quantitative reverse transcription-polymerase chain reaction analysis showed that GPATC2 was widely expressed in 15 human cell lines (representing different lineages) and in 11 different rat tissues, and that the GPATC2 mRNA relative expression level was significantly higher in the testis than it was in other tissues. 293T cells were transiently transfected with GPATC2-p enhanced green fluorescent protein (EGFP)‑N1 or GPATC2-pEGFP-C3 and the nuclei were stained with 4',6'‑diamidino‑2‑phenylindole. The results showed that GPATC2 is predominantly expressed in the nucleus of 293T cells. Overexpression of GPATC2 may inhibit transcription of the NF-κB reporter gene. The role of GPATC2 in proliferation was analyzed with cell counting kit-8, colony-forming efficiency and flow cytometry assays. The results indicated that over‑expression of GPATC2 in 293T cells significantly inhibited cell proliferation by decreasing the number of cells in S phase. By contrast, GPATC2 knockdown by RNA interference exhibited the opposite effect, suggesting that GPATC2 may be involved in inhibiting G1-S phase transition in 293T cells. In conclusion, these results provide novel insight into the breadth of expression of GPATC2 and its role in cell proliferation.

  13. Transfection of neonatal rat Schwann cells with SV-40 large T antigen gene under control of the metallothionein promoter

    PubMed Central

    1987-01-01

    Secondary cultures of Schwann cells were transfected with a plasmid containing the SV-40 T antigen gene expressed under the control of the mouse metallothionein-I promoter. We used the calcium phosphate method for transfection and obtained a transfection efficiency of 0.01%. The colonies were cloned by limited dilution, and these cloned cell lines were carried in medium containing zinc chloride (100 microM). One cloned cell line, which has now been carried for 180 doublings, appears to have a transformed phenotype with a doubling time of 20 h. These cells express SV-40 T antigen while maintaining established Schwann cell properties (positive staining for 217c, Ran-2, A5E3, glial fibrillary acidic protein, presence of 2',3'-cyclic nucleotide phosphohydrolase [CNPase] activity, and the ability to synthesize sulfogalactosylceramide and mRNA for the myelin protein, P0). Removal of zinc chloride from the medium resulted in reduced expression of T antigen and a change in the appearance of the cells to a more bipolar shape, although they still did not exhibit contact inhibition and maintained a doubling time of 20 h. These cells now became Ran-2- negative and showed increases in CNPase activity and in their ability to synthesize sulfogalactosylceramide. The amount of P0 mRNA remained unchanged. Transfected Schwann cells, however, stopped dividing when they contacted either basal lamina or neurites and became bipolar in appearance. The Schwann cells in contact with the neurites then extended processes to wrap around bundles of neurites. Transfection with the SV-40 T antigen gene therefore provides a method for obtaining Schwann cell lines that continue to express properties associated with untransfected cells in culture and may be used to study axon-Schwann cell interaction. PMID:2824529

  14. Functionalized Multi-Wall Carbon Nanotubes Enhance Transfection and Expression Efficiency of Plasmid DNA in Fish Cells.

    PubMed

    Liu, Guanglu; Wang, Yuan; Hu, Yang; Yu, Xiaobo; Zhu, Bin; Wang, Gaoxue

    2016-03-03

    DNA vaccines are considered to be the most promising method against infectious diseases in the aquaculture industry. In the present study, we investigated the potency of ammonium group-functionalized multi-walled carbon nanotubes (MWCNTs) in enhancing the transfection and expression efficiency of plasmid DNA (pEGFP-vp5) in Ctenopharyngodon idellus kidney (CIK) cells. Agarose gel shift assay results show that ammonium group-functionalized carbon nanotubes are able to condense DNA in varying degrees. Scanning electron microscope (SEM) images shows that CIK cells show a great affinity for MWCNTs-NH₃⁺ and the CNTs covering the cell surface tend to orient their tips perpendicularly to the cell surface, and appear to be "needle-pricking the cells". Transmission electron microscope (TEM) images confirmed that MWCNTs-NH₃⁺ penetrate the cell membranes and are widely dispersed in the CIK cell. Real-time PCR was used to detect the transfection efficiency through the expression of the outer capsid protein (VP5). The results showed that the MWCNTs-NH₃⁺:DNA complexes are able to transfect CIK cells effectively at different charge ratio than naked DNA. Subsequent studies confirmed that both functional groups and charge ratio are important factors that determine the transfection efficiency of plasmid DNA. All these results indicated that MWCNTs-NH₃⁺:DNA complexes could be suitable for developing DNA vaccine for the control of virus infection in the aquaculture industry.

  15. Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells

    PubMed Central

    González, Cecilia; Esteban, Rosa; Canals, Carme; Muñiz-Díaz, Eduardo; Nogués, Núria

    2016-01-01

    Background The identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs), which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories. Methods We generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua). High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment. Results TransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies. Conclusions Both stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs. PMID:27603310

  16. Transfection of the Human Heme Oxygenase Gene Into Rabbit Coronary Microvessel Endothelial Cells: Protective Effect Against Heme and Hemoglobin Toxicity

    NASA Astrophysics Data System (ADS)

    Abraham, N. G.; Lavrovsky, Y.; Schwartzman, M. L.; Stoltz, R. A.; Levere, R. D.; Gerritsen, M. E.

    1995-07-01

    Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an ≈3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with >85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

  17. Functionalized Multi-Wall Carbon Nanotubes Enhance Transfection and Expression Efficiency of Plasmid DNA in Fish Cells

    PubMed Central

    Liu, Guanglu; Wang, Yuan; Hu, Yang; Yu, Xiaobo; Zhu, Bin; Wang, Gaoxue

    2016-01-01

    DNA vaccines are considered to be the most promising method against infectious diseases in the aquaculture industry. In the present study, we investigated the potency of ammonium group-functionalized multi-walled carbon nanotubes (MWCNTs) in enhancing the transfection and expression efficiency of plasmid DNA (pEGFP-vp5) in Ctenopharyngodon idellus kidney (CIK) cells. Agarose gel shift assay results show that ammonium group-functionalized carbon nanotubes are able to condense DNA in varying degrees. Scanning electron microscope (SEM) images shows that CIK cells show a great affinity for MWCNTs-NH3+ and the CNTs covering the cell surface tend to orient their tips perpendicularly to the cell surface, and appear to be “needle-pricking the cells”. Transmission electron microscope (TEM) images confirmed that MWCNTs-NH3+ penetrate the cell membranes and are widely dispersed in the CIK cell. Real-time PCR was used to detect the transfection efficiency through the expression of the outer capsid protein (VP5). The results showed that the MWCNTs-NH3+:DNA complexes are able to transfect CIK cells effectively at different charge ratio than naked DNA. Subsequent studies confirmed that both functional groups and charge ratio are important factors that determine the transfection efficiency of plasmid DNA. All these results indicated that MWCNTs-NH3+:DNA complexes could be suitable for developing DNA vaccine for the control of virus infection in the aquaculture industry. PMID:26950121

  18. A new cholesterol derivative suitable for transfecting certain type of cells in the presence of 10% serum.

    PubMed

    Sochanik, A; Kaida, I; Mitrus, I; Rajca, A; Szala, S

    2000-04-01

    We developed a new cationic lipid suitable for use as a DNA carrier in the presence of 10% sera. The novel compound (abbreviated as Arg-Chol) contains cholesterol and a dipeptide consisting of glycine and sterically protected arginine. The efficiency of reporter gene transfection using liposomes based on this new reagent was compared with that of liposomes made with other cationic derivatives of cholesterol. Lipoplexes formulated with the newly synthesized lipid mediate in vitro transfection of B16(F10) murine melanoma cells in the presence of 10% sera more efficiently than in other cell lines and compared with other cholesterol derivatives studied. PMID:10811467

  19. Genetically engineered cardiac pacemaker: Stem cells transfected with HCN2 gene and myocytes—A model

    NASA Astrophysics Data System (ADS)

    Kanani, S.; Pumir, A.; Krinsky, V.

    2008-01-01

    One of the successfully tested methods to design genetically engineered cardiac pacemaker cells consists in transfecting a human mesenchymal stem cell (hMSC) with a HCN2 gene and connecting it to a myocyte. We develop and study a mathematical model, describing a myocyte connected to a hMSC transfected with a HCN2 gene. The cardiac action potential is described both with the simple Beeler Reuter model, as well as with the elaborate dynamic Luo Rudy model. The HCN2 channel is described by fitting electrophysiological records, in the spirit of Hodgkin Huxley. The model shows that oscillations can occur in a pair myocyte-stem cell, that was not observed in the experiments yet. The model predicted that: (1) HCN pacemaker channels can induce oscillations only if the number of expressed I channels is low enough. At too high an expression level of I channels, oscillations cannot be induced, no matter how many pacemaker channels are expressed. (2) At low expression levels of I channels, a large domain of values in the parameter space (n, N) exists, where oscillations should be observed. We denote N the number of expressed pacemaker channels in the stem cell, and n the number of gap junction channels coupling the stem cell and the myocyte. (3) The expression levels of I channels observed in ventricular myocytes, both in the Beeler Reuter and in the dynamic Luo Rudy models are too high to allow to observe oscillations. With expression levels below ˜1/4 of the original value, oscillations can be observed. The main consequence of this work is that in order to obtain oscillations in an experiment with a myocyte-stem cell pair, increasing the values of n, N is unlikely to be helpful, unless the expression level of I has been reduced enough. The model also allows us to explore levels of gene expression not yet achieved in experiments, and could be useful to plan new experiments, aimed at improving the robustness of the oscillations.

  20. Localization and functional analysis of CHIP28k water channels in stably transfected Chinese hamster ovary cells.

    PubMed

    Ma, T; Frigeri, A; Tsai, S T; Verbavatz, J M; Verkman, A S

    1993-10-25

    CHIP28 is a major water transporting protein in erythrocytes and plasma membranes in kidney proximal tubule and thin descending limb of Henle. Chinese hamster ovary cells were stably transfected with the coding sequence of cloned rat kidney CHIP28k using expression vectors containing cytomegalovirus or Rous sarcoma virus promoters. Clonal cell populations expressed a 1.3-kilobase mRNA on Northern blot probed by CHIP28k cDNA and a 28-kDa protein on immunoblot probed by a polyclonal CHIP28 antibody. The clone with greatest expression produced approximately 8 x 10(6) copies of CHIP28k protein/cell. Plasma membrane osmotic water permeability (Pf), measured by stopped-flow light scattering, was 0.004 cm/s in control (vector-transfected) cells (10 degrees C) and 0.014 cm/s in the CHIP28k-transfected cells. Pf in CHIP28k-transfected cells had an activation energy of 4.9 kcal/mol and was reversibly inhibited by HgCl2. CHIP28k expression did not affect the transport of protons and the small polar non-electrolytes urea and formamide. CHIP28k immunoreactivity and function was then determined in subcellular fractions. Pf in 6-carboxyfluorescein-labeled endocytic vesicles, measured by a stopped-flow fluorescence quenching assay, was 0.002 cm/s (control cells) and 0.011 cm/s (CHIP28k-transfected cells); Pf in transfected cells was inhibited by HgCl2. Immunoblotting of fractionated endoplasmic reticulum, Golgi, and plasma membranes revealed high densities of CHIP28k (approximately 5000 monomers/microns 2 in plasma membrane) with different glycosylation patterns; functional water transport activity was present only in Golgi and plasma membrane vesicles. Antibody detection of CHIP28k by confocal fluorescence microscopy and immunogold electron microscopy revealed localization to plasma membrane and intracellular vesicles. These studies establish a stably transfected somatic cell line that strongly expresses functional CHIP28k water channels. As in the original proximal tubule cells

  1. Thermoresponsive polymers as gene delivery vectors: cell viability, DNA transport and transfection studies.

    PubMed

    Twaites, Beverley R; de Las Heras Alarcón, Carolina; Lavigne, Matthieu; Saulnier, Annabelle; Pennadam, Sivanand S; Cunliffe, David; Górecki, Dariusz C; Alexander, Cameron

    2005-11-28

    A range of gene delivery vectors containing the thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAm) was evaluated for effects on cell viability, intracellular trafficking and transgene expression in C2C12 mouse muscle cells. Polymers were complexed with plasmid DNA at pH 7.4 and the ability of the resulting particles to transfect cells was assessed via confocal microscopy and protein expression studies in tissue culture. Cell viability assays indicated that these polymers were toxic at high concentrations when not complexed to DNA or at certain polymer:DNA ratios. Poly(ethyleneimine) co-polymers with side-chain grafted PNIPAm were shown to be less toxic than poly(ethyleneimine) alone or PNIPAm-co-(N,N'-dimethylaminoethylmethacrylate) linear co-polymers and the effects were concentration dependent. Confocal micrographs of labeled polymers and DNA indicated rapid cellular entry for all the complexes but expression of Green Fluorescent Protein was achieved only when the branched PEI-PNIPAm co-polymers were used as vectors. The results indicate that design of appropriate co-polymer components and overall polymer architecture can be used to mediate, and perhaps ultimately control, DNA transport and transgene expression. PMID:16214254

  2. Polyurethane dispersion containing quaternized ammonium groups: An efficient nanosize gene delivery carrier for A549 cancer cell line transfection.

    PubMed

    Yousefpour Marzbali, Mahsa; Yari Khosroushahi, Ahmad; Movassaghpour, AliAkbar; Yeganeh, Hamid

    2016-01-25

    A novel polyurethane containing cationic ammonium groups (QPU) was synthesized and used as vector for gene therapy and cancer gene targeting. The synthesized QPU was characterized by Fourier transform infrared and nuclear magnetic resonance spectroscopy methods. An agarose gel retardation electrophoresis assay was conducted to verify the complete complex formation between QPU and pDNA. The particles size and zeta potential of neat polymers, plasmid DNA, polymers/DNA polyplexes were determined by the dynamic light scattering technique. The polyplexes cytotoxicity was determined using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and its transfection efficiency was examined qualitatively by fluorescent microscopy and quantitatively by flow cytometery methods. The gel retardation assay, particle size and zeta potential measurements were confirmed that the synthesized cationic polymer could condense DNA efficiently in the physiologic condition. QPU polyplexes showed a significantly lower cytotoxicity compared to Polyfect polyplexes in the examined human cancerous (A549) or normal cells (KDR). Based on our findings, the transfection efficiency by QPU was 2.2 fold higher than Polyfect in the A549 cells whereas in the KDR cells, the cell transfection by Polyfect was 18.1 fold higher than QPU. Due to low cytotoxicity for normal cells and high transfection efficiency in cancer cells, the potential applicability of designed QPU as a non-viral gene carrier for targeting of cancer gene therapy was confirmed.

  3. Addition of ‘Charge-Shifting’ Side Chains to Linear Poly(ethyleneimine) Enhances Cell Transfection Efficiency

    PubMed Central

    Liu, Xianghui; Yang, Jennifer W.

    2008-01-01

    We reported recently that the addition of ester-functionalized, ‘charge-shifting’ side chains to linear poly(ethyleneimine) (LPEI) can be used to design polyamines that promote both self-assembly and self-disassembly with DNA in aqueous environments. This investigation sought to characterize the influence of charge-shifting side chains on the ability of LPEI to mediate cell transfection and understand the extent to which increases (or decreases) in levels of transfection could be understood in terms of time-dependent changes in the net charges of these polymers. We report that the addition of ‘charge-shifting’ side chains to LPEI leads to significant increases in levels of LPEI-mediated transfection. In particular, polymer 1e, functionalized with 20 mol% ester-functionalized side chains, mediates levels of transgene expression in vitro up to eight-fold higher than LPEI. Experiments using an amide-functionalized analog of polymer 1e demonstrated that the esters in polymer 1e play an important role in promoting increased levels of transfection. These results, in combination with the results of additional gel electrophoresis experiments, provide support for the view that increases in transfection result from time-dependent changes in the net charge of polymer 1e and the disruption of ionic interactions in polyplexes. Additional support for this view is provided by the results of confocal microscopy experiments and measurements of fluorescence resonance energy transfer, which suggest that polymer 1e promotes the disruption of polyplexes in intracellular environments effectively. The approach reported here provides a means of addressing one important ‘late-stage’ obstacle to polyplex-mediated transfection (polyplex unpackaging). If integrated successfully with methods that have been developed to address other important barriers to transfection, this general approach could lead to the development of multifunctional polyplexes that mimic more effectively the

  4. Two motifs target Batten disease protein CLN3 to lysosomes in transfected nonneuronal and neuronal cells.

    PubMed

    Kyttälä, Aija; Ihrke, Gudrun; Vesa, Jouni; Schell, Michael J; Luzio, J Paul

    2004-03-01

    Batten disease is a neurodegenerative disorder resulting from mutations in CLN3, a polytopic membrane protein, whose predominant intracellular destination in nonneuronal cells is the lysosome. The topology of CLN3 protein, its lysosomal targeting mechanism, and the development of Batten disease are poorly understood. We provide experimental evidence that both the N and C termini and one large loop domain of CLN3 face the cytoplasm. We have identified two lysosomal targeting motifs that mediate the sorting of CLN3 in transfected nonneuronal and neuronal cells: an unconventional motif in the long C-terminal cytosolic tail consisting of a methionine and a glycine separated by nine amino acids [M(X)9G], and a more conventional dileucine motif, located in the large cytosolic loop domain and preceded by an acidic patch. Each motif on its own was sufficient to mediate lysosomal targeting, but optimal efficiency required both. Interestingly, in primary neurons, CLN3 was prominently seen both in lysosomes in the cell body and in endosomes, containing early endosomal antigen-1 along neuronal processes. Because there are few lysosomes in axons and peripheral parts of dendrites, the presence of CLN3 in endosomes of neurons may be functionally important. Endosomal association of the protein was independent of the two lysosomal targeting motifs. PMID:14699076

  5. Green fluorescent protein gene-transfected peafowl somatic cells participate in the development of chicken embryos.

    PubMed

    Xi, Yongmei; Nada, Yoich; Soh, Tomoki; Fujihara, Noboru; Hattori, Masa-Aki

    2004-02-01

    This study was performed to investigate whether the embryonic somatic cells are capable of reconstituting and participating in the embryonic development of chickens to produce chimeras. In order to track the migration behavior of the donor cells, a cell line, originally isolated from an Indian peafowl embryo, was fluorescent-labeled by transfection of the cells with enhanced Green Fluorescent Protein (GFP) and Neomycin resistant (Neo) genes prior to injection into the stage X blastoderm of White Leghorn chickens. The injection was performed with a medium in the presence of 1-5% polyethylene glycol. The development of putative chimeric embryos between the stages three and 24 was examined for GFP expression under fluorescent light. To trace the peafowl cells in the developing chicken embryos, both a species-specific genetic marker originating from the mitochondrial DNA cytochrome b (cyt b) gene and a DNA fragment of GFP gene were used. Of the 185 fertile eggs manipulated, 173 developed into embryos. Fifty-five of them showed positive GFP patches in extra-embryonic tissues, and 15 expressed GFP in intra-embryonic tissues such as those of the head, heart, and gonad. PCR analysis revealed that PCR fragments for the peafowl mitochondrial DNA cyt b and GFP genes were detected in the samples of the GFP positive extra- and intra-embryonic tissues of the chimeras. The present results provide evidence that fluorescent-labeled peafowl embryonic cells carrying GFP and Neo genes are able to participate in the development of chicken embryos to generate chimeras.

  6. Stable expression of transfected Torpedo acetylcholine receptor. cap alpha. subunits in mouse fibroblast L cells

    SciTech Connect

    Claudio, T.

    1987-08-01

    Torpedo californica electric organ cDNA libraries were constructed in lambdagt10 and lambdagt11. Four acetylcholine receptor (AcChoR) subunit cDNA clones were isolated and shown to contain the entire coding region for each of the subunits. When in vitro synthesized AcChoR mRNA was microinjected into Xenopus laevis oocytes, functional cell surface AcChoRs were expressed. A very simple and fast /sup 22/Na-uptake experiment was performed on batches of microinjected oocytes to identify oocytes that were expressing large quantities of functional cell surface AcChoRs for use in single-channel recordings. In addition to the transient expression system, DNA-mediated contransformation is described, which is a method for stably introducing AcChoR cDNAs into the chromosomes of tissue culture cells. Because the AcChoR is composed of four different subunits, it is necessary to integrate four cDNAs into the chromosomes of the same cell before stable expression of a completely functional receptor complex can be established. The authors show that 80% of the cells that integrated the selectable marker gene into their chromosomes also integrated all four AcChoR cDNAs. When Torpedo ..cap alpha..-subunit cDNA inserted into an appropriate expression vector was introduced into cells by transfection, ..cap alpha..-subunit protein was synthesized that migrated on NaDodSO/sub 4//polyacrylamide gels with the same molecular mass as native Torpedo ..cap alpha.. subunits and expressed antigenic determinants similar to those of native Torpedo ..cap alpha.. subunits.

  7. Green fluorescent protein gene-transfected peafowl somatic cells participate in the development of chicken embryos.

    PubMed

    Xi, Yongmei; Nada, Yoich; Soh, Tomoki; Fujihara, Noboru; Hattori, Masa-Aki

    2004-02-01

    This study was performed to investigate whether the embryonic somatic cells are capable of reconstituting and participating in the embryonic development of chickens to produce chimeras. In order to track the migration behavior of the donor cells, a cell line, originally isolated from an Indian peafowl embryo, was fluorescent-labeled by transfection of the cells with enhanced Green Fluorescent Protein (GFP) and Neomycin resistant (Neo) genes prior to injection into the stage X blastoderm of White Leghorn chickens. The injection was performed with a medium in the presence of 1-5% polyethylene glycol. The development of putative chimeric embryos between the stages three and 24 was examined for GFP expression under fluorescent light. To trace the peafowl cells in the developing chicken embryos, both a species-specific genetic marker originating from the mitochondrial DNA cytochrome b (cyt b) gene and a DNA fragment of GFP gene were used. Of the 185 fertile eggs manipulated, 173 developed into embryos. Fifty-five of them showed positive GFP patches in extra-embryonic tissues, and 15 expressed GFP in intra-embryonic tissues such as those of the head, heart, and gonad. PCR analysis revealed that PCR fragments for the peafowl mitochondrial DNA cyt b and GFP genes were detected in the samples of the GFP positive extra- and intra-embryonic tissues of the chimeras. The present results provide evidence that fluorescent-labeled peafowl embryonic cells carrying GFP and Neo genes are able to participate in the development of chicken embryos to generate chimeras. PMID:14743513

  8. Expression of a human placental alkaline phosphatase gene in transfected cells: Use as a reporter for studies of gene expression

    SciTech Connect

    Henthorn, P.; Zervos, P.; Raducha, M.; Harris, H.; Kadesch, T.

    1988-09-01

    The human placental alkaline phosphatase gene has been cloned and reintroduced into mammalian cells. When a plasmid carrying the gene under control of the simian virus 40 early promoter (pSV2Apap) is transfected into a variety of different cell types, placental alkaline phosphatase activity can readily be detected by using whole cell suspensions or cell lysates. Alkaline phosphatase activity can also be visualized directly in individual transfected cells by histochemical staining. The gene is appropriate for use as a reporter in studies of gene regulation since its expression is dependent on the presence of exogenous transcription control elements. The overall assay to detect the expression of the gene is quantitative, very rapid, and inexpensive. Cotransfections of cells with pSV2Apap and a related plasmid carrying the bacterial chloramphenicol acetyltransferase gene (pSV2Acat) indicate that transcription of these two genes is detected with roughly the same sensitivity.

  9. Water-soluble N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride as a nucleic acids vector for cell transfection.

    PubMed

    Faizuloev, Evgeny; Marova, Anna; Nikonova, Alexandra; Volkova, Irina; Gorshkova, Marina; Izumrudov, Vladimir

    2012-08-01

    To endow the cationic polysaccharides with solubility in the whole pH-range without loss of functionality of the amino groups, different chitosan samples were treated with glycidyltrimethylammonium chloride. Each modified unit of the exhaustively alkylated quaternized chitosan (QCht) contained both quaternary and secondary amino groups. The intercalated dye displacement assay and ζ-potential measurements implied stability of QCht polyplexes at physiological conditions and protonation of the secondary amino groups in slightly acidic media which is favorable for transfection according to proton sponge mechanism. The cytotoxicity and transfection efficacy increased with the chain lengthening. Nevertheless, the longest chains of QCht, 250 kDa were less toxic than PEI for COS-1 cells and revealed comparable and even significantly higher transfection activity of siRNA and plasmid DNA, respectively. Thus, highly polymerized QCht (250 kDa) provided the highest level of the plasmid DNA transfection being 5 and 80 times more active than QCht (100 kDa) and QCht (50 kDa), respectively, and 4-fold more effective than PEI, 25 kDa. The established influence of QCht molecular weight on toxicity and transfection efficacy allows elaborating polysaccharide vectors that possess rational balance of these characteristics. PMID:24750918

  10. Magnetic Tweezers-based 3D Microchannel Electroporation for High-Throughput Gene Transfection in Living Cells

    PubMed Central

    Liao, Wei-Ching; Chiang, Chi-Ling; Gallego-Perez, Daniel; Yang, Zhaogang; Lu, Wu; Byrd, John C.; Muthusamy, Natarajan; Lee, L. James.; Sooryakumar, Ratnasingham

    2015-01-01

    We report a novel, high throughput magnetic-tweezers based 3D microchannel electroporation system capable of transfecting 40,000 cells/cm2 on a single-chip for gene therapy, regenerative medicine and intracellular detection of target mRNA for screening cellular heterogeneity. A single cell or an ordered array of individual cells are remotely guided by programmable magnetic fields to poration sites with high (> 90%) cell alignment efficiency to enable various transfection reagents to be delivered simultaneously into the cells. The present technique, in contrast to the conventional vacuum based approach, is significantly gentler on the cellular membrane yielding > 90% cell viability and, moreover, allows transfected cells to be transported for further analysis. Illustrating the versatility of the system, the GATA2 molecular beacon was delivered into leukemia cells to detect the regulation level of the GATA2 gene that is associated with the initiation of leukemia. The uniform delivery and a sharp contrast of fluorescence intensity between GATA2 positive and negative cells demonstrate key aspects of the platform for gene transfer, screening and detection of targeted intracellular markers in living cells. PMID:25469659

  11. Magnetic tweezers-based 3D microchannel electroporation for high-throughput gene transfection in living cells.

    PubMed

    Chang, Lingqian; Howdyshell, Marci; Liao, Wei-Ching; Chiang, Chi-Ling; Gallego-Perez, Daniel; Yang, Zhaogang; Lu, Wu; Byrd, John C; Muthusamy, Natarajan; Lee, L James; Sooryakumar, Ratnasingham

    2015-04-17

    A novel high-throughput magnetic tweezers-based 3D microchannel electroporation system capable of transfecting 40 000 cells/cm(2) on a single chip for gene therapy, regenerative medicine, and intracellular detection of target mRNA for screening cellular heterogeneity is reported. A single cell or an ordered array of individual cells are remotely guided by programmable magnetic fields to poration sites with high (>90%) cell alignment efficiency to enable various transfection reagents to be delivered simultaneously into the cells. The present technique, in contrast to the conventional vacuum-based approach, is significantly gentler on the cellular membrane yielding >90% cell viability and, moreover, allows transfected cells to be transported for further analysis. Illustrating the versatility of the system, the GATA2 molecular beacon is delivered into leukemia cells to detect the regulation level of the GATA2 gene that is associated with the initiation of leukemia. The uniform delivery and a sharp contrast of fluorescence intensity between GATA2 positive and negative cells demonstrate key aspects of the platform for gene transfer, screening and detection of targeted intracellular markers in living cells.

  12. Adenovirus-mediated bone morphogenetic protein-2 gene transfection of bone marrow mesenchymal stem cells combined with nano-hydroxyapatite to construct bone graft material in vitro.

    PubMed

    Li, W C; Wang, D P; Li, L J; Zhu, W M; Zeng, Y J

    2013-04-01

    To study the adhesion, proliferation and expression of bone marrow mesenchymal stem cells (BMSCs) on nano-hydroxyapatite (Nano-HA) bone graft material after transfection of adenovirus-mediated human bone morphogenetic protein-2 expression vector (Ad-BMP-2). BMSCs were transfected using Ad-BMP-2. Immunohistochemistry and Western blot were used to detect BMP-2 expression in transfected cells. After transfection, BMP-2 protein was highly expressed in BMSCs; MTT test assay showed that the Nano-HA bone graft material could not inhibit in vitro proliferation of BMSCs. Ad-BMP-2-transfected BMSCs are well biocompatible with Nano-HA bone graft material, the transfected cells in material can secrete BMP-2 stably for a long time.

  13. Subcellular localization of Regulator of G protein Signaling RGS7 complex in neurons and transfected cells

    PubMed Central

    Liapis, Evangelos; Sandiford, Simone; Wang, Qiang; Gaidosh, Gabriel; Motti, Dario; Levay, Konstantin; Slepak, Vladlen Z.

    2012-01-01

    The R7 family of regulators of G protein signaling (RGS) is involved in many functions of the nervous system. This family includes RGS6, RGS7, RGS9, and RGS11 gene products and is defined by the presence of the characteristic DEP, DHEX, GGL and RGS domains. Here, we examined the subcellular localization of RGS7, the most broadly expressed R7 member. Our immunofluorescence studies of retinal and dorsal root ganglion (DRG) neurons showed that RGS7 concentrated at the plasma membrane of cell bodies, in structures resembling lamellipodia or filopodia along the processes, and at the dendritic tips. At the plasma membrane of DRG neurons, RGS7 co-localized with its known binding partners R7BP, Gαo and Gαq. More than 50% of total RGS7-specific immunofluorescence was present in the cytoplasm, primarily within numerous small puncta that did not co-localize with R7BP. No specific RGS7 or R7BP immunoreactivity was detected in the nuclei. In transfected cell lines, ectopic RGS7 had both diffuse cytosolic and punctate localization patterns. RGS7 also localized in centrosomes. Structure-function analysis showed that the punctate localization was mediated by the DEP/DHEX domains, and centrosomal localization was dependent on the DHEX domain. PMID:22640015

  14. Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells.

    PubMed

    Liapis, Evangelos; Sandiford, Simone; Wang, Qiang; Gaidosh, Gabriel; Motti, Dario; Levay, Konstantin; Slepak, Vladlen Z

    2012-08-01

    The R7 family of regulators of G protein signaling (RGS) is involved in many functions of the nervous system. This family includes RGS6, RGS7, RGS9, and RGS11 gene products and is defined by the presence of the characteristic first found in Disheveled, Egl-10, Pleckstrin (DEP), DEP helical extension (DHEX), Gγ-like, and RGS domains. Herein, we examined the subcellular localization of RGS7, the most broadly expressed R7 member. Our immunofluorescence studies of retinal and dorsal root ganglion neurons showed that RGS7 concentrated at the plasma membrane of cell bodies, in structures resembling lamellipodia or filopodia along the processes, and at the dendritic tips. At the plasma membrane of dorsal root ganglia neurons, RGS7 co-localized with its known binding partners R7 RGS binding protein (R7BP), Gαo, and Gαq. More than 50% of total RGS7-specific immunofluorescence was present in the cytoplasm, primarily within numerous small puncta that did not co-localize with R7BP. No specific RGS7 or R7BP immunoreactivity was detected in the nuclei. In transfected cell lines, ectopic RGS7 had both diffuse cytosolic and punctate localization patterns. RGS7 also localized in centrosomes. Structure-function analysis showed that the punctate localization was mediated by the DEP/DHEX domains, and centrosomal localization was dependent on the DHEX domain.

  15. A high-throughput mammalian cell-based transient transfection assay.

    PubMed

    Noonan, Daniel J; Henry, Kenneth; Twaroski, Michelle L

    2004-01-01

    In eukaryotic organisms gene expression is regulated through a variety of upstream transacting factors (transcription factors) whose primary function appears to be the targeting of coregulatory protein complexes, which interact with basal transcription machinery to define the relative rate of transcription for a specific gene. Understanding the regulatory forces mediating transcription factor activity has been the focus of both academic and industrial research efforts over the past 15 yr, and in this time frame a variety of methodologies have been developed for reconstituting and assaying transcription factor activities in mammalian cell environments. Presented here is a high-throughput version of one of these methodologies that can be readily adapted to the screening of a variety of transcription factors. This technology utilizes co-transfection of mammalian expression and luciferase reporter plasmids to reconstitute transcription events in a mammalian host cell. Included is a detailed protocol for the use of a 96-well plate format, along with a variety of cost-effective measures that can be implemented to facilitate the use of the technology in the average low budget academic laboratory.

  16. Experimental Study of the Effects of Marrow Mesenchymal Stem Cells Transfected with Hypoxia-Inducible Factor-1α Gene

    PubMed Central

    Yang, Jinfu; Tang, Tao; Li, Feng; Zhou, Wenwu; Liu, Jian; Tan, Zhiping; Zheng, Wei; Yang, Yifeng; Zhou, Xinmin; Hu, Jianguo

    2009-01-01

    Objective. To construct the eukaryotic expression vector hypoxia-inducible factor 1α-pcDNA3.1 and to investigate its transfective efficiency into mesenchymal stem cells (MSCs) in vitro and the expression of HIF-1α gene in MSCs. Methods. mRNA of Wistar Rats' myocardial cells was extracted, and cDNA was synthesized with Reverse Transcription Kit, HIF-1α was amplified by polymerase chain reaction (PCR), and constructed into pcDNA3.1. Transfected HIF-1α-pcDNA3.1 into MSCs by liposome mediated method. The expression of HIF-1α in the cells was detected by Western Blot Analysis and ELISA. Results. Eukaryotic expression vector HIF-1α-pcDNA3.1 was constructed successfully. Analyzed by flow cytometer, The MSCs' surfaces mark were CD44+, SH3(CD73)+, CD34−, CD45− and the CD44+ cells and SH3(CD73)+ cells were 94.7% and 97.3%, respectively, showing the high purity of the cultured MSCs. After inducing, the cultured MSCs can differentiate into osteoblasts and adipocytes successfully. In HIF-1α gene transfected MSCs, the expression of HIF-1α mRNA and HIF-1α protein were both increased obviously. Conclusion. HIF-1α was cloned successfully. HIF-1α-pcDNA3.1 can be transfected into MSCs by liposome-mediated method effectively and which resulting stable expression of HIF-1α in transfected MSCs. PMID:19587827

  17. Enhanced plasmid DNA utilization in transiently transfected CHO-DG44 cells in the presence of polar solvents.

    PubMed

    Rajendra, Yashas; Balasubramanian, Sowmya; Kiseljak, Divor; Baldi, Lucia; Wurm, Florian M; Hacker, David L

    2015-01-01

    Although the protein yields from transient gene expression (TGE) with Chinese hamster ovary (CHO) cells have recently improved, the amount of plasmid DNA (pDNA) needed for transfection remains relatively high. We describe a strategy to reduce the pDNA amount by transfecting CHO-DG44 cells with 0.06 μg pDNA/10(6) cells (10% of the optimal amount) in the presence of nonspecific (filler) DNA and various polar solvents including dimethylsufoxide, dimethyl formamide, acetonitrile, dimethyl acetamide (DMA), and hexamethyl phosphoramide (HMP). All of the polar solvents with the exception of HMP increased the production of a recombinant antibody in comparison to the untreated control transfection. In the presence of 0.25% DMA, the antibody yield in a 7-day batch culture was 500 mg/L. This was fourfold higher than the yield from the untreated control transfection. Mechanistic studies revealed that the polar solvents did not affect polyethylenimine-mediated pDNA delivery into cells or nuclei. The steady-state transgene mRNA level was elevated in the presence of each of the polar solvents tested, while the transgene mRNA half-life remained the same. These results indicated that the polar solvents enhanced transgene transcription. When screening a panel of recombinant antibodies and Fc-fusion proteins for production in the presence of the polar solvents, the highest increase in yield was observed following DMA addition for 11 of the 12 proteins. These results are expected to enhance the applicability of high-yielding TGE processes with CHO-DG44 cells by decreasing the amount of pDNA required for transfection.

  18. Effects of the Surface Charge of Stem Cell Membranes and DNA/Polyethyleneimine Nanocomplexes on Gene Transfection Efficiency.

    PubMed

    Kim, Da Yeon; Kwon, Jin Seon; Lee, Jae Hyeok; Jin, Ling Mei; Kim, Jae Ho; Kim, Moon Suk

    2015-03-01

    In this work, we examined the effects of the surface charge of stem cell membranes and DNA/polyethyleneimine (PEI) nanocomplexes on gene transfection efficiency, because PEI was one of the most reliable and efficient carriers, and rat bone marrow mesenchymal stem cells (rBMSCs) and rat muscle-derived stem cells (rMDSCs) were one of the readily accessible and plentiful sources of stem cells. Thus, we compared the efficiency of DNA transfection in rBMSCs and rMDSCs using the PEI as a gene carrier. Transfection efficiency was evaluated on the basis of electrostatic interaction between negatively charged stem cell membranes and positively charged DNA/PEI nanocomplexes. DNA was fully complexed with PEI at negative-to-positive (NIP) charge ratios greater than 2, as confirmed by gel electrophoresis and fluorescence measurements. DNA and PEI formed spherical nanocomplexes ranging in diameter from 150 nm to 500 nm. The positive surface charge of DNA/PEI nanocomplexes increased with an increasing N/P charge ratio, as measured using dynamic light scattering and a single-walled carbon nanotube-based field-effect transistor device. rBMSCs and rMDSCs both carried a negative surface charge, with rBMSCs being more negatively charged. The transfection efficiency of rMDSCs measured using DNA/PEI nanocomplexes was very low (1%-5%) at most of the N/P charge ratios tested, whereas better efficiencies were observed with rBMSCs (1%-17%). Nanocomplexes with high NIP charge ratios were cytotoxic to both rBMSCs and rMDSCs. Collectively, the results indicate that rBMSCs were more effectively transfected with DNA/PEI nanocomplexes than were rMDSCs, reflecting the higher negative charge of rBMSC membranes that facilitate the interaction with positively charged DNA/PEI nanocomplexes. PMID:26307834

  19. SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC

    PubMed Central

    Slusser-Nore, Andrea; Larson-Casey, Jennifer L.; Zhang, Ruowen; Zhou, Xu Dong; Somji, Seema; Garrett, Scott H.; Sens, Donald A.; Dunlevy, Jane R.

    2016-01-01

    Background This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As+3) and cadmium (Cd+2)-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd+2-and As+3-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice. Methods Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As+3-and Cd+2-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres. Results It was shown that the As+3-and Cd+2-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As+3-and Cd+2-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells. Conclusions Tumor initiating cells isolated from SPARC-transfected As+3-and Cd+2-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA. PMID:26783756

  20. Antiangiogenic and Neurogenic Activities of Sleeping Beauty-Mediated PEDF-Transfected RPE Cells In Vitro and In Vivo

    PubMed Central

    Johnen, Sandra; Djalali-Talab, Yassin; Kazanskaya, Olga; Möller, Theresa; Harmening, Nina; Kropp, Martina; Izsvák, Zsuzsanna; Walter, Peter; Thumann, Gabriele

    2015-01-01

    Pigment epithelium-derived factor (PEDF) is a potent multifunctional protein that inhibits angiogenesis and has neurogenic and neuroprotective properties. Since the wet form of age-related macular degeneration is characterized by choroidal neovascularization (CNV), PEDF would be an ideal candidate to inhibit CNV and support retinal pigment epithelial (RPE) cells. However, its short half-life has precluded its clinical use. To deliver PEDF to the subretinal space, we transfected RPE cells with the PEDF gene using the Sleeping Beauty transposon system. Transfected cells expressed and secreted biologically active recombinant PEDF (rPEDF). In cultures of human umbilical vein endothelial cells, rPEDF reduced VEGF-induced cumulative sprouting by ≥47%, decreased migration by 77%, and increased rate of apoptosis at least 3.4 times. rPEDF induced neurite outgrowth in neuroblastoma cells and protected ganglion and photoreceptor cells in organotypic retinal cultures. In a rat model of CNV, subretinal transplantation of PEDF-transfected cells led to a reduction of the CNV area by 48% 14 days after transplantation and decreased clinical significant lesions by 55% and 40% after 7 and 14 days, respectively. We showed that transplantation of pigment epithelial cells overexpressing PEDF can restore a permissive subretinal environment for RPE and photoreceptor maintenance, while inhibiting choroidal blood vessel growth. PMID:26697494

  1. Antiangiogenic and Neurogenic Activities of Sleeping Beauty-Mediated PEDF-Transfected RPE Cells In Vitro and In Vivo.

    PubMed

    Johnen, Sandra; Djalali-Talab, Yassin; Kazanskaya, Olga; Möller, Theresa; Harmening, Nina; Kropp, Martina; Izsvák, Zsuzsanna; Walter, Peter; Thumann, Gabriele

    2015-01-01

    Pigment epithelium-derived factor (PEDF) is a potent multifunctional protein that inhibits angiogenesis and has neurogenic and neuroprotective properties. Since the wet form of age-related macular degeneration is characterized by choroidal neovascularization (CNV), PEDF would be an ideal candidate to inhibit CNV and support retinal pigment epithelial (RPE) cells. However, its short half-life has precluded its clinical use. To deliver PEDF to the subretinal space, we transfected RPE cells with the PEDF gene using the Sleeping Beauty transposon system. Transfected cells expressed and secreted biologically active recombinant PEDF (rPEDF). In cultures of human umbilical vein endothelial cells, rPEDF reduced VEGF-induced cumulative sprouting by ≥47%, decreased migration by 77%, and increased rate of apoptosis at least 3.4 times. rPEDF induced neurite outgrowth in neuroblastoma cells and protected ganglion and photoreceptor cells in organotypic retinal cultures. In a rat model of CNV, subretinal transplantation of PEDF-transfected cells led to a reduction of the CNV area by 48% 14 days after transplantation and decreased clinical significant lesions by 55% and 40% after 7 and 14 days, respectively. We showed that transplantation of pigment epithelial cells overexpressing PEDF can restore a permissive subretinal environment for RPE and photoreceptor maintenance, while inhibiting choroidal blood vessel growth. PMID:26697494

  2. Involvement of Gap Junctions in Tumorigenesis: Transfection of Tumor Cells with Connexin 32 cDNA Retards Growth In vivo

    NASA Astrophysics Data System (ADS)

    Eghbali, B.; Kessler, J. A.; Reid, L. M.; Roy, C.; Spray, D. C.

    1991-12-01

    Gap junction channels provide a pathway for exchange of ions and small molecules between coupled cells, and this exchange is believed to be critical for normal tissue growth and development. As a test for a role of gap junction-mediated intercellular communication in control of cell growth, we have compared growth rates of communication-deficient human tumor cells (SKHep1) with clones stably transfected with cDNA encoding the rat liver gap junction protein connexin 32. In culture, growth rates for parental and transfected clones were similar. However, when sizes of tumors were evaluated following injection of these clones into athymic nude mice, growth rates for two well-coupled clones were significantly lower than for communication-deficient or poorly coupled clones. This study demonstrates that growth rate of these tumor cells in situ is negatively correlated with strength of intercellular communication.

  3. Expression of biologically active human interleukin 1 subpeptides by transfected simian COS cells.

    PubMed Central

    Rosenwasser, L J; Webb, A C; Clark, B D; Irie, S; Chang, L; Dinarello, C A; Gehrke, L; Wolff, S M; Rich, A; Auron, P E

    1986-01-01

    "Interleukin 1" (IL-1) is a term used to describe the family of macrophage-derived proteins that mediate many immune and inflammatory reactions. We have previously described the molecular cloning and sequencing of the cDNA encoding the predominant (neutral) form of human IL-1, which has been designated IL-1 beta. We report here that transfection of simian COS cells with this cDNA clone results in the transcription of IL-1 mRNA and the synthesis of antibody-neutralizable intracellular IL-1 biological activity. In addition, selective deletion of regions of the IL-1 cDNA judged not to be essential for function, on the basis of conserved sequence homology, resulted in localization of a "core" region responsible for a majority of the biological activity. These results permit mapping the active site of IL-1 to a peptide of 6970 molecular weight located within the carboxyl third (between Met-136 and Gln-197) of the IL-1 precursor. Images PMID:3487789

  4. Transfection using DEAE-dextran.

    PubMed

    Gulick, T

    2001-05-01

    Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The basic protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.

  5. Transfection using DEAE-dextran.

    PubMed

    Gulick, Tod

    2003-08-01

    Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The basic protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.

  6. Transfection using DEAE-dextran.

    PubMed

    Gulick, T

    2001-05-01

    Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The Basic Protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.

  7. Preclinical evaluation of autologous dendritic cells transfected with mRNA or loaded with apoptotic cells for immunotherapy of high-risk neuroblastoma.

    PubMed

    Jarnjak-Jankovic, Silvija; Pettersen, Rolf D; Saebøe-Larssen, Stein; Wesenberg, Finn; Olafsen, Mette R K; Gaudernack, Gustav

    2005-08-01

    Children with high-risk neuroblastoma (NB) have a poor clinical outcome. The purpose of the present study was to evaluate different strategies for immunotherapy of high-risk NB based on vaccination with antigen-loaded dendritic cells (DCs). DCs are professional antigen-presenting cells with the ability to induce antitumor T-cell responses. We have compared DCs either loaded with apoptotic tumor cells or transfected with mRNA from the NB cell line HTB11 SK-N-SH, for their capacity to induce T-cell responses in vitro. Monocyte-derived DCs from healthy donors were loaded with tumor-derived antigens in the form of apoptotic cells or mRNA, matured and used to prime autologous T cells in vitro. After 1 week, T-cell responses against antigen-loaded DCs were measured by ELISPOT assay. DCs loaded with apoptotic NB cells or transfected with NB-cell mRNA were both able to efficiently activate autologous T cells. Both T cells of the CD8+ and CD4+ subset were activated. T cells activated by NB mRNA transfected DCs extensively crossreacted with DCs loaded with apoptotic NB cells and vice versa. The results indicate that loading of DCs with apoptotic NB cells or transfection with tumor mRNA represent promising strategies for development of individualized cancer vaccines/cancer gene therapy in treatment of NB.

  8. In vitro and in vivo transfection of melanoma cells B16-F10 mediated by cholesterol-based cationic liposomes.

    PubMed

    Reynier, P; Briane, D; Cao, A; Lievre, N; Naejus, R; Bissieres, P; Salzmann, J L; Taillandier, E

    2002-11-01

    In vitro and in vivo transgene expression in B16-F10 melanoma cells has been investigated using an original cationic liposome prepared with triethyl aminopropane carbamoyl cholesterol iodide (TEAPC-Chol) as carrier. TEAPC-Chol/DOPE (dioleoyl phosphatidyl ethanolamine) liposomes are unilamellar, very stable and not toxic in the used concentration range. The yield in complexation with plasmid DNA can reach 100% even in the presence of fetal calf serum. The transfection level has been evaluated by luminometric measurements of luciferase expression. With TEAPC-Chol/DOPE (1:1) liposomes, a relatively high transfection level in B16-F10 cells has been observed comparing to commercial reagents. For in vivo assays, the transfection level in tumors induced in Nude mice has been optimized by studying the effects of charge ratio, of the helper lipid and of the injection volume. Results showed that TEAPC-Chol/DOPE (1:1) liposomes have improved 10-fold transfection level versus direct gene transfer of free DNA. PMID:12683723

  9. Lipoic acid modified low molecular weight polyethylenimine mediates nontoxic and highly potent in vitro gene transfection.

    PubMed

    Zheng, Meng; Zhong, Yinan; Meng, Fenghua; Peng, Rui; Zhong, Zhiyuan

    2011-12-01

    The clinical success of gene therapy intimately relies on the development of safe and efficient gene carrier systems. We found here that 1.8 kDa polyethylenimine (PEI) following hydrophobic modification with lipoic acid (LA) mediated nontoxic and highly potent in vitro gene transfection in both HeLa and 293T cells. 1.8 kDa PEI-LA conjugates were prepared with controlled degree of substitution (DS) by coupling LA to PEI using carbodiimide chemistry. Gel electrophoresis measurements showed that the DNA binding ability of 1.8 kDa PEI was impaired by lipoylation, in which an N/P ratio of 2/1 and 4-6/1 was required for 1.8 kDa PEI and 1.8 kDa PEI-LA conjugates, respectively, to completely inhibit DNA migration. Interestingly, dynamic light scattering measurements (DLS) revealed that PEI-LA conjugates condensed DNA into much smaller sizes (183-84 nm) than unmodified 1.8 kDa PEI (444-139 nm) at N/P ratios ranging from 20/1 to 60/1. These polyplexes revealed similar surface charges of ca. +22 to +30 mV. 1.8 kDa PEI-LA(2) polyplexes formed at an N/P ratio of 10/1 were stable against exchange with 12-fold excess of negatively charged dextran sodium sulfate (DSS) relative to DNA phosphate groups while 1.8 kDa PEI controls dissociated at 6-fold excess of DSS, indicating that lipoylation of 1.8 kDa PEI resulted in stronger binding with DNA. Importantly, DNA was released from 1.8 kDa PEI-LA(2) polyplexes upon addition of 10 mM dithiothreitol (DTT). Reduction-triggered unpacking of 1.8 kDa PEI-LA(2) polyplexes was also confirmed by DLS. MTT assays demonstrated that all PEI-LA conjugates and polyplexes were essentially nontoxic to HeLa and 293T cells up to a tested concentration of 50 μg/mL and an N/P ratio of 80/1, respectively. The in vitro gene transfection studies in HeLa and 293T cells showed that lipoylation of 1.8 kDa PEI markedly boosted its transfection activity. For example, 1.8 kDa PEI-LA(2) polyplexes displayed 400-fold and 500-fold higher levels of gene expression

  10. Extracellular ATP Causes ROCK I-dependent Bleb Formation in P2X7-transfected HEK293 CellsV⃞

    PubMed Central

    Morelli, Anna; Chiozzi, Paola; Chiesa, Anna; Ferrari, Davide; Sanz, Juana M.; Falzoni, Simonetta; Pinton, Paolo; Rizzuto, Rosario; Olson, Michael F.; Di Virgilio, Francesco

    2003-01-01

    The P2X7 ATP receptor mediates the cytotoxic effect of extracellular ATP. P2X7-dependent cell death is heralded by dramatic plasma membrane bleb formation. Membrane blebbing is a complex phenomenon involving as yet poorly characterized intracellular pathways. We have investigated the effect of extracellular ATP on HEK293 cells transfected with the cytotoxic/pore-forming P2X7 receptor. Addition of ATP to P2X7-transfected, but not to wt P2X7-less, HEK293 cells caused massive membrane blebbing within 1–2 min. UTP, a nucleotide incapable of activating P2X7, had no early effects on cell shape and bleb formation. Bleb formation triggered by ATP was reversible and required extracellular Ca2+ and an intact cytoskeleton. Furthermore, it was completely prevented by preincubation with the P2X blocker oxidized ATP. It was recently observed that the ROCK protein is a key determinant of bleb formation. Preincubation of HEK293-P2X7 cells with the ROCK blocker Y-27632 completely prevented P2X7-dependent blebbing. Although ATP triggered cleavage of the ROCK I isoform in P2X7-transfected HEK293 cells, the wide range caspase inhibitor z-VAD-fluoromethylketone had no effect. These observations suggest that P2X7-dependent plasma membrane blebbing depends on the activation of the serine/threonine kinase ROCK I. PMID:12857854

  11. Stable transfection of protein kinase C alpha cDNA in hormone-dependent breast cancer cell lines

    PubMed Central

    Tonetti, D A; Chisamore, M J; Grdina, W; Schurz, H; Jordan, V C

    2000-01-01

    An inverse relationship between protein kinase C (PKC) activity and oestrogen receptor (ER) expression in human breast cell lines and tumours has been firmly established over the past 10 years. To determine whether specific alterations in PKC expression accompany hormone-independence, we examined the expression of PKC isozymes in the hormone-independent human breast cancer cell clones MCF-7 5C and T47D:C42 compared with their hormone-dependent counterparts, MCF-7 A4, MCF-7 WS8 and T47D:A18 respectively. Both hormone-independent cell clones exhibit elevated PKCα expression and increased basal AP-1 activity compared with the hormone-dependent cell clones. To determine whether PKCα overexpression is sufficient to mediate the hormone-independent phenotype, we stably transfected an expression plasmid containing PKCα cDNA to the T47D:A18 and MCF-7 A4 cell lines. This is the first report of PKCα transfection in T47D cells. In contrast to MCF-7 cells, T47D has the propensity to lose the ER and more readily forms tamoxifen-stimulated tumours in athymic mice. We find that in T47D:A18/PKCα clones, there is concomitant up-regulation of PKC βI and δ, whereas in the MCF-7 A4/PKCα transfectants PKC ɛ is up-regulated. In T47D:A18, but not in MCF-7 A4, PKCα stable transfection is accompanied by down-regulation of ER function whilst basal AP-1 activity is elevated. Our results suggest PKCα overexpression may play a role in growth signalling during the shift from hormone dependent to hormone-independent breast cancers. © 2000 Cancer Research Campaign PMID:10952784

  12. Osteo-/odontogenic differentiation of BMP2 and VEGF gene-co-transfected human stem cells from apical papilla

    PubMed Central

    ZHANG, WEN; ZHANG, XIAOLEI; LING, JUNQI; WEI, XI; JIAN, YUTAO

    2016-01-01

    Stem cells from apical papilla (SCAP) possess clear osteo-/odontogenic differentiation capabilities, and are regarded as the major cellular source for root dentin development. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) serve pivotal roles in the modulation of tooth development and dentin formation. However, the synergistic effects of BMP2 and VEGF on osteo-/odontogenic differentiation of SCAP remain unclear. The current study aimed to investigate the proliferative and osteo-/odontogenic differentiating capabilities of BMP2 and VEGF gene-co-transfected SCAP (SCAP-BMP2-VEGF) in vitro. The basic characteristics of the isolated SCAP were identified by the induction of multipotent differentiation and by flow cytometry. Lentiviral vector-mediated gene transfection was conducted with SCAP in order to construct blank vector-transfected SCAP (SCAP-green fluorescent protein), BMP2 gene-transfected SCAP (SCAP-BMP2), VEGF gene-transfected SCAP (SCAP-VEGF) and SCAP-BMP2-VEGF. The Cell Counting Kit 8 assay was used to analyze the proliferative capacities of the four groups of cells. The expression of osteo-/odontogenic genes and proteins in the cells were evaluated by reverse transcription-quantitative polymerase chain reaction and western blotting. The mineralized nodules formed by the four group cells were visualized by alkaline phosphatase (ALP) staining. Among the four groups of cells, SCAP-VEGF was demonstrated to exhibit increased proliferation, and SCAP-BMP2-VEGF exhibited reduced proliferation during eight days observation. SCAP-BMP2-VEGF exhibited significantly increased expression levels of ALP, osteocalcin, dentin sialophosphoprotein, dentin matrix acidic phosphoprotein gene 1 and dentin sialoprotein than the other three groups at the majority of the time points. Furthermore, the SCAP-BMP2-VEGF group exhibited a significantly greater number of ALP-positive mineralized nodules than the other groups following 16 days culture in

  13. Comparison of Lentiviral Packaging Mixes and Producer Cell Lines for RNAi Applications.

    PubMed

    Albrecht, Christian; Hosiner, Stefanie; Tichy, Brigitte; Aldrian, Silke; Hajdu, Stefan; Nürnberger, Sylvia

    2015-06-01

    Lentiviral transduction is a highly efficient DNA delivery method for RNA interference applications. However, obtaining high lentiviral titers of shRNA and miRNA encoding vectors is challenging, since shRNA and miRNA cassettes have been shown to reduce lentiviral titers. In this study, we compare four commercially available packaging mixes and two producer cell lines in order to optimize lentiviral production for gene silencing experiments. Lentiviral vectors encoding a miRNA sequence and emerald green fluorescence protein were co-transfected with ViraPower™, Lenti-X™ HTX, MISSION(®) Lentiviral or Trans-Lentiviral™ packaging mix in HEK-293T or 293FT cells. After transducing HeLa cells with virus-containing supernatant, lentiviral titers were determined by flow cytomerty. In both cell lines, the highest lentiviral titer was obtained with MISSION(®) Lentiviral packaging mix, followed by ViraPower™, Lenti-X™ HTX, and Trans-Lentiviral™. On average, HEK-293T cells produced 6.2-fold higher lentiviral titers than 293FT cells (p < 0.001). With the combination of MISSION(®) Lentiviral packaging mix and HEK-293T cells, an up to 48.5-fold higher lentiviral titer was reached compared to other packaging mixes and producer cell lines. The optimized selection of packaging mix and cell line described in this work should facilitate the production of high-titer lentiviruses for gene silencing experiments. PMID:25616840

  14. Adipose-derived stem cells transfected with pEGFP-OSX enhance bone formation during distraction osteogenesis.

    PubMed

    Lai, Qing-guo; Sun, Shao-long; Zhou, Xiao-hong; Zhang, Chen-ping; Yuan, Kui-feng; Yang, Zhong-jun; Luo, Sheng-lei; Tang, Xiao-peng; Ci, Jiang-bo

    2014-05-01

    This study was designed to investigate the effects of local delivery of adipose-derived stem cells (ADSCs) transfected with transcription factor osterix (OSX) on bone formation during distraction osteogenesis. New Zealand white rabbits (n=54) were randomly divided into three groups (18 rabbits per group). A directed cloning technique was used for the construction of recombinant plasmid pEGFP-OSX, where EGFP is the enhanced green fluorescence protein. After osteodistraction of the right mandible of all experimental rabbits, rabbits in group A were treated with ADSCs transfected with pEGFP-OSX, group B with ADSCs transfected with pEGFP-N1, and group C with physiological saline. Radiographic and histological examinations were processed after half of the animals within each group were humanely killed by injection of sodium pentothal at Week 2 or 6 after surgery. The distraction bone density was measured as its projectional bone mineral density (BMD). Three parameters were measured, namely, the thickness of new trabeculae (TNT), and the volumes of the newly generated cortical bone (NBV1) and the cancellous bone (NBV2) of the distracted regions. Good bone generation in the distraction areas was found in group A, which had the highest BMD, TNT, and NBV in the distraction zones among the groups. There was no significant difference in bone generation in the distraction areas between groups B and C. The results indicate that the transplantation of ADSCs transfected with pEGFP-OSX can effectively promote bone generation during distraction in vivo. PMID:24793766

  15. Effects of single N-glycosylation site knockout on folding and defibrinogenating activities of acutobin recombinants from HEK293T.

    PubMed

    Tsai, Inn-Ho; Wang, Ying-Ming; Huang, Kai-Fa

    2015-02-01

    Acutobin, the α-fibrinogenase from Deinagkistrodon acutus venom, contains four N-glycosylation sites with disialylated complex-typed glycans. Here, we explore the functional roles of each of the N-glycan by site-directed mutagenesis. The wild-type (ATB-wt) and single glycan-knockout mutants of recombinant acutobin were prepared from HEK293T, demonstrating that mutations at Asn(77), Asn(81) and Asn(100) impaired the folding while the S79A mutant and various Asn(229)-deglycosylated mutants were correctly folded. Based on homology modeling of acutobin and multiple sequence alignment with various venom thrombin-like enzymes, the importance of a hydrophilic environment at each glycosylation site to the enzyme folding could be rationalized. Remarkably, all the mutants showed similar catalytic activities for the chromogenic substrate and similar thermal stabilities as ATB-wt, suggesting that the glycan knockout did not affect the gross conformation and stability of the active sites. Although SDS-PAGE analyses revealed that ATB-wt and the D229-mutant degraded all human fibrinogen subunits faster but less specifically in vitro as compared with other mutants that cleaved only the α-subunit, ATB-wt and D229-mutant were not able to release fibrinogen-peptide A and thus coagulated human plasma slower than the other mutants did. In the mice model, the defibrinogenating effect of ATB-wt was stronger and lasting-longer than those of all the mutants. Taken together, all the glycans contribute to the pharmacokinetics of acutobin and ATB-wt in vivo, and the microenvironment around the Asn(229)-glycan appears to regulate the fibrinogen-chain specificity of acutobin while the N-glycans at positions 77, 81 and 100 are crucial for its folding. PMID:25533529

  16. In vitro anti-tumor immune response induced by dendritic cells transfected with EBV-LMP2 recombinant adenovirus

    SciTech Connect

    Pan Ying; Zhang Jinkun . E-mail: jkzhang126@126.com; Zhou Ling; Zuo Jianmin; Zeng Yi

    2006-09-01

    Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is a high-incidence tumor in southern China. Latent membrane proteins 2 (LMP2) is a subdominant antigen of EBV. The present study was to develop a dendritic cells (DCs)-based cancer vaccine (rAd-LMP2-DC) and to study its biological characteristics and its immune functions. Our results showed that LMP2 gene transfer did not alter the typical morphology of mature DC, and the representative phenotypes of mature DC (CD80, CD83, and CD86) were highly expressed in rAd-LMP2-DCs. The expression of LMP2 in rAd-LPM2-DCs was about 84.54%, which suggested efficient gene transfer. Transfected DCs markedly increased antigen-specific T-cell proliferation. The specific cytotoxicity against NPC cell was significantly higher than that in controls (p < 0.05), and enhanced with increased stimulations by transfected DCs. In addition, phenotypic analysis demonstrated that the LMP2-specific CTLs consisted of both CD4{sup +} and CD8{sup +} T cells. These results showed that development of DC-based vaccine by transfection with malignancy-associated virus antigens could elicit potent CTL response and provide a potential strategy of immunotherapy for EBV-associated NPC.

  17. A mouse model of luciferase-transfected stromal cells of giant cell tumor of bone.

    PubMed

    Lau, Carol P Y; Wong, Kwok Chuen; Huang, Lin; Li, Gang; Tsui, Stephen K W; Kumta, Shekhar Madhukar

    2015-11-01

    A major barrier towards the study of the effects of drugs on Giant Cell Tumor of Bone (GCT) has been the lack of an animal model. In this study, we created an animal model in which GCT stromal cells survived and functioned as proliferating neoplastic cells. A proliferative cell line of GCT stromal cells was used to create a stable and luciferase-transduced cell line, Luc-G33. The cell line was characterized and was found that there were no significant differences on cell proliferation rate and recruitment of monocytes when compared with the wild type GCT stromal cells. We delivered the Luc-G33 cells either subcutaneously on the back or to the tibiae of the nude mice. The presence of viable Luc-G33 cells was assessed using real-time live imaging by the IVIS 200 bioluminescent imaging (BLI) system. The tumor cells initially propagated and remained viable on site for 7 weeks in the subcutaneous tumor model. We also tested in vivo antitumor effects of Zoledronate (ZOL) and Geranylgeranyl transferase-I inhibitor (GGTI-298) alone or their combinations in Luc-G33-transplanted nude mice. ZOL alone at 400 µg/kg and the co-treatment of ZOL at 400 µg/kg and GGTI-298 at 1.16 mg/kg reduced tumor cell viability in the model. Furthermore, the anti-tumor effects by ZOL, GGTI-298 and the co-treatment in subcutaneous tumor model were also confirmed by immunohistochemical (IHC) staining. In conclusion, we established a nude mice model of GCT stromal cells which allows non-invasive, real-time assessments of tumor development and testing the in vivo effects of different adjuvants for treating GCT.

  18. Development and Optimization of AAV hFIX Particles by Transient Transfection in an iCELLis(®) Fixed-Bed Bioreactor.

    PubMed

    Powers, Alicia D; Piras, Bryan A; Clark, Robert K; Lockey, Timothy D; Meagher, Michael M

    2016-06-01

    Adeno-associated virus (AAV) vectors are increasingly popular in gene therapy because they are unassociated with human disease, replication dependent, and less immunogenic than other viral vectors and can infect a variety of cell types. These vectors have been used in over 130 clinical trials, and one AAV product has been approved for treatment of lipoprotein lipase deficiency in Europe. To meet the demand for the increasing quantities of AAV required for clinical trials and treatment, a scalable high-capacity technology is required. Bioreactors meet these requirements but limited options are available for adherent HEK 293T/17 cells. Here we optimize the transient transfection of HEK293T/17 cells for the production of AAV human factor IX in a disposable fixed-bed bioreactor, the iCELLis(®) Nano (PALL Corporation). A fixed bed in the center of the iCELLis bioreactor is surrounded by culture medium that is pumped through the bed from the bottom of the bioreactor so that a thin film of the medium overflows the bed and is replenished with oxygen and depleted of CO2 as it returns to the surrounding medium reservoir. We show that this fixed-bed bioreactor can support as many as 2.5 × 10(8) cells/ml of fixed bed (1.9 × 10(6) cells/cm(2)). By optimizing culture and transfection parameters such as the concentration of DNA for transfection, day of harvest, size of PEI/DNA particles, and transfection medium, and adding an additional medium change to the process, we increased our yield to as high as 9.0 × 10(14) viral particles per square meter of fixed bed. We also show an average GFP transfection of 97% of cells throughout the fixed bed. These yields make the iCELLis a promising scalable technology for the clinical production of AAV gene therapy products. PMID:27229773

  19. Molecular cloning and nucleotide sequence of a transforming gene detected by transfection of chicken B-cell lymphoma DNA

    NASA Astrophysics Data System (ADS)

    Goubin, Gerard; Goldman, Debra S.; Luce, Judith; Neiman, Paul E.; Cooper, Geoffrey M.

    1983-03-01

    A transforming gene detected by transfection of chicken B-cell lymphoma DNA has been isolated by molecular cloning. It is homologous to a conserved family of sequences present in normal chicken and human DNAs but is not related to transforming genes of acutely transforming retroviruses. The nucleotide sequence of the cloned transforming gene suggests that it encodes a protein that is partially homologous to the amino terminus of transferrin and related proteins although only about one tenth the size of transferrin.

  20. Sodium/iodide symporter gene transfection increases radionuclide uptake in human cisplatin-resistant lung cancer cells.

    PubMed

    Chai, W; Yin, X; Ren, L; Cai, M; Long, T; Zhou, M; Tang, Y; Yang, N; Hu, S

    2015-10-01

    The sodium/iodide symporter (NIS) is involved in iodide uptake and has been used for the diagnosis and treatment of thyroid cancer. Transfection of the NIS gene in A549 human lung cancer cells can induce radioactive iodine ((131)I) and radioactive technetium ((99m)Tc) uptake. The aim of the present study was to assess the role of NIS in (99m)Tc and (131)I uptake by the A549/DDP human cisplatin-resistant lung cancer cell line. To do so, recombinant adenovirus, adenovirus-enhanced green fluorescent protein-human NIS (Ad-eGFP-hNIS) and Ad-eGFP-rat NIS (Ad-eGFP-rNIS) vectors were established. These vectors were transfected into A549/DDP cells and xenograft tumors in nude mice. Assessment of (99m)Tc and (131)I uptake was performed. Results showed that the transfection efficiency of Ad-eGFP-hNIS and Ad-eGFP-rNIS in A549/DDP cells was at least 90 % in all experiments, and that the uptake ability of (99m)Tc and (131)I was highly enhanced (14-18 folds for (99m)Tc, and 12-16 folds for (131)I). However, the radionuclide concentration in transfected NIS genes' A549/DDP cells reached a plateau within 30-60 min, indicating that NIS transport led rapidly to (99m)Tc and (131)I saturation in cells. In xenograft tumor models, uptake of (99m)TcO4 (-) was obviously higher in the hNIS and rNIS groups compared with controls. In conclusion, these results support the hypothesis that A549/DDP cells can effectively uptake (99m)Tc and (131)I when transfected with the hNIS and rNIS gene. The rNIS or hNIS gene could be used as an effective method for the effective delivery of radioactive products to specific tissues for imagery and/or treatment.

  1. Comparison of in vitro methods for carboxylesterase activity determination in immortalized cells representative of the intestine, liver and kidney.

    PubMed

    Lamego, Joana; Ferreira, Pedro; Alves, Márcia; Matias, Ana; Simplício, Ana Luisa

    2015-08-01

    Herein we compare the fluorimetric determination of total and specific carboxylesterase activity in immortalized human derived living cells and in cell lysates. The cell lines chosen are representative of metabolism occurring in the intestine (Caco-2 and HT-29), kidney (HEK-293T) and liver (Hep G2). Caco-2 and HT-29, as cells prone to differentiation, were tested along the differentiation time. For evaluation of both methods when distinguishing activity of different carboxylesterases, HEK-293T transfected with the human carboxylestarase-2 (hCES2) were also tested. Application to Caco-2 or HT-29 cells demonstrated higher activity detected in cell lysates than in cell monolayers. The difference is most striking when comparing the methods at different stages of Caco-2 and HT-29 cell maturation, highlighting substrate accessibility as a limiting step in the in vivo hydrolysis rates (possibly limited by plasma and Endoplasmic Reticulum membrane permeability) with increasing relevance as the cells differentiate. Application to Hep G2 or to hCES2 transfected and non-transfected HEK-293T cells, demonstrated a tendency for higher sensitivity in living cell suspensions than that obtained with the cell lysates which indicates the importance of cell environment in the maintenance of enzyme activity. However, quantification of hCES2 activity relative to total esterase, or to total carboxylesterase activity, was not significantly different in any case. The results herein presented help to clarify which method is best suited for evaluation of carboxylesterase activity in vitro depending on the final goal of the study.

  2. Enhanced wound healing by topical administration of mesenchymal stem cells transfected with stromal cell-derived factor-1.

    PubMed

    Nakamura, Yoko; Ishikawa, Hidefumi; Kawai, Katsuya; Tabata, Yasuhiko; Suzuki, Shigehiko

    2013-12-01

    The objective of this study was to investigate the ability of mesenchymal stem cells (MSC) genetically engineered with stromal cell-derived factor-1 (SDF-1) to heal skin wounds. When transfected with SDF-1 plasmid DNA, MSC which were isolated from the bone marrow of rats, secreted SDF-1 for 7 days. In vitro cell migration assay revealed that the SDF-1-engineered MSC (SDF-MSC) enhanced the migration of MSC and dermal fibroblasts to a significantly greater extent than MSC. The SDF-MSC secreted vascular endothelial growth factor, hepatocyte growth factor, and interleukin 6 at a significantly high level. A skin defect model of rats was prepared and MSC and SDF-MSC were applied to the wound to evaluate wound healing in terms of wound size and histological examinations. The wound size decreased significantly faster with SDF-MSC treatment than with MSC and PBS treatments. The length of the neoepithelium and the number of blood vessels newly formed were significantly larger. A cell-tracing experiment with fluorescently labeled cells demonstrated that the percent survival of SDF-MSC in the tissue treated was significantly high compared with that of MSC. It was concluded that SDF-1 genetic engineering is a promising way to promote the wound healing activity of MSC for a skin defect.

  3. Expression of human epidermal growth factor precursor cDNA in transfected mouse NIH 3T3 cells.

    PubMed Central

    Mroczkowski, B; Reich, M; Whittaker, J; Bell, G I; Cohen, S

    1988-01-01

    Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 microM ZnCl2. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is approximately 150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with 125I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself. Images PMID:3257563

  4. Expression of human epidermal growth factor pressures cDNA in transfected mouse NIH 3T3 cells

    SciTech Connect

    Mroczkowski, B.; Reich, M.; Whittaker, J.; Bell, G.I.; Cohen, S.

    1988-01-01

    Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 ..mu..M ZnCl/sub 2/. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is approx. = 150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with /sup 125/I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself.

  5. Experimental research on wild-type p53 plasmid transfected into retinoblastoma cells and tissues using an ultrasound microbubble intensifier.

    PubMed

    Luo, J; Zhou, X; Diao, L; Wang, Z

    2010-01-01

    The transfection efficiency of wild-type p53 (wtp53) was investigated in retinoblastoma (RB) Y79 cells using an ultrasound microbubble technique. A human RB nude mouse xenograft tumour model was also used to investigate whether this technique could deliver wtp53 into solid tumours. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that wtp53 was successfully transfected into Y79 cells in the plasmid with microbubbles and ultrasound group and in the plasmid with liposomes group, but not in the plasmid with ultrasound group or in the untreated control group. Flow cytometry showed that apoptosis was highest in the microbubbles and ultrasound group (25.58%) compared with the plasmid with liposomes group (19.50%), and the other two groups (< 10%). RT-PCR also showed that the wtp53 gene was successfully transfected into solid tumours in the plasmid with microbubbles and ultrasound group. This study provides preliminary evidence in support of a potential new approach to RB gene therapy. PMID:20819437

  6. Experimental research on wild-type p53 plasmid transfected into retinoblastoma cells and tissues using an ultrasound microbubble intensifier.

    PubMed

    Luo, J; Zhou, X; Diao, L; Wang, Z

    2010-01-01

    The transfection efficiency of wild-type p53 (wtp53) was investigated in retinoblastoma (RB) Y79 cells using an ultrasound microbubble technique. A human RB nude mouse xenograft tumour model was also used to investigate whether this technique could deliver wtp53 into solid tumours. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that wtp53 was successfully transfected into Y79 cells in the plasmid with microbubbles and ultrasound group and in the plasmid with liposomes group, but not in the plasmid with ultrasound group or in the untreated control group. Flow cytometry showed that apoptosis was highest in the microbubbles and ultrasound group (25.58%) compared with the plasmid with liposomes group (19.50%), and the other two groups (< 10%). RT-PCR also showed that the wtp53 gene was successfully transfected into solid tumours in the plasmid with microbubbles and ultrasound group. This study provides preliminary evidence in support of a potential new approach to RB gene therapy.

  7. Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells.

    PubMed

    Fitzpatrick, D R; Zamb, T; Parker, M D; van Drunen Littel-van den Hurk, S; Babiuk, L A; Lawman, M J

    1988-11-01

    Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK- cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1-infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1-infected bovine cells. For gI, a deficiency in N-linked glycosylation of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected or BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes. Immunization of mice with the transfected cells elicited BHV-1-specific virus-neutralizing antibody, thus verifying the antigenic authenticity of the recombinant glycoproteins and the important role of gI and gIII as targets of the immune response to BHV-1 in this murine model system.

  8. Histo-blood group A/B versus H status of human carcinoma cells as correlated with haptotactic cell motility: approach with A and B gene transfection.

    PubMed

    Ichikawa, D; Handa, K; Withers, D A; Hakomori, S

    1997-08-01

    In a search for the molecular basis of ABH status of tumors as correlated with malignancy, we studied various malignancy-related phenotypes of high H/Le(y)-expressing tumor cell lines in comparison with phenotypes of the same lines transfected with histo-blood group A or B genes. A and B gene transfectants, prepared independently from different H-active parental cells, showed A or B activity and abolition of H activity. All A and B gene transfectants, regardless of source, were characterized by significantly reduced Matrigel-dependent haptotactic motility. The level of haptotaxis of all transfectants was similar to that of parental cells in the presence of antibodies against human integrin subunits alpha3, alpha6, or beta1. These subunits showed high expression of A or B epitope in the A and B gene transfectants. Enhancement versus reduction of malignancy, associated with deletion versus induction of A/B epitopes, may be due in part to enhanced haptotaxis sustained by alpha3, alpha6, and beta1 integrin receptors, the activities of which are regulated by H or A/B glycosylation. These phenotypic changes provide a rationale for the deletion of A and B epitopes as one criterion defining human tumor malignancy. PMID:9242430

  9. Induction of antitumor immunity with Fas/APO-1 ligand (CD95L)-transfected neuroblastoma neuro-2a cells.

    PubMed

    Shimizu, M; Fontana, A; Takeda, Y; Yagita, H; Yoshimoto, T; Matsuzawa, A

    1999-06-15

    Fas/Apo-1 (CD95)-Fas ligand (FasL) system has been implicated in the suppression and stimulation of immune responses. We examined the induction of antitumor immunity with neuroblastoma Neuro-2a cells transfected with FasL cDNA (Neuro-2a+FasL). Neuro-2a+FasL cells expressed FasL on the cell surface and secreted soluble FasL. Histologic and flow cytometric analyses revealed that Neuro-2a+FasL cells caused neutrophils to infiltrate into the injected site, resulting in strong inflammation. Neutrophil infiltration was inhibited by treatment with anti-FasL mAb and did not occur in Fas-deficient lpr mice. Normal syngeneic mice rejected Neuro-2a+FasL cells after the inflammation and acquired tumor-specific protective immunity. CD8+ T cells were responsible for the antitumor immunity. Neuro-2a+FasL cells formed tumors after far longer latency compared with mock-transfected Neuro-2a+Neo cells in nude mice, and immune competent mice rejected Neuro-2a cells but not sarcoma S713a cells when they were injected with Neuro-2a+FasL cells in a mixture. These results suggest that neutrophils attracted through the Fas-FasL system may impair tumor cells by inflammation at the initial step, followed by development of CD8+ T cell-dependent tumor-specific antitumor immunity, leading to complete eradication of tumor cells. Importantly, the treatment with Neuro-2a+FasL cells exhibited therapeutic efficacy against growing tumors.

  10. Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells.

    PubMed

    Pazzagli, M; Devine, J H; Peterson, D O; Baldwin, T O

    1992-08-01

    The aim of this study was to compare three different luciferase genes by placing them in a single reporter vector and expressing them in the same mammalian cell type. The luciferase genes investigated were the luc genes from the fireflies Photinus pyralis (PP) and Luciola mingrelica (LM) and the lux AB5 gene, a translational fusion of the two subunits of the bacterial luciferase from Vibrio harveyi (VH). The chloramphenicol acetyltransferase (CAT) gene was also included in this study for comparison. The performances of the assay methods of the corresponding enzymes were evaluated using reference materials and the results of the expressed enzymes following transfection were calculated using calibration curves. All of the bioluminescent assays possess high reproducibility both within and between the batches (less than 15%). The comparison of the assay methods shows that firefly luciferases have the highest detection sensitivity (0.05 and 0.08 amol for PP and LM, respectively) whereas the VH bacterial luciferase has 5 amol and CAT 100 amol. On the other hand, the transfection of the various plasmids shows that the content of the expressed enzyme within the cells is much higher for CAT than for the other luciferase genes. VH luciferase is expressed at very low levels in mammalian cells due to the relatively high temperature of growing of the mammalian cells that seems to impair the correct folding of the active enzyme. PP and LM luciferases are both expressed at picomolar level but usually 10 to 70 times less in content with respect to CAT within the transfected cells. On the basis of these results the overall improvement in sensitivity related to the use of firefly luciferases as reporter genes in mammalian cells is about 30 to 50 times with respect to that of CAT. PMID:1443530

  11. In vitro Labeling of Neural Stem Cells with Poly-L-Lysine Coated Super Paramagnetic Nanoparticles for Green Fluorescent Protein Transfection

    PubMed Central

    Albukhaty, Salim; Naderi-Manesh, Hossein; Tiraihi, Taki

    2013-01-01

    Background: The magnetic nanoparticle-based transfection method is a relatively new technique for delivery of functional genes to target tissues. We aimed to evaluate the transfection efficiency of rat neural stem cell (NSC) using poly-L-lysine hydrobromide (PLL)-coated super paramagnetic iron oxide nanoparticles (SPION). Methods: The SPION was prepared and coated with PLL as transfection agent and the transfection efficiency was evaluated in rat NSC using enhanced green fluorescent protein-N1 plasmid containing GFP as a reporter gene. NSC was incubated for 24 h in cell culture media containing 25 µg/ml SPION and in different concentrations of PLL (0.25, 0.50, 0.75, 1 and 2 µg/ml). Cell viability was determined before and after transfection for every concentration using Trypan blue assay. Characterization of prepared uncoated (SPION) and coated (SPION-PLL) complexes were evaluated by a transmission electron microscope and the zeta potential. Results: PLL at 0.75 μg/ml showed optimal results with 25 μg/ml SPION concentration compared with other PLL concentrations (0.25, 0.50, 1 and 2 μg/ml). The 18% efficiency of the transfected cells showed green fluorescence. Conclusion: Transfection with SPION is an efficient, non-viral gene transfere method. PMID:23567848

  12. PACAP receptor pharmacology and agonist bias: analysis in primary neurons and glia from the trigeminal ganglia and transfected cells

    PubMed Central

    Walker, C S; Sundrum, T; Hay, D L

    2014-01-01

    Background and Purpose A major challenge in the development of new medicines targeting GPCRs is the ability to quantify drug action in physiologically relevant models. Primary cell models that closely resemble the clinically relevant in vivo site of drug action are important translational tools in drug development. However, pharmacological studies in these models are generally very limited due to the methodology used. Experimental Approach We used a neuropeptide system to demonstrate the applicability of using highly sensitive signalling assays in primary cells. We quantified the action of pituitary adenylate cyclase-activating peptide (PACAP)-38, PACAP-27 and vasoactive intestinal polypeptide in primary cultures of neurons and glia derived from rat trigeminal ganglia (TG), comparing our observations to transfected cells. Key Results PACAP-responsive receptors in rat trigeminal neurons, glia and transfected PAC1n receptors were pharmacologically distinct. PACAP-38, but not PACAP-27, activated ERK in glia, while both forms stimulated cellular cAMP production. PACAP(6–38) also displayed cell-type-dependent, agonist-specific, antagonism. Conclusions and Implications The complexity of PACAP pharmacology in the TG may help to direct, more effectively, the development of disease treatments targeting the PACAP receptor. We suggest that these methodologies are broadly applicable to other primary cell types of human or animal origin, and that our approach may allow more thorough characterization of ligand properties in physiologically relevant cell types. PMID:24303997

  13. Graphene based gene transfection

    NASA Astrophysics Data System (ADS)

    Feng, Liangzhu; Zhang, Shuai; Liu, Zhuang

    2011-03-01

    Graphene as a star in materials research has been attracting tremendous attentions in the past few years in various fields including biomedicine. In this work, for the first time we successfully use graphene as a non-toxic nano-vehicle for efficient gene transfection. Graphene oxide (GO) is bound with cationic polymers, polyethyleneimine (PEI) with two different molecular weights at 1.2 kDa and 10 kDa, forming GO-PEI-1.2k and GO-PEG-10k complexes, respectively, both of which are stable in physiological solutions. Cellular toxicity tests reveal that our GO-PEI-10k complex exhibits significantly reduced toxicity to the treated cells compared to the bare PEI-10k polymer. The positively charged GO-PEI complexes are able to further bind with plasmid DNA (pDNA) for intracellular transfection of the enhanced green fluorescence protein (EGFP) gene in HeLa cells. While EGFP transfection with PEI-1.2k appears to be ineffective, high EGFP expression is observed using the corresponding GO-PEI-1.2k as the transfection agent. On the other hand, GO-PEI-10k shows similar EGFP transfection efficiency but lower toxicity compared with PEI-10k. Our results suggest graphene to be a novel gene delivery nano-vector with low cytotoxicity and high transfection efficiency, promising for future applications in non-viral based gene therapy.Graphene as a star in materials research has been attracting tremendous attentions in the past few years in various fields including biomedicine. In this work, for the first time we successfully use graphene as a non-toxic nano-vehicle for efficient gene transfection. Graphene oxide (GO) is bound with cationic polymers, polyethyleneimine (PEI) with two different molecular weights at 1.2 kDa and 10 kDa, forming GO-PEI-1.2k and GO-PEG-10k complexes, respectively, both of which are stable in physiological solutions. Cellular toxicity tests reveal that our GO-PEI-10k complex exhibits significantly reduced toxicity to the treated cells compared to the bare PEI

  14. PUMA gene transfection can enhance the sensitivity of epirubicin-induced apoptosis of MCF-7 breast cancer cells.

    PubMed

    Sun, C-G; Zhuang, J; Teng, W-J; Wang, Z; Du, S-S

    2015-05-29

    We explored whether p53 upregulated modulator of apoptosis (PUMA) gene transfection could enhance the sensitivity of epirubicin-induced apoptosis of MCF-7 breast cancer cells. The liposome-mediated recombinant eukaryotic expression vector PU-MA-pCDNA3 and empty vector plasmid were stably transfected into MCF-7 cells. Epirubicin (0.01-100 μM) was applied to MCF-7, MCF-7/PUMA, and MCF-7/pCDNA3 cells for 72 h. The MTT assay was used to calculate the cell survival rate in each group, and the 50% inhibitory concentration (IC50) was calculated. The IC50 values of epirubicin in MCF-7, MCF-7/PUMA, and MCF-7/pCDNA3 cells were 13 ± 1.4, 1.8 ± 0.2, and 10.7 ± 1.3 μM, respectively. The sensitivity of MCF-7/PUMA cells to epirubicin increased 7.2-fold. Epirubicin induced apoptosis in MCF-7 cells dose-dependently, but MCF-7/PUMA cell-induced apoptosis was more significant compared to controls. Low concentrations of epirubicin (0.1 μM) caused low levels of apoptosis of MCF-7/pCDNA3 (1.15 ± 0.26%) and MCF-7 cells (0.9 ± 0.24%), but significantly induced apoptosis of MCF-7/PUMA cells (6.44 ± 1.46%). High epirubicin concentration (1 μM) induced apoptosis in each group, but the epirubicin MCF-7/PUMA apoptosis rate (35.47 ± 9.36%) was significantly higher than that of MCF-7 (12.6 ± 3.73%) and MCF-7/ pCDNA3 (15.2 ± 5.17%) cells (P < 0 01). Flow cytometry and TUNEL assays for apoptosis detection showed similar results. PUMA protein expression in MCF-7/PUMA cells was significantly higher than that in MCF-7 and MCF-7/pCDNA3 cells by Western blot analysis. There-fore, stable transfection of PUMA can significantly enhance epirubicin-induced apoptosis sensitivity of MCF-7 breast cancer cells.

  15. Thyroid and glucocorticoid hormones induce expression of lactase-phlorizin hydrolase gene in CDX-2/HNF-1α co-transfected IEC-6 cells.

    PubMed

    Suzuki, Takuji; Mochizuki, Kazuki; Goda, Toshinao

    2014-01-01

    Thyroid and glucocorticoid hormones and several transcriptional factors such as caudal type homeobox (CDX)-2 and hepatocyte nuclear factor (HNF)-1α are important for the differentiation of small intestinal absorptive cells and the consequent expression of genes related to the digestion/absorption of carbohydrates. In this study, we investigated whether thyroid and glucocorticoid hormones enhanced the expression of lactase-phlorizin hydrolase (LPH) gene, an intestine-specific gene that encodes an enzyme for lactose digestion, in small intestinal stem-like IEC-6 cells co-transfected with CDX-2 and HNF-1α using a retrovirus system. Changes in expression of intestine-specific genes caused by treatment with thyroid and/or glucocorticoid hormones were monitored in empty vector-transfected cells and in CDX-2/HNF-1α co-transfected cells by qRT-PCR. Stable co-transfection with CDX-2 and HNF-1α evoked the expression of the LPH gene in IEC-6 cells. Furthermore, treatment with a thyroid hormone, triiodothyronine, and a glucocorticoid receptor agonist, dexamethasone, significantly enhanced expression of the LPH, CDX-2 and HNF-1α genes in CDX-2/HNF-1α co-transfected IEC-6 cells. These results suggest that thyroid and glucocorticoid hormones synergistically enhance expression of the LPH gene in CDX-2/HNF-1α co-transfected IEC-6 cells. PMID:25744420

  16. Thyroid and glucocorticoid hormones induce expression of lactase-phlorizin hydrolase gene in CDX-2/HNF-1α co-transfected IEC-6 cells.

    PubMed

    Suzuki, Takuji; Mochizuki, Kazuki; Goda, Toshinao

    2014-01-01

    Thyroid and glucocorticoid hormones and several transcriptional factors such as caudal type homeobox (CDX)-2 and hepatocyte nuclear factor (HNF)-1α are important for the differentiation of small intestinal absorptive cells and the consequent expression of genes related to the digestion/absorption of carbohydrates. In this study, we investigated whether thyroid and glucocorticoid hormones enhanced the expression of lactase-phlorizin hydrolase (LPH) gene, an intestine-specific gene that encodes an enzyme for lactose digestion, in small intestinal stem-like IEC-6 cells co-transfected with CDX-2 and HNF-1α using a retrovirus system. Changes in expression of intestine-specific genes caused by treatment with thyroid and/or glucocorticoid hormones were monitored in empty vector-transfected cells and in CDX-2/HNF-1α co-transfected cells by qRT-PCR. Stable co-transfection with CDX-2 and HNF-1α evoked the expression of the LPH gene in IEC-6 cells. Furthermore, treatment with a thyroid hormone, triiodothyronine, and a glucocorticoid receptor agonist, dexamethasone, significantly enhanced expression of the LPH, CDX-2 and HNF-1α genes in CDX-2/HNF-1α co-transfected IEC-6 cells. These results suggest that thyroid and glucocorticoid hormones synergistically enhance expression of the LPH gene in CDX-2/HNF-1α co-transfected IEC-6 cells.

  17. Poly(amido amine)-based multilayered thin films on 2D and 3D supports for surface-mediated cell transfection.

    PubMed

    Hujaya, Sry D; Marchioli, Giulia; Roelofs, Karin; van Apeldoorn, Aart A; Moroni, Lorenzo; Karperien, Marcel; Paulusse, Jos M J; Engbersen, Johan F J

    2015-05-10

    Two linear poly(amido amine)s, pCABOL and pCHIS, prepared by polyaddition of cystamine bisacrylamide (C) with 4-aminobutanol (ABOL) or histamine (HIS), were explored to form alternating multilayer thin films with DNA to obtain functionalized materials with transfection capacity in 2D and 3D. Therefore, COS-7 cells were cultured on top of multilayer films formed by layer-by-layer dipcoating of these polymers with GFP-encoded pDNA, and the effect of the number of layers and cell seeding density on the transfection efficiency was evaluated. Multilayer films with pCABOL were found to be superior to pCHIS in facilitating transfection, which was attributed to higher incorporation of pDNA and release of the transfection agent. High amounts of transfected cells were obtained on pCABOL films, correlating proportionally over a wide range with seeding density. Optimal transfection efficiency was obtained with pCABOL films composed of 10 bilayers. Further increase in the number of bilayers only marginally increased transfection efficiency. Using the optimal multilayer and cell seeding conditions, pCABOL multilayers were fabricated on poly(ε-caprolactone) (PCL), heparinized PCL (PCL-HEP), and poly(lactic acid) (PLA) disks as examples of common biomedical supports. The multilayers were found to completely mask the properties of the original substrates, with significant improvement in cell adhesion, which is especially pronounced for PCL and PLA disks. With all these substrates, transfection efficiency was found to be in the range of 25-50% transfected cells. The pCABOL/pDNA multilayer films can also conveniently add transfection capability to 3D scaffolds. Significant improvement in cell adhesion was observed after multilayer coating of 3D-plotted fibers of PCL (with and without an additional covalent heparin layer), especially for the PCL scaffold without heparin layer and transfection was observed on both 3D PCL and PCL-HEP scaffolds. These results show that layer

  18. Loss of retrovirus production in JB/RH melanoma cells transfected with H-2Kb and TAP-1 genes.

    PubMed

    Li, M; Xu, F; Muller, J; Huang, X; Hearing, V J; Gorelik, E

    1999-01-20

    JB/RH1 melanoma cells, as well as other melanomas of C57BL/6 mice (B16 and JB/MS), express a common melanoma-associated antigen (MAA) encoded by an ecotropic melanoma-associated retrovirus (MelARV). JB/RH1 cells do not express the H-2Kb molecules due to down-regulation of the H-2Kb and TAP-1 genes. When JB/RH1 cells were transfected with the H-2Kb and cotransfected with the TAP-1 gene, it resulted in the appearance of H-2Kb molecules and an increase in their immunogenicity, albeit they lost expression of retrovirus-encoded MAA recognized by MM2-9B6 mAb. Loss of MAA was found to result from a complete and stable elimination of ecotropic MelARV production in the H-2Kb/TAP-1-transfected JB/RH1 cells. Northern blot analysis showed no differences in ecotropic retroviral messages in MelARV-producing and -nonproducing melanoma cells, suggesting that loss of MelARV production was not due to down-regulation of MelARV transcription. Southern blot analysis revealed several rearrangements in the proviral DNA of H-2Kb-positive JB/RH1 melanoma cells. Sequence analysis of the ecotropic proviral DNA from these cells showed numerous nucleotide substitutions, some of which resulted in the appearance of a novel intraviral PstI restriction site and the loss of a HindIII restriction site in the pol region. PCR amplification of the proviral DNAs indicates that an ecotropic provirus found in the H-2Kb-positive cells is novel and does not preexist in the parental H-2Kb-negative melanoma cells. Conversely, the ecotropic provirus of the parental JB/RH1 cells was not amplifable from the H-2Kb-positive cells. Our data indicate that stable loss of retroviral production in the H-2Kb/TAP-1-transfected melanoma cells is probably due to the induction of recombination between a productive ecotropic MelARV and a defective nonecotropic provirus leading to the generation of a defective ecotropic provirus and the loss of MelARV production and expression of the retrovirus-encoded MAA.

  19. RNA interference by osmotic lysis of pinosomes: liposome-independent transfection of siRNAs into mammalian cells.

    PubMed

    Gruber, Jens; Boese, Guido; Tuschl, Thomas; Osborn, Mary; Weber, Klaus

    2004-07-01

    The osmotic lysis of pinosomes procedure has been adapted to deliver small interfering RNAs (siRNAs) into cells in culture. Under hypertonic conditions, siRNAs were internalized into pinosomes. A subsequent osmotic shock in hypotonic buffer disrupted the pinosomes and caused the release of siRNAs into the cell cytoplasm. Both steps could be demonstrated directly using fluorescein-labeled siRNAs and confocal laser-scanning microscopy. Uptake by the pinocytosis/osmotic lysis procedure is concentration- and time-dependent. At an siRNA concentration of 0.4 microM, treatment for 40 or 80 min results in silencing efficiencies of 60% and 90%, respectively, after 44 h. A double treatment resulted in approximately equal silencing efficiencies but in reduced viability. This method has been used on a variety of human and murine cell lines including HEK293, HeLa SS6, and SW3T3 cells. Targets such as lamin A/C and Eg5 were effectively silenced. Novel silencing data are provided for Ki67, one of the few reliable prognostic markers for tumor patients. The new procedure avoids certain technical problems encountered with commercial transfection reagents while yielding silencing efficiencies that are comparable to those obtained with liposome-mediated siRNA transfection.

  20. Expression and phosphorylation of a three-repeat isoform of tau in transfected non-neuronal cells.

    PubMed Central

    Gallo, J M; Hanger, D P; Twist, E C; Kosik, K S; Anderton, B H

    1992-01-01

    The neuronal microtubule-associated protein, tau, is expressed as a set of isoforms containing either three or four tandemly repeated 31-amino-acid motifs in the C-terminal half of the molecule that can bind to microtubules. Three-repeat forms are the only ones expressed early in development. A single three-repeat isoform of tau has been stably expressed in non-neuronal cells which do not express endogenous tau. Chinese hamster ovary (CHO) cells were transfected with a full-length cDNA coding for the foetal form of human tau cloned downstream of the simian virus 40 (SV40) promoter, and a cell line constitutively expressing tau, CHO[pSVtau3], was isolated. Double-label immunofluorescence microscopy reveals that tau co-localizes with the microtubular network of normal or taxol-treated CHO[pSVtau3] cells, without inducing any dramatic change in cell morphology. Tau is expressed in CHO[pSVtau3] cells as three bands in SDS/PAGE recognized by antibodies to tau, the slow-migrating tau species being the most abundant. Tau also appears as three bands in a heat-stable fraction from CHO[pSVtau3] cells, but a single band of enhanced immunoreactivity is detected following treatment of this fraction with alkaline phosphatase. This single band co-migrates with the fast-migrating band of untreated fractions or whole-cell extracts. In conclusion, a three-repeat isoform of tau is capable of binding to microtubules in transfected non-neuronal cells; furthermore, in this system, the protein is phosphorylated in at least two different states inducing a reduced electrophoretic mobility. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:1530572

  1. A Flow-Through Cell Electroporation Device for Rapidly and Efficiently Transfecting Massive Amounts of Cells in vitro and ex vivo

    NASA Astrophysics Data System (ADS)

    Zhao, Deyao; Huang, Dong; Li, Yang; Wu, Mengxi; Zhong, Wenfeng; Cheng, Qiang; Wang, Xiaoxia; Wu, Yidi; Zhou, Xiao; Wei, Zewen; Li, Zhihong; Liang, Zicai

    2016-01-01

    Continuous cell electroporation is an appealing non-viral approach for genetically transfecting a large number of cells. Yet the traditional macro-scale devices suffer from the unsatisfactory transfection efficiency and/or cell viability due to their high voltage, while the emerging microfluidic electroporation devices is still limited by their low cell processing speed. Here we present a flow-through cell electroporation device integrating large-sized flow tube and small-spaced distributed needle electrode array. Relatively large flow tube enables high flow rate, simple flow characterization and low shear force, while well-organized needle array electrodes produce an even-distributed electric field with low voltage. Thus the difficulties for seeking the fine balance between high flow rate and low electroporation voltage were steered clear. Efficient in vitro electrotransfection of plasmid DNA was demonstrated in several hard-to-transfect cell lines. Furthermore, we also explored ex vivo electroporated mouse erythrocyte as the carrier of RNA. The strong ability of RNA loading and short exposure time of freshly isolated cells jointly ensured a high yield of valid carrier erythrocytes, which further successfully delivered RNA into targeted tissue. Both in vitro and ex vivo electrotransfection could be accomplished at high cell processing speed (20 million cells per minute) which remarkably outperforms previous devices.

  2. A Flow-Through Cell Electroporation Device for Rapidly and Efficiently Transfecting Massive Amounts of Cells in vitro and ex vivo

    PubMed Central

    Zhao, Deyao; Huang, Dong; Li, Yang; Wu, Mengxi; Zhong, Wenfeng; Cheng, Qiang; Wang, Xiaoxia; Wu, Yidi; Zhou, Xiao; Wei, Zewen; Li, Zhihong; Liang, Zicai

    2016-01-01

    Continuous cell electroporation is an appealing non-viral approach for genetically transfecting a large number of cells. Yet the traditional macro-scale devices suffer from the unsatisfactory transfection efficiency and/or cell viability due to their high voltage, while the emerging microfluidic electroporation devices is still limited by their low cell processing speed. Here we present a flow-through cell electroporation device integrating large-sized flow tube and small-spaced distributed needle electrode array. Relatively large flow tube enables high flow rate, simple flow characterization and low shear force, while well-organized needle array electrodes produce an even-distributed electric field with low voltage. Thus the difficulties for seeking the fine balance between high flow rate and low electroporation voltage were steered clear. Efficient in vitro electrotransfection of plasmid DNA was demonstrated in several hard-to-transfect cell lines. Furthermore, we also explored ex vivo electroporated mouse erythrocyte as the carrier of RNA. The strong ability of RNA loading and short exposure time of freshly isolated cells jointly ensured a high yield of valid carrier erythrocytes, which further successfully delivered RNA into targeted tissue. Both in vitro and ex vivo electrotransfection could be accomplished at high cell processing speed (20 million cells per minute) which remarkably outperforms previous devices. PMID:26728941

  3. STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation.

    PubMed

    Yang, Xuechao; van der Kraan, Peter M; van den Dolder, Juliette; Walboomers, X Frank; Bian, Zhuan; Fan, Mingwen; Jansen, John A

    2007-11-01

    Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and dentin formation,in this study STRO-1-selected rat dental pulp-derived stem cells were transfected with the adenoviral mediated human BMP-2 gene. Subsequently, the cells were evaluated for their odontogenic differentiation ability in medium not containing dexamethasone or other stimuli. Cultures were investigated using light microscopy and scanning electron microscopy (SEM) and evaluated for cell proliferation, alkaline phosphatase(ALP) activity, and calcium content. Real-time polymerase chain reaction (PCR) was performed for gene expression of Alp, osteocalcin, collagen type I, bone sialoprotein, dentin sialophosphoprotein, and dentin matrix acidic phosphoprotein 1. Finally, an oligo-microarray was used to profile the expression of odontogenesis-related genes. Results of ALP activity, calcium content, and real-time PCR showed that only BMP2-transfected cells had the ability to differentiate into the odontoblast phenotype and to produce a calcified extracellular matrix. SEM and oligo-microarray confirmed these results. In contrast, the non-transfected cells represented a less differentiated cell phenotype. Based on our results, we concluded that the adenovirus can transfect STRO-1 selected cells with high efficacy. After BMP2 gene transfection, these cells had the ability to differentiate into odontoblast phenotype, even without the addition of odontogenic supplements to the medium. PMID:17824831

  4. Stably transfected human cell lines as fluorescent screening assay for nuclear factor KB activation dependent gene expression

    NASA Astrophysics Data System (ADS)

    Hellweg, Christine E.; Baumstark-Khan, Christa; Horneck, Gerda

    2004-06-01

    Activation of the Nuclear Factor kappaB (NF-kappaB) pathway as a possible antiapoptotic route represents one important cellular stress response. For identifying conditions which are capable to modify this pathway, a screening assay for detection of NF-kappaB-dependent gene activation using the reporter proteins Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) has been developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing four copies of the NF-kappaB response element. Treatment with tumor necrosis factor alpha (TNF-alpha) gave rise to substantial EGFP / d2EGFP expression in up to 90 % of the cells and was therefore used to screen different stably transfected clones for induction of NF-kappaB dependent gene expression. The time course of d2EGFP expression after treatment with TNF-alpha or phorbol ester was measured using flow cytometry. Cellular response to TNF-alpha was faster than to phorbol ester. Treatment of cells with TNF-alpha and DMSO revealed antagonistic interactions of these substances in the activation NF-kappaB dependent gene expression. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening.

  5. Human lysosomal acid phosphatase is transported as a transmembrane protein to lysosomes in transfected baby hamster kidney cells.

    PubMed Central

    Waheed, A; Gottschalk, S; Hille, A; Krentler, C; Pohlmann, R; Braulke, T; Hauser, H; Geuze, H; von Figura, K

    1988-01-01

    BHK cells transfected with human lysosomal acid phosphatase (LAP) cDNA (CT29) expressed 70-fold higher enzyme activities of acid phosphatase than non-transfected BHK cells. The CT29-LAP was synthesized in BHK cells as a heterogeneously glycosylated precursor that was tightly membrane associated. Transfer to the trans-Golgi was associated with a small increase in size (approximately 7 kd) and partial processing of the oligosaccharides to complex type structures. CT29-LAP was transferred into lysosomes as shown by subcellular fractionation, immunofluorescence and immunoelectron microscopy. Lack of mannose-6-phosphate residues suggested that transport does not involve mannose-6-phosphate receptors. Part of the membrane-associated CT29-LAP was processed to a soluble form. The mechanism that converts CT29-LAP into a soluble form was sensitive to NH4Cl, and reduced the size of the polypeptide by 7 kd. In vitro translation of CT29-derived cRNA in the presence of microsomal membranes yielded a CT29-LAP precursor that is protected from proteinase K except for a small peptide of approximately 2 kd. In combination with the sequence data available for LAP, these observations suggest that CT29-LAP is synthesized and transported to lysosomes as a transmembrane protein. In the lysosomes, CT29-LAP is released from the membrane by proteolytic cleavage, which removes a C-terminal peptide including the transmembrane domain and the cytosolic tail of 18 amino acids. Images PMID:3056714

  6. Molecular cloning, sequencing, and expression of functional bovine herpesvirus 1 glycoprotein gIV in transfected bovine cells.

    PubMed Central

    Tikoo, S K; Fitzpatrick, D R; Babiuk, L A; Zamb, T J

    1990-01-01

    The gene encoding bovine herpesvirus 1 (BHV-1) glycoprotein gIV was mapped, cloned, and sequenced. The gene is situated between map units 0.892 and 0.902 and encodes a predicted protein of 417 amino acids with a signal sequence cleavage site between amino acids 18 and 19. Comparison of the BHV-1 amino acid sequence with the homologous glycoproteins of other alphaherpesviruses, including herpes simplex virus type 1 glycoprotein gD, revealed significant homology in the amino-terminal half of the molecules, including six invariant cysteine residues. The identity of the open reading frame was verified by expression of the authentic recombinant BHV-1 gIV in bovine cells by using eucaryotic expression vectors pRSDneo (strong, constitutive promoter) and pMSG (weak, dexamethasone-inducible promoter). Constitutive expression of gIV proved toxic to cells, since stable cell lines could only be established when the gIV gene was placed under the control of an inducible promoter. Expression of gIV was cell associated and localized predominantly in the perinuclear region, although nuclear and plasma membrane staining was also observed. Radioimmunoprecipitation revealed that the recombinant glycoprotein was efficiently processed and had a molecular weight similar to that of the native form of gIV expressed in BHV-1-infected bovine cells. Recombinant gIV produced in the transfected bovine cells induced cell fusion, polykaryon formation, and nuclear fusion. In addition, expression of gIV interfered with BHV-1 replication in the transfected bovine cells. Images PMID:2168991

  7. Efficient delivery and functional expression of transfected modified mRNA in human embryonic stem cell-derived retinal pigmented epithelial cells.

    PubMed

    Hansson, Magnus L; Albert, Silvia; González Somermeyer, Louisa; Peco, Rubén; Mejía-Ramírez, Eva; Montserrat, Núria; Izpisua Belmonte, Juan Carlos

    2015-02-27

    Gene- and cell-based therapies are promising strategies for the treatment of degenerative retinal diseases such as age-related macular degeneration, Stargardt disease, and retinitis pigmentosa. Cellular engineering before transplantation may allow the delivery of cellular factors that can promote functional improvements, such as increased engraftment or survival of transplanted cells. A current challenge in traditional DNA-based vector transfection is to find a delivery system that is both safe and efficient, but using mRNA as an alternative to DNA can circumvent these major roadblocks. In this study, we show that both unmodified and modified mRNA can be delivered to retinal pigmented epithelial (RPE) cells with a high efficiency compared with conventional plasmid delivery systems. On the other hand, administration of unmodified mRNA induced a strong innate immune response that was almost absent when using modified mRNA. Importantly, transfection of mRNA encoding a key regulator of RPE gene expression, microphthalmia-associated transcription factor (MITF), confirmed the functionality of the delivered mRNA. Immunostaining showed that transfection with either type of mRNA led to the expression of roughly equal levels of MITF, primarily localized in the nucleus. Despite these findings, quantitative RT-PCR analyses showed that the activation of the expression of MITF target genes was higher following transfection with modified mRNA compared with unmodified mRNA. Our findings, therefore, show that modified mRNA transfection can be applied to human embryonic stem cell-derived RPE cells and that the method is safe, efficient, and functional.

  8. Efficient delivery and functional expression of transfected modified mRNA in human embryonic stem cell-derived retinal pigmented epithelial cells.

    PubMed

    Hansson, Magnus L; Albert, Silvia; González Somermeyer, Louisa; Peco, Rubén; Mejía-Ramírez, Eva; Montserrat, Núria; Izpisua Belmonte, Juan Carlos

    2015-02-27

    Gene- and cell-based therapies are promising strategies for the treatment of degenerative retinal diseases such as age-related macular degeneration, Stargardt disease, and retinitis pigmentosa. Cellular engineering before transplantation may allow the delivery of cellular factors that can promote functional improvements, such as increased engraftment or survival of transplanted cells. A current challenge in traditional DNA-based vector transfection is to find a delivery system that is both safe and efficient, but using mRNA as an alternative to DNA can circumvent these major roadblocks. In this study, we show that both unmodified and modified mRNA can be delivered to retinal pigmented epithelial (RPE) cells with a high efficiency compared with conventional plasmid delivery systems. On the other hand, administration of unmodified mRNA induced a strong innate immune response that was almost absent when using modified mRNA. Importantly, transfection of mRNA encoding a key regulator of RPE gene expression, microphthalmia-associated transcription factor (MITF), confirmed the functionality of the delivered mRNA. Immunostaining showed that transfection with either type of mRNA led to the expression of roughly equal levels of MITF, primarily localized in the nucleus. Despite these findings, quantitative RT-PCR analyses showed that the activation of the expression of MITF target genes was higher following transfection with modified mRNA compared with unmodified mRNA. Our findings, therefore, show that modified mRNA transfection can be applied to human embryonic stem cell-derived RPE cells and that the method is safe, efficient, and functional. PMID:25555917

  9. Efficient synthesis and cell-transfection properties of a new multivalent cationic lipid for nonviral gene delivery.

    PubMed

    Ewert, Kai; Ahmad, Ayesha; Evans, Heather M; Schmidt, Hans-Werner; Safinya, Cyrus R

    2002-11-01

    Lipid-mediated delivery of DNA into cells holds great promise both for gene therapy and basic research applications. This paper describes the efficient and facile synthesis and the characterization of a new multivalent cationic lipid with a double-branched headgroup structure for gene delivery applications. The synthetic scheme can be extended to give cationic lipids of different charge, spacer, or lipid chain length. The chemical and physical properties of self-assembled complexes of the cationic liposomes (CLs) with DNA give indications of why multivalent cationic lipids possess superior transfection properties. The lipid bears a headgroup with five charges in the fully protonated state, which is attached to an unsaturated double-chain hydrophobic moiety based on 3,4-dihydroxybenzoic acid. Liposomes consisting of the new multivalent lipid and the neutral lipid 1,2-dioleoyl-sn-glycerophosphatidylcholine (DOPC) were used to prepare complexes with DNA. Investigations of the structures of these complexes by optical microscopy and small-angle X-ray scattering reveal a lamellar L(alpha)(C) phase of CL-DNA complexes with the DNA molecules sandwiched between bilayers of the lipids. Experiments using plasmid DNA containing the firefly luciferase reporter gene show that these complexes efficiently transfect mammalian cells. When compared to the monovalent cationic lipid 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP), the higher charge density of the membranes of CL-DNA complexes achievable with the new multivalent lipid greatly increases transfection efficiency in the regime of small molar ratios of cationic to neutral lipid. This is desired to minimize the known toxicity effects of cationic lipids.

  10. Tumor mRNA-transfected dendritic cells stimulate the generation of CTL that recognize neuroblastoma-associated antigens and kill tumor cells: immunotherapeutic implications.

    PubMed

    Morandi, Fabio; Chiesa, Sabrina; Bocca, Paola; Millo, Enrico; Salis, Annalisa; Solari, Massimo; Pistoia, Vito; Prigione, Ignazia

    2006-10-01

    Several observations suggest a potential role of T-cell-mediated immunity in the control of neuroblastoma (NB). However, the generation of NB-specific cytotoxic T lymphocytes (CTL) on T-cell priming with tumor mRNA-transfected dendritic cells (DC) has never been investigated before. In the present study, the feasibility of this strategy has been analyzed, both in healthy donors and in NB patients. Monocyte-derived DC were raised from three human leukocyte antigen (HLA) A2+ NB patients and seven HLA-A1+ or HLA-A2+ healthy donors transfected with mRNA from four NB cell lines and cocultured with autologous CD8+ lymphocytes. Expanded CTL expressed an effector/memory phenotype and a T cytotoxic 1-like profile of cytokine secretion. CTL specificity was demonstrated by interferon-gamma release on incubation with HLA-matched NB cell lines. The latter cell lines, but not autologous T-cell blasts, were lysed by CTL in an HLA-restricted manner. Cytotoxicity was found to involve the release of granzyme B. When tested for reactivity against NB-associated antigens, CTL from normal individuals recognized anaplastic lymphoma-associated kinase (ALK) and preferentially expressed antigen of melanoma (PRAME) peptides only, whereas patients' CTL reacted also to survivin, telomerase, and tyrosine hydroxylase peptides. This study demonstrates that DC transfected with NB mRNA induce the generation of patients' CTL specific for different NB-associated antigens, supporting the feasibility of NB T-cell immunotherapy.

  11. HIV-1 Virus-Like Particles Produced by Stably Transfected Drosophila S2 Cells: a Desirable Vaccine Component

    PubMed Central

    Yang, Lifei; Song, Yufeng; Li, Xiaomin; Huang, Xiaoxing; Liu, Jingjing; Ding, Heng; Zhu, Ping

    2012-01-01

    The development of a successful vaccine against human immunodeficiency virus type 1 (HIV-1) likely requires immunogens that elicit both broadly neutralizing antibodies against envelope spikes and T cell responses that recognize multiple viral proteins. HIV-1 virus-like particles (VLP), because they display authentic envelope spikes on the particle surface, may be developed into such immunogens. However, in one way or the other current systems for HIV-1 VLP production have many limitations. To overcome these, in the present study we developed a novel strategy to produce HIV-1 VLP using stably transfected Drosophila S2 cells. We cotransfected S2 cells with plasmids encoding HIV-1 envelope, Gag, and Rev proteins and a selection marker. After stably transfected S2 clones were established, HIV-1 VLP and their immunogenicity in mice were carefully evaluated. Here, we report that HIV-1 envelope proteins are properly cleaved, glycosylated, and incorporated into VLP with Gag. The amount of VLP released into culture supernatants is comparable to those produced by insect cells infected with recombinant baculoviruses. Moreover, cryo-electron microscopy tomography revealed average 17 spikes per purified VLP, and antigenic epitopes on the spikes were recognized by the broadly neutralizing antibodies 2G12, b12, VRC01, and 4E10 but not by PG16. Finally, mice primed with DNA and boosted with VLP in the presence of CpG exhibited anti-envelope antibody responses, including ELISA-binding, neutralizing, antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated viral inhibition, as well as envelope and Gag-specific CD8 T cell responses. Thus, we conclude that HIV-1 VLP produced by the S2 expression system has many desirable features to be developed into a vaccine component against HIV-1. PMID:22553333

  12. Non-viral transfection of goat germline stem cells by nucleofection results in production of transgenic sperm after germ cell transplantation.

    PubMed

    Zeng, W; Tang, L; Bondareva, A; Luo, J; Megee, S O; Modelski, M; Blash, S; Melican, D T; Destrempes, M M; Overton, S A; Gavin, W G; Ayres, S; Echelard, Y; Dobrinski, I

    2012-04-01

    Germline stem cells (GSCs) can be used for large animal transgenesis, in which GSCs that are genetically manipulated in vitro are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objectives of this study were to explore a non-viral approach for transgene delivery into goat GSCs and to investigate the efficiency of nucleofection in producing transgenic sperm. Four recipient goats received fractionated irradiation at 8 weeks of age to deplete endogenous GSCs. Germ cell transplantations were performed 8-9 weeks post-irradiation. Donor cells were collected from testes of 9-week-old goats, enriched for GSCs by Staput velocity sedimentation, and transfected by nucleofection with a transgene construct harboring the human growth hormone gene under the control of the goat beta-casein promoter (GBC) and a chicken beta-globin insulator (CBGI) sequence upstream of the promoter. For each recipient, transfected cells from 10 nucleofection reactions were pooled, mixed with non-transfected cells to a total of 1.5 × 10(8) cells in 3 ml, and transplanted into one testis (n = 4 recipients) by ultrasound-guided cannulation of the rete testis. The second testis of each recipient was removed. Semen was collected, starting at 9 months after transplantation, for a period of over a year (a total of 62 ejaculates from four recipients). Nested genomic PCR for hGH and CBGI sequences demonstrated that 31.3% ± 12.6% of ejaculates were positive for both hGH and CBGI. This study provides proof-of-concept that non-viral transfection (nucleofection) of primary goat germ cells followed by germ cell transplantation results in transgene transmission to sperm in recipient goats.

  13. Low molecular weight polyethylenimine grafted N-maleated chitosan for gene delivery: properties and in vitro transfection studies.

    PubMed

    Lu, Bo; Xu, Xiao-Ding; Zhang, Xian-Zheng; Cheng, Si-Xue; Zhuo, Ren-Xi

    2008-10-01

    To develop chitosan-based efficient gene vectors, chitosans with different molecular weights were chemically modified with low molecular weight polyethylenimine. The molecular weight and composition of polyethylenimine grafted N-maleated chitosan (NMC-g-PEI) copolymers were characterized using gel permeation chromatography (GPC) and (1)H NMR, respectively. Agarose gel electrophoresis assay showed that NMC-g-PEI had good binding ability with DNA, and the particle size of the NMC-g-PEI/DNA complexes was 200-400 nm, as determined by a Zeta sizer. The nanosized complexes observed by scanning electron microscopy (SEM) exhibited a compact and spherical morphology. The NMC-g-PEI copolymers showed low cytotoxicity and good transfection activity, comparable to PEI (25 KDa) in both 293T and HeLa cell lines, except for NMC 50K-g-PEI. The results indicated that the molecular weight of NMC-g-PEI has an important effect on cytotoxicity and transfection activity, and low molecular weight NMC-g-PEI has a good potential as efficient nonviral gene vectors.

  14. Humic acid inhibits HBV-induced autophagosome formation and induces apoptosis in HBV-transfected Hep G2 cells

    PubMed Central

    Pant, Kishor; Yadav, Ajay K.; Gupta, Parul; Rathore, Abhishek Singh; Nayak, Baibaswata; Venugopal, Senthil K.

    2016-01-01

    Hepatitis B Virus (HBV) utilizes several mechanisms to survive in the host cells and one of the main pathways being autophagosome formation. Humic acid (HA), one of the major components of Mineral pitch, is an Ayurvedic medicinal food, commonly used by the people of the Himalayan regions of Nepal and India for various body ailments. We hypothesized that HA could induce cell death and inhibit HBV-induced autophagy in hepatic cells. Incubation of Hep G2.2.1.5 cells (HepG2 cells stably expressing HBV) with HA (100 μM) inhibited both cell proliferation and autophagosome formation significantly, while apoptosis induction was enhanced. Western blot results showed that HA incubation resulted in decreased levels of beclin-1, SIRT-1 and c-myc, while caspase-3 and β-catenin expression were up-regulated. Western blot results showed that HA significantly inhibited the expression of HBx (3-fold with 50 μM and 5-fold with 100 μM) compared to control cells. When HA was incubated with HBx-transfected Hep G2 cells, HBx-induced autophagosome formation and beclin-1 levels were decreased. These data showed that HA induced apoptosis and inhibited HBV-induced autophagosome formation and proliferation in hepatoma cells. PMID:27708347

  15. Intracellular Availability of pDNA and mRNA after Transfection: A Comparative Study among Polyplexes, Lipoplexes, and Lipopolyplexes.

    PubMed

    Gonçalves, Cristine; Akhter, Sohail; Pichon, Chantal; Midoux, Patrick

    2016-09-01

    Intracellular availability of nucleic acids from synthetic vectors is critical and directly influences the transfection efficiency (TE). Herein, we evaluated the TE of polymer- and lipid-based nanoplexes (polyplexes, lipoplexes and lipopolyplexes) of EGFP-encoding mRNA and pDNA. To determine the translation and transcription efficiency of each nucleic acid nanoplex, in vitro expression was measured in HEK293T7 cells that permit gene expression in the cytoplasmic region. Globally, mRNA transfection profile was well corroborative with cytoplasmic transfection of pT7-pDNA as well as with nuclear transfection of pCMV-DNA. Irrespective of the nucleic acid, high TE was observed with histidinylated l-polyethylenimine (His-lPEI) polyplexes and dioleyl succinyl paromomycin/O,O-dioleyl-N-histamine phosphoramidate (DOPS/MM27) lipoplexes. Moreover, His-lPEI polyplexes yielded higher in vitro expression of EGFP for pDNA than for mRNA. Furthermore, a significant enhancement in the TE in the presence of an excess of His-lPEI was observed indicating that this polymer promotes cytosolic delivery. Compared to other nanoplexes, His-lPEI polyplex showed high intracellular availability of DNA and mRNA along with low cytotoxicity, owing to its rapid (complete or partial) unpacking in the cytosol and/or endosomes. This study gives an insight that, whether with mRNA or pDNA, enhancing nanoplex unpacking in the endosomes and cytosol would improve the delivery of nucleic acid in the cytosol and particularly in the case of pDNA where a sufficient available amount of pDNA in the cytoplasm would definitely improve its transport toward the nucleus. PMID:27486998

  16. Transfection mechanisms of polyplexes, lipoplexes, and stealth liposomes in α₅β₁ integrin bearing DLD-1 colorectal cancer cells.

    PubMed

    Adil, Maroof M; Erdman, Zachary S; Kokkoli, Efrosini

    2014-04-01

    Receptor targeted, PEGylated transfection agents can improve stability and delivery specificity of current cationic lipid and polymer based nonviral gene delivery vehicles, but their mode of transfection is poorly understood. We therefore investigated the transfection mechanisms of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipoplexes, branched polyethylenimine (bPEI) polyplexes, and bPEI encapsulated in either PEGylated (stealth) nontargeted liposomes or PR_b peptide (targeted to α5β1 integrin) functionalized stealth liposomes in DLD-1 colorectal cancer cells in vitro with gene expression assays, flow cytometry and confocal microscopy. DOTAP/DOPE and PR_b functionalized stealth liposomes mediated higher gene expression compared to nontargeted stealth liposomes and bPEI. However DOTAP/DOPE was internalized slowly leading to lower levels of DNA uptake. In contrast, despite high internalization of bPEI polyplexes, gene expression levels were low as DNA was unable to escape from the endosomes. Nontargeted stealth liposomes also mediated low gene expression due to low amounts of DNA internalized and slow internalization kinetics. PR_b functionalized stealth liposomes struck an optimal balance among these transfection agents with efficient transfection arising from fast integrin mediated internalization kinetics, high amounts of DNA uptake, and endosomal escape. We found α5β1 integrin to be a valuable target for gene delivery and that the caveolar endocytic pathway may offer an advantage to receptor targeted PEGylated transfection agents in DLD-1 cells.

  17. Sodium-iodide symporter (NIS)-mediated accumulation of [(211)At]astatide in NIS-transfected human cancer cells.

    PubMed

    Carlin, Sean; Mairs, Robert J; Welsh, Phil; Zalutsky, Michael R

    2002-10-01

    The cellular expression of the sodium iodide symporter (NIS) has been shown to confer iodide-concentrating capacity in non-thyroid cell types. We examined the role of NIS in the uptake of the alpha-particle emitting radiohalide [(211)At]astatide in the UVW human glioma cell line transfected to express NIS. [(211)At]Astatide uptake is shown to be NIS-dependent, with characteristics similar to [(131)I]iodide uptake. These studies suggest [(211)At]astatide as a possible alternative radionuclide to [(131)I]iodide for NIS-based endoradiotherapy, and provide a model for the study of [(211)At]astatide behavior at a cellular level. PMID:12381453

  18. Structural characterization of a new lipid/DNA complex showing a selective transfection efficiency in ovarian cancer cells

    NASA Astrophysics Data System (ADS)

    Caracciolo, G.; Pozzi, D.; Caminiti, R.; Congiu Castellano, A.

    2003-04-01

    We investigated, for the first time, by using Energy Dispersive X-ray Diffraction, the structure of a new ternary cationic liposome formulated with dioleoyl trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE) and cholesterol (Chol) (DDC) which has been recently found to have a selective high gene transfer ability in ovarian cancer cells. Our structural results provide a further experimental support to the widely accepted statement that there is not a simple and direct correlation between structure and transfection efficiency and that the factors controlling cationic lipid/DNA (CL-DNA) complexes-mediated gene transfer depend not only on the formulations of the cationic liposomes and their thermodynamic phase, but also significantly on the cell properties.

  19. Testosterone modulates mitochondrial aconitase in the full-length human androgen receptor-transfected PC-3 prostatic carcinoma cells.

    PubMed

    Juang, H-H; Hsieh, M-L; Tsui, K-H

    2004-08-01

    In vitro studies indicated that dihydrotestosterone (DHT) stimulates the enzymatic activity of the mitochondrial aconitase (mACON) in androgen-sensitive prostatic carcinoma cells, LNCaP. Cell proliferation assay determined that DHT doubles the optimal proliferation response of LNCaP cells. The androgen-insensitive human prostatic carcinoma cells, PC-3, were overexpressed in the human androgen receptor to assess the involvement of the native androgen receptor in the regulation by DHT of mACON gene expression. A stable-transfected clone that expresses the full-length androgen receptor was selected and termed PCAR9. The results revealed that DHT-treated PCAR9 cells paradoxically not only reduced the enzymatic activity of mACON but also blocked the biosynthesis of intracellular ATP attenuating cell proliferation. Transient gene expression assay indicated that DHT divergently regulates the promoter activity of the mACON gene in LNCaP and PCAR9 cells. This study suggested that DHT regulates mACON gene expression and the proliferation of cells in a receptor-dependent model through modulation by unidentified non-receptor factors. PMID:15291747

  20. Transfection of shRNA-encoding Minivector DNA of a few hundred base pairs to regulate gene expression in lymphoma cells

    PubMed Central

    Zhao, N; Fogg, J M; Zechiedrich, L; Zu, Y

    2011-01-01

    This work illustrates the utility of Minivector DNA, a non-viral, supercoiled gene therapy vector incorporating short hairpin RNA from an H1 promoter. Minivector DNA is superior to both plasmid DNA and small interfering RNA (siRNA) in that it has improved biostability while maintaining high cell transfection efficiency and gene silencing capacity. Minivector DNAs were stable for over 48 h in human serum, as compared with only 0.5 and 2 h for siRNA and plasmid, respectively. Although all three nucleic acids exhibited similar transfection efficiencies in easily transfected adhesion fibroblasts cells, only Minivector DNAs and siRNA were capable of transfecting difficult-to-transfect suspension lymphoma cells. Minivector DNA and siRNA were capable of silencing the gene encoding anaplastic lymphoma kinase, a key pathogenic factor of human anaplastic large cell lymphoma, and this silencing caused inhibition of the lymphoma cells. Based on these results, Minivector DNAs are a promising new gene therapy tool. PMID:20962872

  1. Analysis of arsenic metabolites in HepG2 and AS3MT-transfected cells.

    PubMed

    Watanabe, Takayuki; Ohta, Yuki; Mizumura, Ayano; Kobayashi, Yayoi; Hirano, Seishiro

    2011-06-01

    It has been suggested that arsenic (+3 oxidation state) methyltransferase (AS3MT) plays a critical role in methylation of arsenic, and that arsenic-glutathione conjugate is a substrate for AS3MT-catalyzed methylation of arsenic. However, the mechanism of arsenic methylation in cells is not fully understood. Here, we have constructed T-REx-CHO-hAS3MTtr cells that transiently overexpress human AS3MT in response to tetracycline. The decreases in cell viability after exposure to sodium arsenite were greater in tetracycline-treated cells (tet(+) cells) than in untreated cells (tet(-) cells). Concentration of total cellular arsenic was significantly higher in tet(+) cells than in tet(-) cells. Speciation analyses of arsenic metabolites in whole cell lysates and cell culture medium were performed using both HepG2 cells and T-REx-CHO-hAS3MTtr cells. Speciation analyses of arsenic metabolites in lysates of T-REx-CHO-hAS3MTtr cells revealed that dimethylated arsenicals were the predominant arsenic metabolites in tet(+) cells, while methylated metabolites were not found in tet(-) cells. In contrast, less amount of methylated arsenic metabolites were found in the HepG2 cell lysates, and monomethylated trivalent arsenicals were the predominant methylated arsenic metabolites. Arsenate was found in the culture medium after 24 h culture with arsenite. A larger amount of arsenate was found in the culture medium of tet(+) or tet(-) cells compared to HepG2 cells. These findings indicated that AS3MT expression enhanced the cytotoxic effect of arsenite in tet(+) cells because these cells accumulated more arsenic metabolites than did the tet(-) cells, and accordingly, the tet(+) cells were more susceptible to arsenic than were the tet(-) cells. Oxidation--reduction of arsenic may be implicated in the toxic effects of arsenite.

  2. Increased cytotoxicity and bystander effect of 5-fluorouracil and 5′-deoxy-5-fluorouridine in human colorectal cancer cells transfected with thymidine phosphorylase

    PubMed Central

    Evrard, A; Cuq, P; Ciccolini, J; Vian, L; Cano, J-P

    1999-01-01

    5-Fluorouracil (5-FU) and 5′-deoxy-5-fluorouridine (5′-DFUR), a prodrug of 5-FU, are anticancer agents activated by thymidine phosphorylase (TP). Transfecting the human TP cDNA into cancer cells in order to sensitize them to these pyrimidine antimetabolites may be an important approach in human cancer gene therapy research. In this study, an expression vector containing the human TP cDNA (pcTP5) was transfected into LS174T human colon carcinoma cells. Eight stable transfectants were randomly selected and analysed. The cytotoxic effects of 5-FU and 5′-DFUR were higher in TP-transfected cells as compared to wild-type cells. The maximal decreases in the IC50 were 80-fold for 5-FU and 40-fold for 5′-DFUR. The increase in sensitivity to these pyrimidines of TP-transfected cells significantly correlated with the increase in both TP activity and TP expression. Transfected clone LS174T-c2 but not wild-type cells exhibited formation of [3H]FdUMP from [3H]5-FU. In addition the LS174T-c2 clone enhanced the cytotoxic effect of 5′-DFUR, but also that of 5-FU, towards co-cultured parental cells. For both anti-cancer agents, this bystander effect did not require cell–cell contact. These results show that both 5-FU or 5′-DFUR could be used together with a TP-suicide vector in cancer gene therapy. © 1999 Cancer Research Campaign PMID:10468288

  3. Amino acid sequence and posttranslational modifications of human factor VII sub a from plasma and transfected baby hamster kidney cells

    SciTech Connect

    Thim, L.; Bjoern, S.; Christensen, M.; Nicolaisen, E.M.; Lund-Hansen, T.; Pedersen, A.H.; Hedner, U. )

    1988-10-04

    Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VII{sub a}, participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca{sup 2+} and tissue factor. Three types of potential posttranslational modifications exist in the human factor VII{sub a} molecule, namely, 10 {gamma}-carboxylated, N-terminally located glutamic acid residues, 1 {beta}-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VII{sub a} as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VII{sub a}. By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradation, the protein backbone of recombinant factor VII{sub a} was found to be identical with human factor VII{sub a}. Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VII{sub a}. In the recombinant factor VII{sub a}, asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VII{sub a} and human plasma factor VII{sub a}. These results show that factor VII{sub a} as produced in the transfected baby hamster kidney cells is very similar to human plasma factor VII{sub a} and that this cell line thus might represent an alternative source for human factor VII{sub a}.

  4. Changes in the phenotype of human small cell lung cancer cell lines after transfection and expression of the c-myc proto-oncogene.

    PubMed Central

    Johnson, B E; Battey, J; Linnoila, I; Becker, K L; Makuch, R W; Snider, R H; Carney, D N; Minna, J D

    1986-01-01

    Small cell lung cancer growing in cell culture possesses biologic properties that allow classification into two categories: classic and variant. Compared with classic small cell lung cancer cell lines, variant lines have altered large cell morphology, shorter doubling times, higher cloning efficiencies in soft agarose, and very low levels of L dopa decarboxylase production and bombesin-like immunoreactivity. C-myc is amplified and expressed in some small cell lung cancer cell lines and all c-myc amplified lines studied to date display the variant phenotype. To investigate if c-myc amplification and expression is responsible for the variant phenotype, a normal human c-myc gene was transfected into a cloned classic small cell lung cancer cell line not amplified for or expressing detectable c-myc messenger RNA (mRNA). Clones were isolated with one to six copies of c-myc stably integrated into DNA that expressed c-myc mRNA. In addition, one clone with an integrated neo gene but a deleted c-myc gene was isolated and in this case c-myc was not expressed. C-myc expression in transfected clones was associated with altered large cell morphology, a shorter doubling time, and increased cloning efficiency, but no difference in L dopa decarboxylase levels and bombesin-like immunoreactivity. We conclude increased c-myc expression observed here in transfected clones correlates with some of the phenotypic properties distinguishing c-myc amplified variants from unamplified classic small cell lung cancer lines. Images PMID:3016030

  5. Evaluation of FTA(®) card for the rescue of infectious foot-and-mouth disease virus by chemical transfection of extracted RNA in cultured cells.

    PubMed

    Biswal, Jitendra K; Subramaniam, Saravanan; Ranjan, Rajeev; Pattnaik, Bramhadev

    2016-08-01

    Foot-and-mouth disease (FMD) is a highly contagious epidemic disease of transboundary importance. Inadequate storage and shipment of suspected clinical samples can compromise the ability to detect and characterise FMD virus (FMDV) in endemic countries, thereby, leading to the loss of valuable virological and epidemiological data. This study, investigates the potential of using FTA(®) cards for dry transportation of clinical samples and subsequent recovery of infectious FMDV by chemical transfection of FTA(®) card fixed RNA as an alternative to the conventional cell culture based virus isolation method. A higher proportion of infectious FMDV was rescued from clinical samples (cell culture isolates, tongue epithelial suspension and impression smears) by the FTA(®) card fixed RNA transfection method (76%) compared to the conventional cell culture based virus isolation (56%), suggesting a better performance of the current RNA transfection procedure. Furthermore, it was possible to rescue live virus by the transfection of RNA extracted from FTA(®) card impregnated with clinical samples that had been stored at varying temperature (4-37 °C) up to a period of six weeks. The VP1 sequence data and antigenic relationships with the vaccine strains, between viruses rescued by FTA(®) card fixed RNA transfection and conventional cell culture, were comparable. Therefore, these results support the use of the FTA(®) card for the economic, dry, non-hazardous transport of FMD suspected clinical samples from the site of collection to national/international reference laboratories. PMID:27321701

  6. Evaluation of FTA(®) card for the rescue of infectious foot-and-mouth disease virus by chemical transfection of extracted RNA in cultured cells.

    PubMed

    Biswal, Jitendra K; Subramaniam, Saravanan; Ranjan, Rajeev; Pattnaik, Bramhadev

    2016-08-01

    Foot-and-mouth disease (FMD) is a highly contagious epidemic disease of transboundary importance. Inadequate storage and shipment of suspected clinical samples can compromise the ability to detect and characterise FMD virus (FMDV) in endemic countries, thereby, leading to the loss of valuable virological and epidemiological data. This study, investigates the potential of using FTA(®) cards for dry transportation of clinical samples and subsequent recovery of infectious FMDV by chemical transfection of FTA(®) card fixed RNA as an alternative to the conventional cell culture based virus isolation method. A higher proportion of infectious FMDV was rescued from clinical samples (cell culture isolates, tongue epithelial suspension and impression smears) by the FTA(®) card fixed RNA transfection method (76%) compared to the conventional cell culture based virus isolation (56%), suggesting a better performance of the current RNA transfection procedure. Furthermore, it was possible to rescue live virus by the transfection of RNA extracted from FTA(®) card impregnated with clinical samples that had been stored at varying temperature (4-37 °C) up to a period of six weeks. The VP1 sequence data and antigenic relationships with the vaccine strains, between viruses rescued by FTA(®) card fixed RNA transfection and conventional cell culture, were comparable. Therefore, these results support the use of the FTA(®) card for the economic, dry, non-hazardous transport of FMD suspected clinical samples from the site of collection to national/international reference laboratories.

  7. Biologic effect and immunoisolating behavior of BMP-2 gene-transfected bone marrow-derived mesenchymal stem cells in APA microcapsules.

    PubMed

    Ding, H F; Liu, R; Li, B G; Lou, J R; Dai, K R; Tang, T T

    2007-11-01

    We investigated the encapsulation of BMP-2 gene-modified mesenchymal stem cells (MSCs) in alginate-poly-L-lysine (APA) microcapsules for the persistent delivery of bone morphogenic protein-2 (BMP-2) to induce bone formation. An electrostatic droplet generator was employed to produce APA microcapsules containing encapsulated beta-gal or BMP-2 gene-transfected bone marrow-derived MSCs. We found that X-gal staining was still positive 28 days after encapsulation. Encapsulated BMP-2 gene-transfected cells were capable of constitutive delivery of BMP-2 proteins for at least 30 days. The encapsulated BMP-2 gene-transfected MSCs or the encapsulated non-gene transfer MSCs (control group) were cocultured with the undifferentiated MSCs. The gene products from the encapsulated BMP-2 cells could induce the undifferentiated MSCs to become osteoblasts that had higher alkaline phosphatase (ALP) activity than those in the control group (p<0.05). The APA microcapsules could inhibit the permeation of fluorescein isothiocyanate-conjuncted immunoglobulin G. Mixed lymphocyte reaction also indicates that the APA microcapsules could prevent the encapsulated BMP-2 gene-transfected MSCs from initiating the cellular immune response. These results demonstrated that the nonautologous BMP-2 gene-transfected stem cells are of potential utility for enhancement of bone repair and bone regeneration in vivo.

  8. Chitosan-gelatin scaffolds for tissue engineering: physico-chemical properties and biological response of buffalo embryonic stem cells and transfectant of GFP-buffalo embryonic stem cells.

    PubMed

    Thein-Han, W W; Saikhun, J; Pholpramoo, C; Misra, R D K; Kitiyanant, Y

    2009-11-01

    The favorable cellular response of newly developed cell line, buffalo embryonic stem (ES) cells to three-dimensional biodegradable chitosan-gelatin composite scaffolds with regard to stem-cell-based tissue engineering is described. Chitosan-gelatin composites were characterized by a highly porous structure with interconnected pores, and the mechanical properties were significantly enhanced. Furthermore, X-ray diffraction study indicated increased amorphous content in the scaffold on the addition of gelatin to chitosan. To develop a transfectant of green fluorescence protein (GFP)-buffalo ES cell, transfection of GFP plasmid to the cell was carried out via the electroporation procedure. In comparison with pure chitosan, cell spreading and proliferation were greater in highly visualized GFP-expressing cell-chitosan-gelatin scaffold constructs. The relative comparison of biological response involving cell proliferation and viability on the scaffolds suggests that blending of gelatin in chitosan improved cellular efficiency. Studies involving scanning electron and fluorescence microscopy, histological observations and flow cytometer analysis of the constructs implied that the polygonal cells attached to and penetrated the pores, and proliferated well, while maintaining their pluripotency during the culture period for 28days. Chitosan-gelatin scaffolds were cytocompatible with respect to buffalo ES cells. The study underscores for the first time that chitosan-gelatin scaffolds are promising candidates for ES-cell-based tissue engineering.

  9. Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity.

    PubMed

    Omokoko, Tana A; Luxemburger, Uli; Bardissi, Shaheer; Simon, Petra; Utsch, Magdalena; Breitkreuz, Andrea; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard (51)Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the (51)Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies. PMID:27057556

  10. Use of CYP2E1-transfected human liver cell lines in elucidating the actions of ethanol.

    PubMed

    Lakshman, Raj; Cederbaum, Arthur I; Hoek, Jan B; Konishi, Masahiro; Koop, Dennis; Donohu, Terrence M

    2005-09-01

    This article represents the proceedings of a symposium at the 2004 RSA Meeting held in Vancouver, Canada. The chairs were Arthur I. Cederbaum and Raj Lakshman. The presentations were (1) ethanol regulates 2,6-sialyltransferase (2,6-ST) gene expression posttranscriptionally by the interaction of a cytosolic binding protein with 2,6-ST mRNA in CYP2E1- and ADH-transfected HepG2 cells, by Raj Lakshman; (2) nature versus nurture: HepG2-E47 cells as a tool to investigate mechanisms of ethanol-mediated potentiation of cell killing, by Jan B. Hoek; (3) ethanol up-regulates profibrogenic connective tissue growth factor gene expression in HepG2 cells via cytochrome P-450 2E1-mediated ethanol oxidation, by Masahiro Konishi; (4) role of calcium and calcium-activated enzymes in CYP2E1-dependent toxicity, by Arthur I Cederbaum; (5) the use of cell lines to characterize the role of CYP2E1 in the metabolism of farnesol, by Dennis Koop; and (6) studies with HepG2 cells that express the two major ethanol-metabolizing enzymes, by Terrence M. Donohue. PMID:16205373

  11. Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity

    PubMed Central

    Omokoko, Tana A.; Luxemburger, Uli; Bardissi, Shaheer; Simon, Petra; Utsch, Magdalena; Breitkreuz, Andrea; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard 51Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the 51Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies. PMID:27057556

  12. The efficiency of membrane transport of vitamin B6 coupled to poly(ester amine) gene transporter and transfection in cancer cells.

    PubMed

    Pandey, Shambhavi; Garg, Pankaj; Lim, Ki Taek; Kim, Jangho; Choung, Yun-Hoon; Choi, Yun-Jaie; Choung, Pill-Hoon; Cho, Chong-Su; Chung, Jong Hoon

    2013-05-01

    Vitamin B6 (VB6) plays an essential role as a coenzyme in various cellular metabolic functions, including DNA biosynthesis for cellular growth and proliferation. VB6 is taken up by cells through facilitated diffusion via VB6 transporting membrane carrier (VTC). In this study, we demonstrated that the VB6-coupled poly(ester amine) (VBPEA) gene transporter utilizes this uptake mechanism, leading to enhanced vector transport inside the rapidly proliferating cancer cells with relatively high affinity. Physicochemical characterization, cell viability assays, and transfection studies showed VBPEA to meet the standards of a good transfection agent. Competitive inhibition of VBPEA uptake by its structural analog 4'-deoxypyridoxine hydrochloride revealed the involvement of VB6 specific transporting membrane carrier in VBPEA internalization in tumor cells. VBPEA elicit higher transfection levels in lung cancer cells than in normal lung cells, indicating that cancer cells which have a high demand for VB6, have a higher affinity for VB6-coupled vector. VB6 coupling to the gene transporter is important to enforce a high level of VTC-mediated endocytosis compared to VB6 alone. This system illustrated how understanding of the VB6 membrane transporter specificity allowed for the design of a VB6-coupled gene transporter with accelerated transfection activity in cancer cells owing to an advanced mode of internalization. PMID:23425622

  13. The role of helper lipids in the intracellular disposition and transfection efficiency of niosome formulations for gene delivery to retinal pigment epithelial cells.

    PubMed

    Ojeda, Edilberto; Puras, Gustavo; Agirre, Mireia; Zarate, Jon; Grijalvo, Santiago; Eritja, Ramon; DiGiacomo, Luca; Caracciolo, Giulio; Pedraz, Jose-Luis

    2016-04-30

    In this work, we carried out a comparative study of four different niosome formulations based on the same cationic lipid and non-ionic tensoactive. The niosomes prepared by oil-in-water emulsion technique (o/w) only differed in the helper lipid composition: squalene, cholesterol, squalane or no helper lipid. Niosomes and nioplexes elaborated upon the addition of pCMS-EGFP reporter plasmid were characterized in terms of size, zeta potential and polydispersity index. The capacity of the niosomes to condense, release and protect the DNA against enzymatic degradation was evaluated by agarose gel electrophoresis. In vitro experiments were carried out to evaluate transfection efficiency and cell viability in retinal pigment epithelial cells. Moreover, uptake and intracellular trafficking studies were performed to further understand the role of the helper lipids in the transfection process. Interestingly, among all tested formulations, niosomes elaborated with squalene as helper lipid were the most efficient transfecting cells. Such transfection efficiency could be attributed to their higher cellular uptake and the particular entry pathways used, where macropinocytosis pathway and lysosomal release played an important role. Therefore, these results suggest that helper lipid composition is a crucial step to be considered in the design of niosome formulation for retinal gene delivery applications since clearly modulates the cellular uptake, internalization mechanism and consequently, the final transfection efficiency.

  14. Extracellular matrix and hormones transcriptionally regulate bovine. beta. -casein 5 prime sequences in stably transfected mouse mammary cells

    SciTech Connect

    Schmidhauser, C. Bissell, M.J. ); Myers, C.A.; Casperson, G.F. )

    1990-12-01

    Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-2D has been developed in which more than 35% of the cells express {beta}-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete {beta}-casein undirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine {beta}-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency. This regulation occurered primarily at the transcriptional level and was dependent on the length of the 5{prime} flanking region of the {beta}-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous {beta}-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.

  15. Single Cell Analysis to locate the Restriction Point with respect to E2F Expression

    NASA Astrophysics Data System (ADS)

    Pimienta, R.; Johnson, A.

    2011-12-01

    The restriction point is a G1-phase checkpoint that regulates passage through the cell cycle and is misregulated in all known types of cancer. The Rb-E2F switch is thought to be one of the most relevant molecular mechanisms which regulate the restriction point in mammalian cells. However, recent experiments have brought the timing of the restriction point into question. In previous studies, cells were analyzed as populations and this prevented an accurate determination of the restriction point. By creating and analyzing an E2F-GFP reporter in single cells, we can pinpoint the timing of E2F activation and determine whether it coincides with the restriction point. Using calcium phosphate and Fugene,we transfected human embryonic kidney (293T) cells with a CMV-GFP plasmid and an E2F-GFP reporter. Based on our results, it appears that calcium phosphate is more effective than Fugene at transfecting mammalian cells. The calcium phosphate transfection had 9.59% more fluorescent cells than Fugene. However, this result only occurred with the CMV-GFP plasmid and not the E2F-GFP reporter, which was not properly expressed in human embryonic kidney (293T) cells. We will continue troubleshooting to fix this reporter as we proceed with our research. Once the reporter is properly cloned, we will transfect it into retinal pigmented epithelial (RPE1-hTERT) cells using the calcium phosphate method. RPE1-hTERT cells are an immortalized with telomerase and are more close to normal cells than tumor-derived cell lines. Through this research we will better comprehend commitment to the mammalian cell cycle.

  16. Utility of large-scale transiently transfected cells for cell-based high-throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists.

    PubMed

    Chen, Jun; Lake, Marc R; Sabet, Reza S; Niforatos, Wende; Pratt, Steve D; Cassar, Steven C; Xu, Jing; Gopalakrishnan, Sujatha; Pereda-Lopez, Ana; Gopalakrishnan, Murali; Holzman, Thomas F; Moreland, Robert B; Walter, Karl A; Faltynek, Connie R; Warrior, Usha; Scott, Victoria E

    2007-02-01

    Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources. Moreover, significant variability invariably occurs in cell yield, viability, expression levels, and target activities. In particular, stable expression of targets such as transient receptor potential A1 (TRPA1) causes toxicity, cell line degeneration, and loss of functional activity. Therefore, in an effort to identify TRPA1 antagonists, the authors used large-scale transiently transfected (LSTT) cells, enabling rapid establishment of assays suitable for HTS. LSTT cells, which could- be stored frozen for a long period of time (e.g., at least 42 weeks), retained TRPA1 protein expression and could be easily revived to produce robust and consistent signals in calcium influx and electrophysiological assays. Using cells from a single transfection, a chemical library of 700,000 compounds was screened, and TRPA1 antagonists were identified. The use of LSTT circumvented issues associated with stable TRPA1 expression, increased flexibility and consistency, and greatly reduced labor and cost. This approach will also be applicable to other pharmaceutical targets.

  17. Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection.

    PubMed

    Van Pham, Phuc; Thi-My Nguyen, Phuoc; Thai-Quynh Nguyen, Anh; Minh Pham, Vuong; Nguyen-Tu Bui, Anh; Thi-Tung Dang, Loan; Gia Nguyen, Khue; Kim Phan, Ngoc

    2014-06-01

    Numerous studies have sought to identify diabetes mellitus treatment strategies with fewer side effects. Mesenchymal stem cell (MSC) therapy was previously considered as a promising therapy; however, it requires the cells to be trans-differentiated into cells of the pancreatic-endocrine lineage before transplantation. Previous studies have shown that PDX-1 expression can facilitate MSC differentiation into insulin-producing cells (IPCs), but the methods employed to date use viral or DNA-based tools to express PDX-1, with the associated risks of insertional mutation and immunogenicity. Thus, this study aimed to establish a new method to induce PDX-1 expression in MSCs by mRNA transfection. MSCs were isolated from human umbilical cord blood and expanded in vitro, with stemness confirmed by surface markers and multipotentiality. MSCs were transfected with PDX-1 mRNA by nucleofection and chemically induced to differentiate into IPCs (combinatorial group). This IPC differentiation was then compared with that of untransfected chemically induced cells (inducer group) and uninduced cells (control group). We found that PDX-1 mRNA transfection significantly improved the differentiation of MSCs into IPCs, with 8.3±2.5% IPCs in the combinatorial group, 3.21±2.11% in the inducer group and 0% in the control. Cells in the combinatorial group also strongly expressed several genes related to beta cells (Pdx-1, Ngn3, Nkx6.1 and insulin) and could produce C-peptide in the cytoplasm and insulin in the supernatant, which was dependent on the extracellular glucose concentration. These results indicate that PDX-1 mRNA may offer a promising approach to produce safe IPCs for clinical diabetes mellitus treatment.

  18. Construction and characterization of a human T-cell lymphotropic virus type 3 infectious molecular clone.

    PubMed

    Chevalier, Sébastien Alain; Ko, Nga Ling; Calattini, Sara; Mallet, Adeline; Prévost, Marie-Christine; Kehn, Kylene; Brady, John N; Kashanchi, Fatah; Gessain, Antoine; Mahieux, Renaud

    2008-07-01

    We and others have uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3). We have now generated an HTLV-3 proviral clone. We established that gag, env, pol, pro, and tax/rex as well as minus-strand mRNAs are present in cells transfected with the HTLV-3 clone. HTLV-3 p24(gag) protein is detected in the cell culture supernatant. Transfection of 293T-long terminal repeat (LTR)-green fluorescent protein (GFP) cells with the HTLV-3 clone promotes formation of syncytia, a hallmark of Env expression, together with the appearance of fluorescent cells, demonstrating that Tax is expressed. Viral particles are visible by electron microscopy. These particles are infectious, as demonstrated by infection experiments with purified virions.

  19. Expression of transfected vimentin genes in differentiating murine erythroleukemia cells reveals divergent cis-acting regulation of avian and mammalian vimentin sequences.

    PubMed Central

    Ngai, J; Bond, V C; Wold, B J; Lazarides, E

    1987-01-01

    We studied the expression of transfected chicken and hamster vimentin genes in murine erythroleukemia (MEL) cells. MEL cells normally repress the levels of endogenous mouse vimentin mRNA during inducermediated differentiation, resulting in a subsequent loss of vimentin filaments. Expression of vimentin in differentiating MEL cells reflects the disappearance of vimentin filaments during mammalian erythropoiesis in vivo. In contrast, chicken erythroid cells express high levels of vimentin mRNA and vimentin filaments during terminal differentiation. We demonstrate here that chicken vimentin mRNA levels increase significantly in differentiating transfected MEL cells, whereas similarly transfected hamster vimentin genes are negatively regulated. In conjunction with in vitro nuclear run-on transcription experiments, these results suggest that the difference in vimentin expression in avian and mammalian erythropoiesis is due to a divergence of cis-linked vimentin sequences that are responsible for transcriptional and posttranscriptional regulation of vimentin gene expression. Transfected chicken vimentin genes produce functional vimentin protein and stable vimentin filaments during MEL cell differentiation, further demonstrating that the accumulation of vimentin filaments is determined by the abundance of newly synthesized vimentin. Images PMID:3481037

  20. Role of calpain-9 and PKC-delta in the apoptotic mechanism of lumen formation in CEACAM1 transfected breast epithelial cells.

    PubMed

    Chen, Charng-Jui; Nguyen, Tung; Shively, John E

    2010-02-15

    CEACAM1-4S (carcinoembryonic antigen-related cell adhesion molecule 1) is a type I membrane protein with a short (12-amino acid) cytoplasmic tail. Wild type CEACAM1-4S-transfected MCF7 cells form glands with lumena when grown in 3D culture, while null mutations of two putative phosphorylation sites (T457A and S459A) in the cytoplasmic domain fail to undergo lumen formation. When gene chip analysis was performed on mRNA isolated from both wild type and T457A,S459A mutated CEACAM1-4S-transfected MCF7 cells grown in 3D culture, calpain-9 (CAPN9) was identified out of over 400 genes with a >2 log 2 difference as a potential inducer of lumen formation. Inhibition of CAPN9 expression in MCF7/CEACAM1-4S cells by RNAi or by calpeptin or PD150606 inhibited lumen formation. Transfection of CAPN9 into wild type MCF7 cells restores lumen formation demonstrating that calpain-9 may play a critical role in lumen formation. Additionally, we demonstrate that the apoptosis related kinase, PKC-delta, is activated by proteolytic cleavage during lumen formation exclusively in wild type CEACAM1-4S-transfected MCF7 cells grown in 3D culture and that lumen formation is inhibited by either RNAi to PKC-delta or by the PKC-delta inhibitor rottlerin.

  1. Transfected human neuropeptide Y cDNA expression in mouse pituitary cells. Inducible high expression, peptide characterization, and secretion.

    PubMed

    Dickerson, I M; Dixon, J E; Mains, R E

    1987-10-01

    An expression vector was constructed that placed the cDNA for human neuropeptide Y (NPY) under the control of the mouse metallothionein promoter and was used to transfect the AtT-20 mouse anterior pituitary corticotrope cell line. AtT-20 cells normally process the pro-ACTH/endorphin precursor but do not produce detectable levels of NPY. The resulting AtT-20/NPY cell line (Mt.NPY1a) was used to study the ability of the corticotrope cells to synthesize, process, and secrete the foreign proNPY-related peptide products. The stable cell line created contains approximately 40 copies of proNPY cDNA per cell. NPY mRNA levels and proNPY synthesis were increased at least 35-fold when maximally induced with cadmium; proNPY synthesis was also induced by glucocorticoids. Upon induction the NPY secretion rate was equimolar to that of the endogenous peptides. ProNPY, NPY, and the COOH-terminal peptide produced by this cell line had molecular weight and amino acid-labeling pattern predicted from cDNA sequence data and from previous isolation of NPY-related molecules from NPY-producing cells. The structures of secreted proNPY, NPY, and COOH-terminal peptide, as well as determination of the site of proteolytic cleavage between NPY and the COOH-terminal peptide, were determined by tryptic mapping and Edman degradation of secreted biosynthetically labeled peptide products. The proNPY molecule appears to be processed in the same pathway responsible for cleavage of the endogenous pro-ACTH/endorphin precursor. Secretion of proNPY-derived peptides paralleled secretion of endogenous pro-ACTH/endorphin-derived products, under both basal and stimulated conditions. With induction proNPY expression there is a dose-dependent inhibition of both proNPY and pro-ACTH/endorphin proteolytic processing.

  2. Aquaporin-4 antibody testing: direct comparison of M1-AQP4-DNA-transfected cells with leaky scanning versus M23-AQP4-DNA-transfected cells as antigenic substrate

    PubMed Central

    2014-01-01

    Background Neuromyelitis optica (NMO, Devic syndrome) is associated with antibodies to aquaporin-4 (NMO-IgG/AQP4-Ab) in the majority of cases. NMO-IgG/AQP4-Ab seropositivity in patients with NMO and its spectrum disorders has important differential diagnostic, prognostic and therapeutic implications. So-called cell-based assays (CBA) are thought to provide the best AQP4-Ab detection rates. Objective To compare directly the AQP4-IgG detection rates of the currently most widely used commercial CBA, which employs cells transfected with a full-length (M1)-human AQP4 DNA in a fashion that allows leaky scanning (LS) and thus expression of M23-AQP4 in addition to M1-AQP, to that of a newly developed CBA from the same manufacturer employing cells transfected with human M23-AQP4-DNA. Methods Results from 368 serum samples that had been referred for routine AQP4-IgG determination and had been tested in parallel in the two assays were compared. Results Seventy-seven out of 368 samples (20.9%) were positive for NMO-IgG/AQP4-Ab in at least one assay. Of these, 73 (94.8%) were positive in both assays. A single sample (1.3%) was exclusively positive in the novel assay; three samples (3.9%) were unequivocally positive only in the ‘classic’ assay due to high background intensity in the novel assay. Both median fluorescence intensity and background intensity were higher in the new assay. Conclusions This large study did not reveal significant differences in AQP4-IgG detection rates between the ‘classic’ CBA and a new M23-DNA-based CBA. Importantly, our results largely re-affirm the validity of previous studies that had used the ‘classic’ AQP4-CBA to establish NMO-IgG/AQP4-Ab seropositivity rates in NMO and in a variety of NMO spectrum disorders. PMID:25074611

  3. The effect of aloe emodin-encapsulated nanoliposome-mediated r-caspase-3 gene transfection and photodynamic therapy on human gastric cancer cells.

    PubMed

    Li, Kai-Ting; Duan, Qin-Qin; Chen, Qing; He, Juan-Wen; Tian, Si; Lin, Hai-Dan; Gao, Qing; Bai, Ding-Qun

    2016-02-01

    Gastric carcinoma (GC) has high incidence and mortality rates in China. Surgery and chemotherapy are the main treatments. Photodynamic therapy (PDT) has become a new treatment modality, appearing in recent experimental studies and clinical trials in various tumors. This study explores the combined effect of gene transfection with PDT on GC cells using aloe emodin (AE)-encapsulated nanoliposomes, which acted as gene carrier as well as one photosensitizer (PS). AE-encapsulated nanoliposomes (nano-AE) were prepared by reverse evaporation method. Electron microscopy and nano-ZS90 analyzer were used to detect its morphology, size, and wavelength. Western blot was used to detect the expression of the caspase-3 after transfection. MTT assay and flow cytometry were employed to determine the cytotoxic and apoptotic rates, respectively. Hoechst 33342 staining was adopted to detect the morphological changes in death gastric cancer cells. Cellular reactive oxygen species (ROS) contents were measured by DCFH-DA staining. Outcomes demonstrated that the nano-AE has good properties as gene delivery carriers as well as a PS. The group in which the recombinant plasmid of r-caspase-3 was transfected had higher protein expression of the caspase-3 than controls, meanwhile the proliferation rates of the transfected cells were inhibited by the nano-AE-mediated PDT in an energy-dependent manner. In addition, in the transfected cells, the death rate increased to 77.3% as assessed 12 h after PDT (6.4 J/cm(2) ). Hochest 33342 staining also revealed that the death rate increased significantly in the transfected group compared with other groups. Compared to control groups, the production of ROS in nano-AE PDT group had quadrupled in SGC-7901 cells as early as 1 h after PDT, while it is similar to the group of nano-AE transfection and PDT. Nano-AE-mediated r-caspase-3 gene transfection coupled with PDT could inhibit the proliferation rate and increase the apoptotic rate remarkably in human

  4. Electric pulse current stimulation increases electrophysiological properties of If current reconstructed in mHCN4-transfected canine mesenchymal stem cells

    PubMed Central

    FENG, YUANYUAN; LUO, SHOUMING; YANG, PAN; SONG, ZHIYUAN

    2016-01-01

    The ‘funny’ current, also known as the If current, play a crucial role in the spontaneous diastolic depolarization of sinoatrial node cells. The If current is primarily induced by the protein encoded by the hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) gene. The functional If channel can be reconstructed in canine mesenchymal stem cells (cMSCs) transfected with mouse HCN4 (mHCN4). Biomimetic studies have shown that electric pulse current stimulation (EPCS) can promote cardiogenesis in cMSCs. However, whether EPCS is able to influence the properties of the If current reconstructed in mHCN4-transfected cMSCs remains unclear. The present study aimed to investigate the effects of EPCS on the If current reconstructed in mHCN4-transfected cMSCs. The cMSCs were transfected with the lentiviral vector pLentis-mHCN4-GFP. Following transfection, these cells were divided into two groups: mHCN4-transfected cMSCs (group A), and mHCN4-transfected cMSCs induced by EPCS (group B). Using a whole cell patch-clamp technique, the If current was recorded, and group A cMSCs showed significant time and voltage dependencies and sensitivity to extracellular Cs+. The half-maximal activation (V1/2) value was −101.2±4.6 mV and the time constant of activation was 324±41 msec under −160 mV. In the group B cells the If current increased obviously and activation curve moved to right. The absolute value of V1/2 increased significantly to −92.4±4.8 mV (P<0.05), and the time constant of activation diminished under the same command voltage (251±44 vs. 324±41, P<0.05). In addition, the mRNA and protein expression levels of HCN4, connexin 43 (Cx43) and Cx45 were upregulated in group B compared with group A, as determined by reverse transcription-quantitative polymerase chain reaction and western blot analyses. Transmission electron micrographs also confirmed the increased gap junctions in group B. Collectively, these results indicated that reconstructed If channels

  5. Transformation of human epidermal cells by transfection with plasmid containing simian virus 40 DNA linked to a neomycin gene is a defined medium

    SciTech Connect

    Su, R.T.; Yen-Chu Chang )

    1989-01-01

    A human epidermal cell culture was transformed by transfection with a recombinant plasmid containing simian virus 40 DNA with a deletion at the origin and an antibiotic (neomycin or G418) marker. A calcium phosphate-mediated DNA transfection method was optimized for introducing exogenous DNA into cells maintained in a fully defined medium. The transformed cells were propagated for more than 200 population doublings and did not appear to go through a crisis period. The growth characteristics of the transformed cells were similar to those found in normal epidermal cells. Transformed cells initially transfected with the recombinant plasmid could be propagated for more than 30 passages. Actively growing cells could then be repeatedly selected from cell populations based upon their neomycin (G418)-resistant phenotype for at least another 30 passages. Simian virus 40 T-antigen and extrachromosomal DNA containing plasmid- and SV40-specific DNA sequences were detected in the transformed cells. Because of their nononcogenic phenotype and defined growth requirements, the transformed cells provide a model for examining structural changes during cell proliferation and differentiation, and for exploring the multistage carcinogenesis of human epithelial cells.

  6. Physico-Chemical Characteristics of Lipoplexes Influence Cell Uptake Mechanisms and Transfection Efficacy

    PubMed Central

    Resina, Sarah; Prevot, Paul; Thierry, Alain R.

    2009-01-01

    Background Formulation of DNA/cationic lipid complexes (lipoplexes) designed for nucleic acid delivery mostly results in positively charged particles which are thought to enter cells by endocytosis. We recently developed a lipoplex formulation called Neutraplex that allows preparation of both cationic and anionic stable complexes with similar lipid content and ultrastructure. Methodology/Principal Findings To assess whether the global net charge could influence cell uptake and activity of the transported oligonucleotides (ON), we prepared lipoplexes with positive and negative charges and compared: (i) their physicochemical properties by zeta potential analysis and dynamic light scattering, (ii) their cell uptake by fluorescence microscopy and flow cytometry, and (iii) the biological activity of the transported ON using a splicing correction assay. We show that positively or negatively charged lipoplexes enter cells cells using both temperature-dependent and -independent uptake mechanisms. Specifically, positively charged lipoplexes predominantly use a temperature-dependent transport when cells are incubated OptiMEM medium. Anionic lipoplexes favour an energy-independent transport and show higher ON activity than cationic lipoplexes in presence of serum. However, lipoplexes with high positive global net charge and OptiMEM medium give the highest uptake and ON activity levels. Conclusions These findings suggest that, in addition to endocytosis, lipoplexes may enter cell via a temperature-independent mechanism, which could be mediated by lipid mixing. Such characteristics might arise from the specific lipoplex ultrastructure and should be taken into consideration when developing lipoplexes designed for in vivo or ex vivo nucleic acid transfer. PMID:19557145

  7. Combination of LC3 shRNA plasmid transfection and genistein treatment inhibited autophagy and increased apoptosis in malignant neuroblastoma in cell culture and animal models.

    PubMed

    Mohan, Nishant; Chakrabarti, Mrinmay; Banik, Naren L; Ray, Swapan K

    2013-01-01

    Malignant neuroblastoma is an extracranial solid tumor that usually occurs in children. Autophagy, which is a survival mechanism in many solid tumors including malignant neuroblastoma, deters the efficacy of conventional chemotherapeutic agents. To mimic starvation, we used 200 nM rapamycin that induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells in cell culture and animal models. Combination of microtubule associated protein light chain 3 short hairpin RNA (LC3 shRNA) plasmid transfection and genistein (GST) treatment was tested for inhibiting rapamycin-induced autophagy and promoting apoptosis. The best synergistic efficacy caused the highest decrease in cell viability due to combination of 50 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated IMR-32 cells. Quantitation of acidic vesicular organelles confirmed that combination of LC3 shRNA plasmid transfection and GST treatment prevented rapamycin-induced autophagy due to down regulation of autophagy promoting marker molecules (LC3 II, Beclin 1, TLR-4, and Myd88) and upregulation of autophagy inhibiting marker molecules (p62 and mTOR) in both cell lines. Apoptosis assays showed that combination therapy most effectively activated mitochondrial pathway of apoptosis in human malignant neuroblastoma in cell culture and animal models. Collectively, our current combination of LC3 shRNA plasmid transfection and GST treatment could serve as a promising therapeutic strategy for inhibiting autophagy and increasing apoptosis in human malignant neuroblastoma in cell culture and animal models.

  8. A combination of targeted toxin technology and the piggyBac-mediated gene transfer system enables efficient isolation of stable transfectants in nonhuman mammalian cells.

    PubMed

    Sato, Masahiro; Inada, Emi; Saitoh, Issei; Matsumoto, Yuko; Ohtsuka, Masato; Miura, Hiromi; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

    2015-01-01

    Isolation of cells harboring exogenous DNA is typically achieved by the introduction of plasmids, but its efficiency remains still low. In this study, we developed a novel strategy to obtain stable transfectants efficiently. Porcine embryonic fibroblasts were transfected with two plasmids: (i) pTransIEnd, which comprises the ubiquitous promoter, the piggyBac (PB) transposase gene, an internal ribosomal entry site, the Clostridium perfringens-derived endo-β-galactosidase C (EndoGalC) gene, and a poly(A) tail and (ii) a PB-based plasmid, termed pT-EGFP, which contains enhanced green fluorescent protein (EGFP) expression unit flanked by PB acceptor sites. The PB transposase can accelerate the chromosomal integration of transposon vectors. EndoGalC expression results in removal of a cell surface α-Gal epitope, which is specifically recognized by Bandeiraea simplicifolia isolectin-B4 (IB4). Four days after transfection, cells were treated with IB4SAP (IB4 conjugated to saporin, which eliminates any α-Gal epitope-expressing cells) for a short period, followed by standard culture for approximately 10 days. Several colonies emerged, most of which were positive for EGFP expression and lacked TransIEnd. These results indicated that the proposed approach is useful and efficient for obtaining stable transfectants without the use of drug-resistance genes, and offers a novel route for gene manipulation in cultured nonhuman mammalian cells. PMID:25345906

  9. A combination of targeted toxin technology and the piggyBac-mediated gene transfer system enables efficient isolation of stable transfectants in nonhuman mammalian cells.

    PubMed

    Sato, Masahiro; Inada, Emi; Saitoh, Issei; Matsumoto, Yuko; Ohtsuka, Masato; Miura, Hiromi; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

    2015-01-01

    Isolation of cells harboring exogenous DNA is typically achieved by the introduction of plasmids, but its efficiency remains still low. In this study, we developed a novel strategy to obtain stable transfectants efficiently. Porcine embryonic fibroblasts were transfected with two plasmids: (i) pTransIEnd, which comprises the ubiquitous promoter, the piggyBac (PB) transposase gene, an internal ribosomal entry site, the Clostridium perfringens-derived endo-β-galactosidase C (EndoGalC) gene, and a poly(A) tail and (ii) a PB-based plasmid, termed pT-EGFP, which contains enhanced green fluorescent protein (EGFP) expression unit flanked by PB acceptor sites. The PB transposase can accelerate the chromosomal integration of transposon vectors. EndoGalC expression results in removal of a cell surface α-Gal epitope, which is specifically recognized by Bandeiraea simplicifolia isolectin-B4 (IB4). Four days after transfection, cells were treated with IB4SAP (IB4 conjugated to saporin, which eliminates any α-Gal epitope-expressing cells) for a short period, followed by standard culture for approximately 10 days. Several colonies emerged, most of which were positive for EGFP expression and lacked TransIEnd. These results indicated that the proposed approach is useful and efficient for obtaining stable transfectants without the use of drug-resistance genes, and offers a novel route for gene manipulation in cultured nonhuman mammalian cells.

  10. Transient transfection of macrophage migration inhibitory factor small interfering RNA disrupts the biological behavior of oral squamous carcinoma cells.

    PubMed

    Zeng, Jie; Quan, Jingjing; Xia, Xuefeng

    2016-01-01

    Macrophage migration inhibitory factor (MIF) is closely associated with tumorigenesis. The present study aimed to investigate the effects of MIF on the proliferation, migration and colony formation of oral squamous cell carcinoma (OSCC), and to quantify the protein expression levels of MIF in OSCC tissue samples. Firstly, small interfering (si)RNA was used to knock down the gene expression of MIF in Tca8113, HN5 and SCC25 OSCC cells. Secondly, proliferation, migration and colony formation of the OSCC cells were determined by MTT, transmigration and colony formation assays, respectively. Western blotting was performed to detect changes in the protein expression levels of the epithelial mesenchymal transition markers, Twist‑related protein 1 (Twist1), matrix metalloproteinase (MMP)‑2 and MMP‑9. Finally, immunohistochemistry was used to examine the protein expression of MIF in OSCC tissue samples. The results demonstrated that siRNA against MIF significantly downregulated the expression levels of MIF in all OSCC cells, and decreased their proliferation and migration ability. Colony formation ability was also inhibited in the OSCC cells following transfection with MIF siRNA. Furthermore, western blotting demonstrated that the protein expression of Twist1 was decreased similarly to those of MIF. The protein expression of MMP‑2 revealed no change, whereas that of MMP‑9 decreased. The protein expression of MIF was detected in OSCC tissue samples with staining predominantly located in the cell membrane and cytoplasm. The present study demonstrated that MIF may be important in the pathogenesis and progression of OSCC, and indicated its potential therapeutic value. PMID:26549761

  11. High-efficiency loading, transfection, and fusion of cells by electroporation in two-phase polymer systems.

    PubMed

    Hui, S W; Stoicheva, N; Zhao, Y L

    1996-08-01

    A method to concentrate drugs, DNA, or other materials with target cells in two-phase polymer systems for high-efficiency electroloading is described. The two-phase polymer system is utilized for cell and loading material selection, as well as for cell aggregation before electrofusion. The phase mixing of several water-soluble polymers is characterized, and the polyethylene glycol-Dextran (PEG m.w. 8,000 + Dextran m.w. 71,000) mixture is selected to illustrate the advantage of the two-phase systems. Fluorescently labeled Dextran or DNA is loaded into Chinese hamster ovary (CHO) and JTL cells, using electroporation in either the two-phase polymer system or the conventional single-phase suspension. The loading efficiency is 4 to 30 times higher for the two-phase system, with the best advantage at lower applied field range. Transfections of CHO, COS, Melan C, and JTL lymphoid cells using pSV-beta-galactosidase (for CHO and COS), pBK-RSV-tyrosinase, and pCP4-fucosidase plasmids, respectively, by electroporation in the two-phase polymer system and the conventional single-phase electroporation method, are compared. The former method is far superior to the latter in terms of efficiency. The threshold and optimal field strengths for the former are significantly lower than those for the latter method, so the former method is more favorable in terms of equipment requirement and safety. Electrofusion efficiency in the two-phase system is comparable to that in polyethylene glycol suspension alone and is a significant improvement from the conventional electrofusion method with dielectrophoresis. The two-phase polymer method is, therefore, a valuable technique for gene delivery to a limited cell source, as in ex vivo gene therapy.

  12. Differentiation of human induced pluripotent stem cells into insulin-like cell clusters with miR-186 and miR-375 by using chemical transfection.

    PubMed

    Shaer, Anahita; Azarpira, Negar; Karimi, Mohammad Hosein

    2014-09-01

    Diabetes mellitus is characterized by either the inability to produce insulin or insensitivity to insulin secreted by the body. Islet cell replacement is an effective approach for diabetes treatment; however, it is not sufficient for all the diabetic patients. MicroRNAs (miRNAs) are a class of small noncoding RNAs that play an important role in mediating a broad and expanding range of biological activities, such as pancreas development. The present study aimed to develop a protocol to efficiently differentiate human induced pluripotent stem (iPS) cells into islet-like cell clusters (ILCs) in vitro by using miR-186 and miR-375. The human iPS colonies were transfected with hsa-miR-186 and hsa-miR-375 by using siPORT™ NeoFX™ Transfection Agent, and the differentiation was compared to controls. Total RNA was extracted 24 and 48 h after transfection. The gene expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, Glucagon, and OCT4 were then evaluated through real-time qPCR. On the third day, the potency of the clusters was assessed in response to high glucose levels. Dithizone (DTZ) was used to identify the existence of the β-cells. Besides, the presence of insulin and NGN3 proteins was investigated by immunocytochemistry. Morphological changes were observed on the first day after the chemical transfection, and cell clusters were formed on the third day. The expression of pancreatic specific transcription factors was increased on the first day and significantly increased on the second day. The ILCs were positive for insulin and NGN3 proteins in the immunocytochemistry. Besides, the clusters were stained with DTZ and secreted insulin in glucose challenge test. Overexpression of miR-186 and miR-375 can be an alternative strategy for producing ILCs from the iPS cells in a short time. This work provides a new approach by using patient-specific iPSCs for β-cell replacement therapy in diabetic patients. PMID:25059983

  13. Differentiation of human induced pluripotent stem cells into insulin-like cell clusters with miR-186 and miR-375 by using chemical transfection.

    PubMed

    Shaer, Anahita; Azarpira, Negar; Karimi, Mohammad Hosein

    2014-09-01

    Diabetes mellitus is characterized by either the inability to produce insulin or insensitivity to insulin secreted by the body. Islet cell replacement is an effective approach for diabetes treatment; however, it is not sufficient for all the diabetic patients. MicroRNAs (miRNAs) are a class of small noncoding RNAs that play an important role in mediating a broad and expanding range of biological activities, such as pancreas development. The present study aimed to develop a protocol to efficiently differentiate human induced pluripotent stem (iPS) cells into islet-like cell clusters (ILCs) in vitro by using miR-186 and miR-375. The human iPS colonies were transfected with hsa-miR-186 and hsa-miR-375 by using siPORT™ NeoFX™ Transfection Agent, and the differentiation was compared to controls. Total RNA was extracted 24 and 48 h after transfection. The gene expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, Glucagon, and OCT4 were then evaluated through real-time qPCR. On the third day, the potency of the clusters was assessed in response to high glucose levels. Dithizone (DTZ) was used to identify the existence of the β-cells. Besides, the presence of insulin and NGN3 proteins was investigated by immunocytochemistry. Morphological changes were observed on the first day after the chemical transfection, and cell clusters were formed on the third day. The expression of pancreatic specific transcription factors was increased on the first day and significantly increased on the second day. The ILCs were positive for insulin and NGN3 proteins in the immunocytochemistry. Besides, the clusters were stained with DTZ and secreted insulin in glucose challenge test. Overexpression of miR-186 and miR-375 can be an alternative strategy for producing ILCs from the iPS cells in a short time. This work provides a new approach by using patient-specific iPSCs for β-cell replacement therapy in diabetic patients.

  14. Influence of polymer architecture and molecular weight of poly(2-(dimethylamino)ethyl methacrylate) polycations on transfection efficiency and cell viability in gene delivery.

    PubMed

    Synatschke, Christopher V; Schallon, Anja; Jérôme, Valérie; Freitag, Ruth; Müller, Axel H E

    2011-12-12

    Nonviral gene delivery with the help of polycations has raised considerable interest in the scientific community over the past decades. Herein, we present a systematic study on the influence of the molecular weight and architecture of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) on the transfection efficiency and the cytotoxicity in CHO-K1 cells. A library of well-defined homopolymers with a linear and star-shaped topology (3- and 5-arm stars) was synthesized via atom transfer radical polymerization (ATRP). The molecular weights of the polycations ranged from 16 to 158 kDa. We found that the cytotoxicity at a given molecular weight decreased with increasing number of arms. For a successful transfection a minimum molecular weight was necessary, since the polymers with a number-average molecular weight, M(n), below 20 kDa showed negligible transfection efficiency at any of the tested polyelectrolyte complex compositions. From the combined analysis of cytotoxicity and transfection data, we propose that polymers with a branched architecture and an intermediate molecular weight are the most promising candidates for efficient gene delivery, since they combine low cytotoxicity with acceptable transfection results.

  15. Viral susceptibility, transfection and growth of SPB--a fish neural progenitor cell line from the brain of snubnose pompano, Trachinotus blochii (Lacépède).

    PubMed

    Wen, C-M; Ku, C-C; Wang, C-S

    2013-07-01

    This study investigates the susceptibilities of the SPB cell line to fish viruses including giant seaperch iridovirus (GSIV-K1), red sea bream iridovirus (RSIV-Ku), grouper nervous necrosis virus (GNNV-K1), chum salmon reovirus (CSV) and eel herpesvirus (HVA). GSIV-K1, RSIV-Ku and CSV replicated well in SPB cells, with a significant cytopathic effect and virus production. However, the cells were HVA and GNNV refractory. To examine the ability of SPB cells to stably express foreign protein, expression vectors encoding GNNV B1 and B2 fused to enhanced green fluorescent protein (EGFP) and GSIV ORF35L fused to DsRed were constructed and introduced by transfection into SPB cells. Stable transfectants displayed different morphologies compared with SPB and with each other. EGFP-B1 was predominantly localized in the nuclei, EFPF-B2 was distributed throughout the cytoplasm and nucleus, and granular 35L-DsRed was localized with secreted vesicles. The expression of EFPF-B2 in SPB cells produced blebs on the surface, but the cells showing stable expression of EGFP, EGFP-B1 or 35L-DsRed showed normal morphologies. Results show the SPB cells and the transfected cells grow well at temperatures between 20 and 35 °C and with serum-dependent growth. SPB cells are suitable for studies on foreign protein expression and virology.

  16. A comprehensive analysis of transfection-assisted delivery of iron oxide nanoparticles to dendritic cells

    PubMed Central

    Toki, Shinji; Omary, Reed A.; Wilson, Kevin; Gore, John C.; Peebles, R. Stokes; Pham, Wellington

    2014-01-01

    Polylysine (PL) has been used to facilitate dendritic cell (DC) uptake of super paramagnetic iron oxide (SPIO) nanoparticles for use in magnetic resonance imaging (MRI). In this work, we examined the effect of PL on cell toxicity and induction of cell maturation as manifested by the up-regulation of surface molecules. We found that PL became toxic to bone marrow-derived DCs (BMDCs) at the 10 μg/ ml threshold. Incubation of BMDCs with 20 μg/ml of PL for 1 h resulted in approximately 90% cell death. However, addition of SPIO nanoparticles rescued DCs from PL-induced death as the combination of SPIO with PL did not cause cytotoxicity until the PL concentration was 1000 μg/ml. Prolonged exposure to PL induced BMDC maturation as noted by the expression of surface molecules such as MHC class II, CD40, CCR7 and CD86. However, the combination of SPIO and PL did not induce BMDC maturation at 1 h. However prolonged exposure to SPIO nanoparticles induced CD40 expression and protein expression of TNFα and KC. The data suggest that the use of PL to enhance the labeling of DCs with SPIO nanoparticles is a dedicated work. Appropriate calibration of the incubation time and concentrations of PL and SPIO nanoparticles is crucial to the development of MRI technology for noninvasive imaging of DCs in vivo. PMID:23747738

  17. INFLUENCE OF TEMPERATURE ON AN ESTROGEN-RESPONSIVE RAINBOW TROUT CELL TRANSFECTION ASSAY

    EPA Science Inventory

    One uncertainty in extrapolating estrogenic effects in mammalian systems to those in fish and wildlife is the influence that temperature has on these effects. A reporter gene assay in cultured rainbow trout cell lines was used to determine the influence of temperature on the exp...

  18. Differentiation, not determination, regulates muscle gene activation: transfection of troponin I genes into multipotential and muscle lineages of 10T1/2 cells.

    PubMed Central

    Konieczny, S F; Emerson, C P

    1985-01-01

    Transcription of quail skeletal muscle troponin I (TnI) genes was examined after stable transfection into multipotential 10T1/2 mouse cells and into determined myoblast lineages derived by 5-azacytidine conversion. Transfected TnI and endogenous mouse muscle genes were inactive both in multipotential 10T1/2 and in proliferating myoblasts but were activated coordinately and to high levels when myoblast lineages differentiated, regardless of whether TnI genes were transfected before or after myoblast lineage determination. We conclude that the TnI gene contains evolutionarily conserved control sequences that activate its transcription in response to differentiation-specific regulatory signals. Myoblast lineage determination, therefore, does not appear to act directly on TnI and other muscle genes but likely establishes a regulatory control system that mediates expression of differentiation-specific transcription signals. Images PMID:2426582

  19. Tamoxifen-resistant fibroblast growth factor-transfected MCF-7 cells are cross-resistant in vivo to the antiestrogen ICI 182,780 and two aromatase inhibitors.

    PubMed

    McLeskey, S W; Zhang, L; El-Ashry, D; Trock, B J; Lopez, C A; Kharbanda, S; Tobias, C A; Lorant, L A; Hannum, R S; Dickson, R B; Kern, F G

    1998-03-01

    Although the antiestrogen tamoxifen has been the mainstay of therapy for estrogen receptor (ER)-positive breast cancer, successful treatment of responsive tumors is often followed by the acquisition of tamoxifen resistance. Subsequently, only 30-40% of patients have a positive response to second hormonal therapies. This lack of response might be explained by mechanisms for tamoxifen resistance that sensitize ER pathways to small amounts of estrogenic activity present in tamoxifen or that bypass ER pathways completely. To elucidate one possible mechanism of tamoxifen resistance, we treated ovariectomized tumor-bearing mice injected with fibroblast growth factor (FGF)-transfected MCF-7 breast carcinoma cells with the steroidal antiestrogen ICI 182,780 or one of two aromatase inhibitors, 4-OHA or letrozole. These treatments did not slow estrogen-independent growth or prevent metastasis of tumors produced by FGF-transfected MCF-7 cells in ovariectomized nude mice. FGF-transfected cells had diminished responses to ICI 182,780 in vitro, suggesting that autocrine activity of the transfected FGF may be replacing estrogen as a mitogenic stimulus for tumor growth. ER levels in FGF transfectants were not down-regulated, and basal levels of transcripts for estrogen-induced genes or of ER-mediated transcription of estrogen response element (ERE) luciferase reporter constructs in the FGF expressing cells were not higher than parental cells, implying that altered hormonal responses are not due to down-regulation of ER or to FGF-mediated activation of ER. These studies indicate that estrogen independence may be achieved through FGF signaling pathways independent of ER pathways. If so, therapies directed at the operative mechanism might produce a therapeutic response or allow a response to a second course of antiestrogen treatment.

  20. Recombination of homologous DNA fragments transfected into mammalian cells occurs predominantly by terminal pairing.

    PubMed Central

    Anderson, R A; Eliason, S L

    1986-01-01

    The mechanism by which double-strand cleavages stimulate the joining of plasmid DNA fragments introduced into cultured mammalian cells was investigated by cotransfecting pairs of plasmids encoding deletion mutations in a dominant selectable gene into LMtk- cells. Plasmid recombination substrates were produced by creating deletions of different sizes within the neo coding region of the pSV2neo plasmid. Complementing pairs of deleted plasmid DNAs were linearized at specific unique sites before cotransfection into mouse LMtk- cells by the calcium phosphate precipitation method. Cleaving one donor plasmid produced a 4- to 10-fold stimulation in the production of colonies able to survive in medium containing G-418. The linearization of the second plasmid further increased the efficiency by another factor of 6 to 15 when the cut was made on the opposite side of the homology, approximately equidistant from the center of the overlap. Fifty-seven individual G-418-resistant colonies representing the products of individual crosses were isolated, and the genomic DNAs containing the presumably integrated, functional recombinant neo genes were analyzed on Southern blots. A band consistent with the exchange of markers flanking the neo gene was present in 90% of the DNAs examined. In only one case was the pattern indicative of either a double crossover or a gene conversion event. These results support the idea that homologous extrachromosomal DNA fragments are joined through annealing of overlapping single-stranded ends. This DNA-joining phenomenon may represent the activity of cellular DNA repair enzymes; its relationship to genetic recombination occurring at the chromosomal level remains to be determined. Images PMID:3023971

  1. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection.

    PubMed

    Xu, Y; Szoka, F C

    1996-05-01

    To understand how DNA is released from cationic liposome/DNA complexes in cells, we investigated which biomolecules mediate release of DNA from a complex with cationic liposomes. Release from monovalent[1,2-dioleoyl-3(1)-1(trimethylammonio)propane] or multivalent (dioctadecylamidoglycylspermine) lipids was quantified by an increase of ethidium bromide (EtBr) fluorescence. Plasmid sensitivity to DNAse I degradation was examined using changes in plasmid migration on agarose gel electrophoresis. Physical separation of the DNA from the cationic lipid was confirmed and quantified on sucrose density gradients. Anionic liposomes containing compositions that mimic the cytoplasmic-facing monolayer of the plasma membrane (e.g. phosphatidylserine) rapidly released DNA from the complex. Release occurred near a 1/1 charge ratio (-/+) and was unaffected by ionic strength or ion type. Water soluble molecules with a high negative linear charge density such as dextran sulfate or heparin also released DNA. However, ionic water soluble molecules such as ATP, tRNA, DNA, poly(glutamic acid), spermidine, spermine, or histone did not, even at 100-fold charge excess (-/+). On the basis of these results, we propose that after the cationic lipid/DNA complex is internalized into cells by endocytosis it destabilizes the endosomal membrane. Destabilization induces flip-flop of anionic lipids from the cytoplasmic-facing monolayer, which laterally diffuse into the complex and form a charge neutral ion pair with the cationic lipids. This results in displacement of the DNA from the cationic lipid and release of the DNA into cytoplasm. This mechanism accounts for a variety of observations on cationic lipid/DNA complex-cell interactions.

  2. Effect of targeted ovarian cancer therapy using amniotic fluid mesenchymal stem cells transfected with enhanced green fluorescent protein-human interleukin-2 in vivo

    PubMed Central

    YOU, QI; YAO, YUAN; ZHANG, YUANLONG; FU, SONGBIN; DU, MEI; ZHANG, GUANGMEI

    2015-01-01

    The aim of the present study was to investigate the effect of using amniotic fluid mesenchymal stem cells (AF-MSCs) in targeted ovarian cancer therapy in vivo. AF-MSCs were isolated from human second trimester AF and a plasmid, enhanced green fluorescent protein-human interleukin-2 (pEGFP-hIL-2) was formed. The plasmid was stably transfected into the AF-MSCs and the cells were intravenously injected into ovarian cancer nude mice models. Following stable transfection of the vector, tumor formation, and the expression and activity of hIL-2 were investigated, and microscopic pathological examinations of the tumor were performed. It was found that AF-MSCs exhibited high motility during migration in vivo, and the vector, pEGFP-hIL-2 can be stably transfected into AF-MSCs. Following stable transfection, this type of stem cell is able to successfully transport the therapeutic gene, IL-2, migrate to the ovarian cancer tumor site to secrete the functional IL-2 and treat the tumor. Thus, AF-MSCs may serve as transporters for therapeutic genes targeting ovarian tumor sites and, therefore, be involved in the treatment of tumors. PMID:26179662

  3. Effect of targeted ovarian cancer therapy using amniotic fluid mesenchymal stem cells transfected with enhanced green fluorescent protein-human interleukin-2 in vivo.

    PubMed

    You, Qi; Yao, Yuan; Zhang, Yuanlong; Fu, Songbin; Du, Mei; Zhang, Guangmei

    2015-10-01

    The aim of the present study was to investigate the effect of using amniotic fluid mesenchymal stem cells (AF-MSCs) in targeted ovarian cancer therapy in vivo. AF-MSCs were isolated from human second trimester AF and a plasmid, enhanced green fluorescent protein‑human interleukin‑2 (pEGFP‑hIL‑2) was formed. The plasmid was stably transfected into the AF‑MSCs and the cells were intravenously injected into ovarian cancer nude mice models. Following stable transfection of the vector, tumor formation, and the expression and activity of hIL‑2 were investigated, and microscopic pathological examinations of the tumor were performed. It was found that AF‑MSCs exhibited high motility during migration in vivo, and the vector, pEGFP‑hIL‑2 can be stably transfected into AF‑MSCs. Following stable transfection, this type of stem cell is able to successfully transport the therapeutic gene, IL-2, migrate to the ovarian cancer tumor site to secrete the functional IL-2 and treat the tumor. Thus, AF-MSCs may serve as transporters for therapeutic genes targeting ovarian tumor sites and, therefore, be involved in the treatment of tumors.

  4. Activation of Estrogen Receptor Transfected into a Receptor-Negative Brest Cancer Cell Line Decreases the Metastatic and Invasive Potential of the Cells

    NASA Astrophysics Data System (ADS)

    Garcia, Marcel; Derocq, Danielle; Freiss, Gilles; Rochefort, Henri

    1992-12-01

    Breast cancers containing estrogen receptors are responsive to antiestrogen treatment and have a better prognosis than estrogen receptor-negative tumors. The loss of estrogen and progesterone receptors appears to be associated with a progression to less-differentiated tumors. We transfected the human estrogen receptor into the estrogen receptor-negative metastatic breast cancer cell line MDA-MB-231 in an attempt to restore their sensitivity to antiestrogens. Two stable sublines of MDA-MB-231 cells (HC1 and HE5) expressing functional estrogen receptors were studied for their ability to grow and invade in vitro and to metastasize in athymic nude mice. The number and size of lung metastases developed by these two sublines in ovariectomized nude mice was not markedly altered by tamoxifen but was inhibited 3-fold by estradiol. Estradiol also significantly inhibited in vitro cell proliferation of these sublines and their invasiveness in Matrigel, a reconstituted basement membrane, whereas the antiestrogens 4-hydroxytamoxifen and ICI 164,384 reversed these effects. These results show that estradiol inhibits the metastatic ability of estrogen receptornegative breast cancer cells following transfection with the estrogen receptor, whereas estrogen receptor-positive breast cancers are stimulated by estrogen, indicating that factors other than the estrogen receptor are involved in progression toward hormone independence. Reactivation or transfer of the estrogen receptor gene can therefore be considered as therapeutic approaches to hormone-independent cancers

  5. Adoptive transfer of cytotoxic T lymphocytes induced by CD86-transfected tumor cells suppresses multi-organ metastases of C1300 neuroblastoma in mice.

    PubMed

    Enomoto, A; Kato, K; Yagita, H; Okumura, K

    1997-06-01

    In this study, we examined the therapeutic antitumor effect of cytotoxic T lymphocytes (CTL) generated against CD86-transfected mouse neuroblastoma C1300. We first generated the transfectant, CD86 + C1300, expressing a high level of mouse CD86 on the cell surface. While CD86 + C1300 cells were rejected in syngeneic A/J mice when inoculated subcutaneously, neither vaccination nor any therapeutic antitumor effect was obtained, implying that C1300 may be a poorly immunogenic tumor. However, in vitro stimulation of splenocytes from either C1300-bearing or CD86 + C1300-rejecting mice with CD86 + C1300 cells resulted in remarkable CTL activity against C1300 cells. The CTL activity induced by CD86 + C1300 was mediated by T cell receptor/CD3 and CD8 and was further enhanced by the addition of interleukin-2. Intravenous inoculation of C1300 cells led to multiple organ metastases including the liver, lung, kidney, ovary, lymph node and bone marrow. To examine the therapeutic effect of CTL in this metastasis model, CTL induced by parental or CD86 + C1300 cells were administrated into C1300-bearing mice. Adoptive transfer of CD86 + C1300-induced CTL resulted in marked elimination of multi-organ metastases and prolonged survival in almost all mice, 70% of which survived indefinitely. These results indicate that adoptive transfer of CTL induced by CD86-transfected tumor cells in vitro would be effective and useful for tumor immunotherapy against poorly immunogenic tumors.

  6. Induction of an antitumor response using dendritic cells transfected with DNA constructs encoding the HLA-A*02:01-restricted epitopes of tumor-associated antigens in culture of mononuclear cells of breast cancer patients.

    PubMed

    Sennikov, Sergey Vital'evich; Shevchenko, Julia Alexandrovna; Kurilin, Vasilii Vasil'evich; Khantakova, Julia Nikolaevna; Lopatnikova, Julia Anatol'evna; Gavrilova, Elena Vasil'evna; Maksyutov, Rinat Amirovich; Bakulina, Anastasiya Yur'evna; Sidorov, Sergey Vasil'evich; Khristin, Alexander Alexandrovich; Maksyutov, Amir Zakievich

    2016-02-01

    Advances in oncoimmunology related to the definition of the basic mechanisms of the formation of antitumor immune response, as well as the opening of tumor-associated antigens recognized by immune cells, allowed to start developing ways to influence the effector cells of the immune system to generate effective antitumor cytotoxic response. We investigated the possibility to stimulate an antitumor response in a culture of mononuclear cells of breast cancer patients by dendritic cells transfected with HLA-A*02:01-restricted DNA constructs. We isolated dendritic cells from peripheral blood monocytes and delivered our constructs to these cells by magnetic transfection. Additionally, a series of experiments with loading of dendritic cells with autologous tumor cell lysate antigens was conducted. We have shown that dendritic cells transfected with the HLA-A*02:01-restricted DNA constructs are effective in inducing an antitumor response in a culture of mononuclear cells of breast cancer patients. Dendritic cells transfected with DNA constructor dendritic cells loaded with lysate antigens revealed a comparable stimulated cytotoxic response of mononuclear cells to these two ways of antigen delivery. We conclude that using DNA constructs in conjunction with patient stratification by HLA type allows the application of transfected DCs as an effective method to stimulate antitumor immunity in vitro.

  7. Exogenous Nkx2.5- or GATA-4-transfected rabbit bone marrow mesenchymal stem cells and myocardial cell co-culture on the treatment of myocardial infarction in rabbits

    PubMed Central

    LI, PU; ZHANG, LEI

    2015-01-01

    The present study aimed to investigate the effects of Nkx2.5 or GATA-4 transfection with myocardial extracellular environment co-culture on the transformation of bone marrow mesenchymal stem cells (BMSCs) into differentiated cardiomyocytes. Nkx2.5 or GATA-4 were transfected into myocardial extracellular environment co-cultured BMSCs, and then injected into the periphery of infarcted myocardium of a myocardial infarction rabbit model. The effects of these gene transfections and culture on the infarcted myocardium were observed and the results may provide an experimental basis for the efficient myocardial cell differentiation of BMSCs. The present study also suggested that these cells may provide a source and clinical basis for myocardial injury repair via stem cell transplantation. The present study examined whether Nkx2.5 or GATA-4 exogenous gene transfection with myocardial cell extracellular environment co-culture were able to induce the differentiation of BMSCs into cardiac cells. In addition, the effect of these transfected BMSCs on the repair of the myocardium following myocardial infarction was determined using New Zealand rabbit models. The results demonstrated that myocardial cell differentiation was significantly less effective following exogenous gene transfection of Nkx2.5 or GATA-4 alone compared with that of transfection in combination with extracellular environment co-culture. In addition, the results of the present study showed that exogenous gene transfection of Nkx2.5 or GATA-4 into myocardial cell extracellular environment co-cultured BMSCs was able to significantly enhance the ability to repair, mitigating the death of myocardial cells and activation of the myocardium in rabbits with myocardial infarction compared with those of the rabbits transplanted with untreated BMSCs. In conclusion, the exogenous Nkx2.5 and GATA-4 gene transfection into myocardial extracellular environment co-cultured BMSCs induced increased differentiation into myocardial

  8. Exogenous Nkx2.5- or GATA-4-transfected rabbit bone marrow mesenchymal stem cells and myocardial cell co-culture on the treatment of myocardial infarction in rabbits.

    PubMed

    Li, Pu; Zhang, Lei

    2015-08-01

    The present study aimed to investigate the effects of Nkx2.5 or GATA-4 transfection with myocardial extracellular environment co-culture on the transformation of bone marrow mesenchymal stem cells (BMSCs) into differentiated cardiomyocytes. Nkx2.5 or GATA-4 were transfected into myocardial extracellular environment co-cultured BMSCs, and then injected into the periphery of infarcted myocardium of a myocardial infarction rabbit model. The effects of these gene transfections and culture on the infarcted myocardium were observed and the results may provide an experimental basis for the efficient myocardial cell differentiation of BMSCs. The present study also suggested that these cells may provide a source and clinical basis for myocardial injury repair via stem cell transplantation. The present study examined whether Nkx2.5 or GATA-4 exogenous gene transfection with myocardial cell extracellular environment co-culture were able to induce the differentiation of BMSCs into cardiac cells. In addition, the effect of these transfected BMSCs on the repair of the myocardium following myocardial infarction was determined using New Zealand rabbit models. The results demonstrated that myocardial cell differentiation was significantly less effective following exogenous gene transfection of Nkx2.5 or GATA-4 alone compared with that of transfection in combination with extracellular environment co-culture. In addition, the results of the present study showed that exogenous gene transfection of Nkx2.5 or GATA-4 into myocardial cell extracellular environment co-cultured BMSCs was able to significantly enhance the ability to repair, mitigating the death of myocardial cells and activation of the myocardium in rabbits with myocardial infarction compared with those of the rabbits transplanted with untreated BMSCs. In conclusion, the exogenous Nkx2.5 and GATA-4 gene transfection into myocardial extracellular environment co-cultured BMSCs induced increased differentiation into myocardial

  9. Ligand-induced adhesion to activated endothelium and to vascular cell adhesion molecule-1 in lymphocytes transfected with the N-formyl peptide receptor.

    PubMed

    Honda, S; Campbell, J J; Andrew, D P; Engelhardt, B; Butcher, B A; Warnock, R A; Ye, R D; Butcher, E C

    1994-04-15

    Binding of FMLP to the neutrophil N-formyl peptide receptor (FPR) transmits signals through pertussis toxin-sensitive G proteins triggering Ca2+ flux, superoxide production, granule exocytosis, and neutrophil aggregation and adhesion involving the beta 2 (CD18) integrins. Expression of the FPR in mouse fibroblasts or human kidney cells has been shown to confer an N-formyl peptide-inducible Ca2+ flux in transfectants. Here we demonstrate that the transfected receptor can also support ligand-induced alterations in cellular adhesion. We established stable transfectants of mouse L1-2 pre-B cells with cDNA for human FPR (L1-2 FPR cells). The transfectants bind N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with 1.4 x 10(5) sites per cell and a dissociation constant of 3.3 nM. Stimulation with FMLP induces a transient Ca2+ flux. FMLP also triggers adhesion of L1-2 FPR cells to TNF-alpha- or LPS-activated bEnd3 cells (mouse brain-derived endothelial cells) and to purified mouse VCAM-1. Binding is inhibited by Abs to VCAM-1 and to the alpha-chain of its lymphocyte receptor (the alpha 4 beta 1 integrin, VLA-4). Stimulation with FMLP does not induce a change in cell surface expression of alpha 4. Induced adhesion to VCAM-1 is rapid, detectable at the earliest times measurable (30 to 60 s after FMLP addition), and is inhibited by pertussis toxin. We conclude that FPR can mediate integrin activation not only in neutrophils but also in lymphocytes, and can trigger rapid adhesion via lymphocyte alpha 4 beta 1. The adhesion of lymphocytes is critical to their migration and targeting; our results suggest the possibility of manipulating adhesive responses through expression of chemoattractant receptors in lymphoid cells engineered for cellular therapy, allowing targeted adhesion and potentially migration in response to locally administered ligands.

  10. Ligand-induced adhesion to activated endothelium and to vascular cell adhesion molecule-1 in lymphocytes transfected with the N-formyl peptide receptor.

    PubMed

    Honda, S; Campbell, J J; Andrew, D P; Engelhardt, B; Butcher, B A; Warnock, R A; Ye, R D; Butcher, E C

    1994-04-15

    Binding of FMLP to the neutrophil N-formyl peptide receptor (FPR) transmits signals through pertussis toxin-sensitive G proteins triggering Ca2+ flux, superoxide production, granule exocytosis, and neutrophil aggregation and adhesion involving the beta 2 (CD18) integrins. Expression of the FPR in mouse fibroblasts or human kidney cells has been shown to confer an N-formyl peptide-inducible Ca2+ flux in transfectants. Here we demonstrate that the transfected receptor can also support ligand-induced alterations in cellular adhesion. We established stable transfectants of mouse L1-2 pre-B cells with cDNA for human FPR (L1-2 FPR cells). The transfectants bind N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with 1.4 x 10(5) sites per cell and a dissociation constant of 3.3 nM. Stimulation with FMLP induces a transient Ca2+ flux. FMLP also triggers adhesion of L1-2 FPR cells to TNF-alpha- or LPS-activated bEnd3 cells (mouse brain-derived endothelial cells) and to purified mouse VCAM-1. Binding is inhibited by Abs to VCAM-1 and to the alpha-chain of its lymphocyte receptor (the alpha 4 beta 1 integrin, VLA-4). Stimulation with FMLP does not induce a change in cell surface expression of alpha 4. Induced adhesion to VCAM-1 is rapid, detectable at the earliest times measurable (30 to 60 s after FMLP addition), and is inhibited by pertussis toxin. We conclude that FPR can mediate integrin activation not only in neutrophils but also in lymphocytes, and can trigger rapid adhesion via lymphocyte alpha 4 beta 1. The adhesion of lymphocytes is critical to their migration and targeting; our results suggest the possibility of manipulating adhesive responses through expression of chemoattractant receptors in lymphoid cells engineered for cellular therapy, allowing targeted adhesion and potentially migration in response to locally administered ligands. PMID:7511663

  11. Adoptive immunotherapy with MUC1-mRNA transfected dendritic cells and cytotoxic lymphocytes plus gemcitabine for unresectable pancreatic cancer

    PubMed Central

    2014-01-01

    Background We previously reported the clinical efficacy of adoptive immunotherapy (AIT) with dendritic cells (DCs) pulsed with mucin 1 (MUC1) peptide and cytotoxic T lymphocytes (CTLs). We also reported that gemcitabine (GEM) enhances anti-tumor immunity by suppressing regulatory T cells. Therefore, in the present study, we performed combination therapy with AIT and GEM for patients with unresectable or recurrent pancreatic cancer. Patients and methods Forty-two patients with unresectable or recurrent pancreatic cancer were treated. DCs were generated by culture with granulocyte macrophage colony-stimulating factor and interleukin-4 and then exposed to tumor necrosis factor-α. Mature DCs were transfected with MUC1-mRNA by electroporation (MUC1-DCs). MUC1-CTLs were induced by co-culture with YPK-1, a human pancreatic cancer cell line, and then with interleukin-2. Patients were treated with GEM, while MUC1-DCs were intradermally injected, and MUC1-CTLs were intravenously administered. Results Median survival time (MST) was 13.9 months, and the 1-year survival rate was 51.1%. Of 42 patients, one patient had complete response (2.4%), three patients had partial response (7.1%) and 22 patients had stable disease (52.4%). The disease control ratio was 61.9%. The MST and 1-year survival rate of 35 patients who received more than 1 × 107 MUC1-DCs per injection was 16.1 months and 60.3%, respectively. Liver metastasis occurred in only 5 patients among 35 patients without liver metastasis before treatment. There were no severe toxicities associated with AIT. Conclusion AIT with MUC1-DCs and MUC1-CTLs plus GEM may be a feasible and effective treatment for pancreatic cancer. PMID:24947606

  12. 1α, 25-Dihydroxyvitamin D regulates hypoxia-inducible factor-1α in untransformed and Harvey-ras transfected breast epithelial cells.

    PubMed

    Jiang, Yan; Zheng, Wei; Teegarden, Dorothy

    2010-12-01

    The purpose of this study was to determine the mechanism by which 1α, 25-dihydroxyvitamin D (1,25(OH)(2)D) alters hypoxia-inducible factor-1α (HIF-1α) protein in untransformed and Harvey-ras (H-ras) oncogene transfected MCF10A breast epithelial cells. Treatment with 1,25(OH)(2)D (10nM) increased both mRNA (2.55±0.6-fold vs. vehicle, p=0.03) and protein levels (2.37±0.3-fold vs. vehicle, p<0.0001) of HIF-1α in MCF10A cells in 12h, which remained elevated at 24h. However, in H-ras transfected MCF10A cells, 1,25(OH)(2)D treatment increased HIF-1α protein level (2.08±0.38-fold vs. vehicle, p=0.05) at 12h, with no change in mRNA level and HIF-1α protein level returned to baseline after 24h. A transcription inhibitor prevented the 1,25(OH)(2)D induction of HIF-1α protein and mRNA levels in MCF10A cells, but failed to alter the induction of HIF-1α protein level in H-ras transfected MCF10A cells. On the other hand, inhibition of proteasomal degradation prevented the 1,25(OH)(2)D-induced HIF-1α protein level in H-ras transfected MCF10A but not in MCF10A cells. These results support that 1,25(OH)(2)D regulates HIF-1α protein level via transcriptional regulation in MCF10A cells in contrast to through proteosomal degradation with the presence of H-ras oncogene in MCF10A cells.

  13. Establishment and characterization of Epstein-Barr virus gp350-expressing transfected human lymphoid (Raji) cell clones.

    PubMed

    Khyatti, M; Patel, P C; Menezes, J

    1991-06-19

    Gp350, a late Epstein-Barr-virus (EBV) glycoprotein expressed on both the envelope of viral particles and EBV-producing cells, is also the candidate for the development of an anti-EBV subunit vaccine. This glycoprotein is thought to play an important role in anti-EBV immunity. However, studies on the role of this viral antigen in cellular cytotoxicity and other immune functions have been hampered by the lack of a suitable model expressing gp350. We describe here a study in which we successfully transfected a gp350-negative cell line resistant to natural-killer(NK)-cell activity (i.e., Raji) with a recombinant plasmid (pZIP-MA) containing the EBV-gp350 and the neomycin resistance gene. Three clones with a stable and strong expression of gp350 on their surface membrane, as demonstrated using a gp350-specific (i.e., 2LI0) monoclonal antibody (MAb) were isolated, characterized and used as targets in an antibody-dependent cellular cytotoxicity (ADCC) assay. However, gp350 expression on 2 of the 3 isolated clones was not recognized by an anti-gp350 MAb (72AI) which is specific to a unique gp350 epitope with a dual function (i.e., involved in both EBV binding to its target cell receptors and in inducing virus-neutralizing antibody). We have also found that gp350 expression on our 3 selected clones does not affect EBV-receptor (CR2) density. Our model of gp350-expressing, NK-cell-activity-resistant targets revealed very useful in determining that gp350 serves as a target antigen for EBV-specific ADCC. These gp350-expressing cell clones appear to represent a valuable tool for diagnostic purposes (i.e., for detecting and titrating gp350 antibodies in patients with EBV-associated diseases). Our approach should also prove useful for studying the expression of other cell-surface-associated viral and tumor antigens and their role in specific cellular immunity and immunosurveillance. PMID:1646179

  14. Dendritic Cells Transfected with scFv from Mab 7.B12 Mimicking Original Antigen gp43 Induces Protection against Experimental Paracoccidioidomycosis

    PubMed Central

    Ferreira, Karen S.; Maranhão, Andrea Q.; Garcia, Maria C. C.; Brígido, Marcelo M.; Santos, Suelen S.; Lopes, José D.; Almeida, Sandro R.

    2011-01-01

    Paracoccidioidomycosis (PCM), endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis), which primarily attacks lung tissue. Dendritic cells (DCs) are able to initiate a response in naïve T cells, and they also participate in Th-cell education. Furthermore, these cells have been used for therapy in several disease models. Here we transfected DCs with a plasmid (pMAC/PS-scFv) encoding a single chain variable fragment (scFv) of an anti-Id antibody that is capable of mimicking gp43, the main antigenic component of P. brasiliensis. First, Balb/c mice were immunized subcutaneously with pMAC/PS-scFv and, after seven days, scFv protein was presented to the regional lymph nodes cells. Moreover, we showed that the DCs transfected with scFv were capable of efficiently activating proliferation of total lymph node cells and inducing a decrease in lung infection. Therefore, our results suggested that the use of scFv-transfected DCs may be a promising therapy in the paracoccidioidomycosis (PCM) model. PMID:21249212

  15. Continuous expression and replication of the hepatitis delta virus genome in Hep G2 hepatoblastoma cells transfected with cloned viral DNA.

    PubMed

    Chen, P J; Kuo, M Y; Chen, M L; Tu, S J; Chiu, M N; Wu, H L; Hsu, H C; Chen, D S

    1990-07-01

    To establish stable cell clones allowing continuous replication of hepatitis delta virus (HDV), Hep G2, a hepatoblastoma cell line containing no hepatitis B virus (HBV) DNA sequences, was transfected with a recombinant plasmid containing a tandem trimer of HDV cDNA (driven by the simian virus 40 late promoter) and a neomycin-resistance gene. After selection with the neomycin analogue G418, at least two of the resistant clones were shown to have intact delta antigen by specific immunoblotting, and the delta antigen was located in the cell nucleus by immunofluorescence. Transfected cloned viral DNAs were found to be integrated into cell chromosomes. Replication of the HDV genome was demonstrated by the presence of not only genomic and antigenomic HDV RNAs but also HDV RNAs in multimeric and circular forms. In addition, a 0.8-kilobase antigenomic RNA containing a poly(A) tail and encoding the delta-antigen open reading frame was documented. Continuous replication and transcription of the HDV genome was thus achieved in these transfected cell lines. The results confirmed that replication of HDV was unassisted by HBV. Stable passage of such cell lines strongly suggests that HDV lacks direct cytopathicity in hepatocytes. These clones should be useful in studying the details of the HDV life cycle and the relationship between HDV and its helper virus, HBV.

  16. The response of HEK293 cells transfected with bovine TLR2 to established pathogen-associated molecular patterns and to bacteria causing mastitis in cattle.

    PubMed

    Farhat, Katja; Sauter, Kay-Sara; Brcic, Marija; Frey, Joachim; Ulmer, Artur J; Jungi, Thomas W

    2008-10-15

    Toll-like receptors (TLRs) are key sensors of pathogen-associated molecular patterns (PAMPs). Their role in immunity is difficult to examine in species of veterinary interest, due to restricted access to the knockout technology and TLR-specific antibodies. An alternative approach is to generate cell lines transfected with various TLRs and to examine the recognition of PAMPs or relevant bacteria. In this report, we examined whether recognition of various PAMPs and mastitis-causing bacteria is achieved by transfection of recombinant bovine TLR2 (boTLR2). Therefore, human embryonic kidney (HEK) 293 cells were transfected by whole boTLR2. A clonal analysis of stably transfected cells disclosed variable recognition of several putative TLR2 agonists although expressing similar amounts of the transgene and endogenous TLR6. One clone (clone 25) reacted by copious interleukin-8 (IL-8) production to several stimulants of TLR2 such as di-palmitoylated cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam2), a biochemical preparation of lipoteichoic acid from Staphylococcus aureus, a commercial preparation of peptidoglycan from S. aureus, and heat-killed Listeria monocytogenes (HKLM). TLR2-dependent induction of IL-8 release was stronger in medium containing human serum albumin than in medium containing fetal calf serum. Clone 25 cells responded to high concentrations of S. aureus and to Escherichia coli causing mastitis, but not to Streptococcus uberis and to Streptococcus agalactiae which also cause mastitis. Stimulation by S. aureus was relatively weak when compared (i) with stimulation of the same cells by HKLM and PAMPs derived from S. aureus, (ii) with a clone stably transfected with TLR4 and MD-2 and stimulated by E. coli causing mastitis, and (iii) with interferon-gamma-costimulated bovine macrophages stimulated by S. aureus and S. agalactiae. Thus, clone 25 is suitable for studying the interaction of putative TLR2 agonists with bovine TLR2-transfected cells, provides a cell to

  17. Human concentrative nucleoside transporter 3 transfection with ultrasound and microbubbles in nucleoside transport deficient HEK293 cells greatly increases gemcitabine uptake.

    PubMed

    Paproski, Robert J; Yao, Sylvia Y M; Favis, Nicole; Evans, David; Young, James D; Cass, Carol E; Zemp, Roger J

    2013-01-01

    Gemcitabine is a hydrophilic clinical anticancer drug that requires nucleoside transporters to cross plasma membranes and enter cells. Pancreatic adenocarcinomas with low levels of nucleoside transporters are generally resistant to gemcitabine and are currently a clinical problem. We tested whether transfection of human concentrative nucleoside transporter 3 (hCNT3) using ultrasound and lipid stabilized microbubbles could increase gemcitabine uptake and sensitivity in HEK293 cells made nucleoside transport deficient by pharmacologic treatment with dilazep. To our knowledge, no published data exists regarding the utility of using hCNT3 as a therapeutic gene to reverse gemcitabine resistance. Our ultrasound transfection system--capable of transfection of cell cultures, mouse muscle and xenograft CEM/araC tumors--increased hCNT3 mRNA and (3)H-gemcitabine uptake by >2,000- and 3,400-fold, respectively, in dilazep-treated HEK293 cells. Interestingly, HEK293 cells with both functional human equilibrative nucleoside transporters and hCNT3 displayed 5% of (3)H-gemcitabine uptake observed in cells with only functional hCNT3, suggesting that equilibrative nucleoside transporters caused significant efflux of (3)H-gemcitabine. Efflux assays confirmed that dilazep could inhibit the majority of (3)H-gemcitabine efflux from HEK293 cells, suggesting that hENTs were responsible for the majority of efflux from the tested cells. Oocyte uptake transport assays were also performed and provided support for our hypothesis. Gemcitabine uptake and efflux assays were also performed on pancreatic cancer AsPC-1 and MIA PaCa-2 cells with similar results to that of HEK293 cells. Using the MTS proliferation assay, dilazep-treated HEK293 cells demonstrated 13-fold greater resistance to gemcitabine compared to dilazep-untreated HEK293 cells and this resistance could be reversed by transfection of hCNT3 cDNA. We propose that transfection of hCNT3 cDNA using ultrasound and microbubbles may be a

  18. Measurement of CD8+ and CD4+ T Cell Frequencies Specific for EBV LMP1 and LMP2a Using mRNA-Transfected DCs.

    PubMed

    Sohn, Dae-Hee; Sohn, Hyun-Jung; Lee, Hyun-Joo; Lee, Seon-Duk; Kim, Sueon; Hyun, Seung-Joo; Cho, Hyun-Il; Cho, Seok-Goo; Lee, Suk-Kyeong; Kim, Tai-Gyu

    2015-01-01

    An EBV-specific cellular immune response is associated with the control of EBV-associated malignancies and lymphoproliferative diseases, some of which have been successfully treated by adoptive T cell therapy. Therefore, many methods have been used to measure EBV-specific cellular immune responses. Previous studies have mainly used autologous EBV-transformed B-lymphoblastoid cell lines (B-LCLs), recombinant viral vectors transfected or peptide pulsed dendritic cells (DCs) as stimulators of CD8(+) and CD4(+) T lymphocytes. In the present study, we used an interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay by using isolated CD8(+) and CD4(+) T cells stimulated with mRNA-transfected DCs. The frequency of latent membrane protein 1 (LMP1)-specific IFN-γ producing CD4(+) T cells was significantly higher than that of LMP2a. The frequency of IFN-γ producing CD4(+) T cells was significantly correlated with that of CD8(+) T cells in LMP1-specific immune responses (r = 0.7187, Pc < 0.0001). To determine whether there were changes in LMP1- or LMP2a-specific immune responses, subsequent peripheral blood mononuclear cells (PBMCs) samples were analyzed. Significant changes were observed in 5 of the 10 donors examined, and CD4(+) T cell responses showed more significant changes than CD8(+) T cell responses. CD8(+) and CD4(+) T cells from EBV-seropositive donors secreted only the Th1 cytokines IFN-γ, TNF-α, and IL-2, while Th2 (IL-4) and Th17 (IL-17a) cytokines were not detected. CD4(+) T cells secreted significantly higher cytokine levels than did CD8(+) T cells. Analysis of EBV-specific T cell responses using autologous DCs transfected with mRNA might provide a comprehensive tool for monitoring EBV infection and new insights into the pathogenesis of EBV-associated diseases. PMID:26023769

  19. Kinetic mechanism and characterization of human beta-galactosidase precursor secreted by permanently transfected Chinese hamster ovary cells.

    PubMed

    Zhang, S; McCarter, J D; Okamura-Oho, Y; Yaghi, F; Hinek, A; Withers, S G; Callahan, J W

    1994-11-15

    Chinese hamster ovary cell clones permanently transfected with the cDNA for human lysosomal beta-galactosidase secrete the enzyme precursor into the cell medium, from which it is purified to apparent homogeneity in a single step by affinity chromatography. The purified precursor is fully active, displays the same pH optimum and Km values as the mature placental enzyme, and has an intact C-terminus. The intact enzyme when chromatographed on a Sephacryl S-200 molecular-sieve column elutes as a 105,500 Da monomer, whereas on SDS/PAGE gels the polypeptide migrates as an 88 kDa polypeptide. A time course of digestion with glycopeptide-N-glycanase shows the gradual conversion of the precursor from an 88 to a 72 kDa protein, suggesting the presence of five N-linked oligosaccharides in the protein. The precursor is readily taken up in a mannose-6-phosphate-dependent manner into beta-galactosidase-deficient, GM1-gangliosidosis fibroblasts, and the enzyme activity is returned to normal levels. We show that the stereochemical course of enzymic hydrolysis involves the retention of the beta-configuration at the anomeric centre, suggesting a double-displacement mechanism. Furthermore, the enzyme is rapidly and irreversibly inactivated in the presence of the mechanism-based inactivator 2,4-dinitrophenyl-2-deoxy-2-fluoro-beta-D-galactopyranoside, which implicates a covalent intermediate. The enzyme is also inactivated by 1-ethyl-3(3-dimethylamino-propyl)carbodi-imide and by phenylglyoxal, which implicates carboxylate and arginine residues respectively in the active site. We conclude that the beta-galactosidase precursor is functionally identical to the mature lysosomal form of the enzyme and serves as an excellent enzyme source for investigation of structure-function relationships in the protein.

  20. Experimental endostatin-GFP gene transfection into human retinal vascular endothelial cells using ultrasound-targeted cationic microbubble destruction

    PubMed Central

    Xu, Yan; Xie, Zongyuan; Zhou, Yu; Zhou, Xiyuan; Li, Pan; Wang, Zhigang

    2015-01-01

    Purpose The purpose of this study was to investigate whether ultrasound-targeted cationic microbubble destruction could effectively deliver endostatin-green fluorescent protein (ES-GFP) plasmids to human retinal vascular endothelial cells (HRECs). Methods Cationic microbubbles (CMBs) were prepared and then compared with neutral microbubbles (NMBs) and liposomes. First, the two types of microbubbles were characterized in terms of size and zeta potential. The cell viability of the HRECs was measured using the 3-(4,5-dimthylthiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) assay. The transcription and expression of endostatin, VEGF, Bcl-2, and Bcl-xl were measured via quantitative real-time PCR (qPCR) and western blotting, respectively. Results CMBs differed significantly from NMBs in terms of the zeta potential, but no differences in size were detected. Following ultrasound-targeted microbubble destruction (UTMD)-mediated gene therapy, the transcription and expression of endostatin were highest in the CMB group (p<0.05), while the transcription and expression of VEGF, Bcl-2, and Bcl-xl were lowest compared with the other groups. Moreover, the inhibition of HREC growth was enhanced following treatment with CMBs compared with NMBs or liposomes in vitro (p<0.01). Conclusions This study demonstrated that ultrasound-mediated cationic microbubbles could enhance the transfection efficiency of ES-GFP, which had obvious impacts on the inhibition of the growth process of HRECs in vitro. These results suggest that the combination of UTMD and ES-GFP compounds might be a useful tool for gene therapy targeting retinal neovascularization. PMID:26321867

  1. The cyclin-dependent kinase inhibitor p21(cip1/waf1) enhances the cytotoxicity of ganciclovir in HSV-tk transfected ovarian carcinoma cells.

    PubMed

    Ziller, Christelle; Lincet, Hubert; Muller, Christian D; Staedel, Cathy; Behr, Jean-Paul; Poulain, Laurent

    2004-08-20

    Suicide gene therapy could be an attractive addition to the treatment of ovarian carcinomas, for which acquired chemoresistance frequently results in treatment failure. Here we show that transfection of the HSV-tk gene, followed by incubation with up to 1 mM ganciclovir fails to induce cell death in SKOV3 chemoresistant human ovarian carcinoma cells. However, co-transfection of HSV-tk with Cip1/Waf1 encoding the p21(cip1/waf1) inhibitor of cdks, allows 100 microM ganciclovir to eradicate the population of tumor cells. Potentiation of a drug by co-transfer of HSV-tk with Cip1/Waf1could thus represent another therapeutic approach for tumours that are resistant to conventional therapy. PMID:15246560

  2. A Lentiviral Vector Expressing Desired Gene Only in Transduced Cells: An Approach for Suicide Gene Therapy.

    PubMed

    Mohammadi, Zahra; Shariati, Laleh; Khanahmad, Hossein; Kolahdouz, Mahsa; Kianpoor, Fariborz; Ghanbari, Jahan Afrooz; Hejazi, Zahra; Salehi, Mansoor; Nikpour, Parvaneh; Tabatabaiefar, Mohammad Amin

    2015-09-01

    Suicide gene therapy is a therapeutic strategy, in which cell suicide inducing transgenes are introduced into target cells. Inserting a toxin-encoding gene into a lentiviral vector leads to decreased efficiency of virus production due to lethal effect of toxin on packaging cells. In this study, we designed and constructed a transfer vector to express the toxin in transduced cells but not in packaging cells. Plasmid pLenti-F/GFP was constructed by cutting out R 5'LTR-R 3'LTR fragment with the AflII restriction endonuclease from a plasmid pLenti4-GW/H1/TO-laminshRNA, followed by ligating R 5'LTR-R 3'LTR fragment, constructed by three PCR stages. The promoter and GFP CDS were inserted in opposite strand. For lentiviral production, the HEK293T cell line was co-transfected with the PMD2G, psPAX2, and pLenti-F/GFP plasmids (envelope, packaging, and transfer plasmids).Viral vector titers were assayed. The HEK293T cell line was transduced with this virus. PCR was performed to confirm the presence of the promoter fragment between the R and U5 in 3'LTR. The lentivirus titers were approximately 2 × 10(5). The GFP expression was seen in 51 % of the HEK293T cells transduced with lentivirus. The PCR product size was 1440 bp confirming the promoter fragment position between the R and U5 in 3'LTR. The strategy enables us to use a broad spectrum of toxin genes in gene therapy and helps avoid the death of the packaging cells with lentiviral vectors carrying a toxin-encoding gene, thereby increasing the efficiency of viral production in packaging cells.

  3. Intracellular Delivery of Bioactive Cargos to Hard-to-Transfect Cells Using Carbon Nanosyringe Arrays under an Applied Centrifugal g-Force.

    PubMed

    Choi, Minsuk; Lee, Sang Ho; Kim, Won Bae; Gujrati, Vipul; Kim, Daejin; Lee, Jinju; Kim, Jae-Il; Kim, Hyungjun; Saw, Phei Er; Jon, Sangyong

    2016-01-01

    There is considerable interest in developing a common, universal platform for delivering biomacromolecules such as proteins and RNAs into diverse cells with high efficiency. Here, it is shown that carbon nanosyringe arrays (CNSAs) under an applied centrifugal g-force (cf-CNSAs) can deliver diverse bioactive cargos directly into the cytosol of hard-to-transfect cells with relatively high efficiency and reproducibility. The cf-CNSA platform, an optimized version of a previous CNSA-mediated intracellular delivery platform that adds a g-force feature, exhibits more rapid and superior delivery of cargos to various hard-to-transfect cells than is the case in the absence of g-force. Active species, including small interfering RNAs, plasmids, and proteins are successfully transported across plasma membrane barriers into various cells. By overcoming the limitations of currently available transfection methods, the cf-CNSA platform paves the way to universal delivery of a variety of cargos, facilitating the analysis of cellular responses in diverse cell types.

  4. Transfection of fetal rat intestinal epithelial cells by viral oncogenes: establishment and characterization of the E1A-immortalized SLC-11 cell line.

    PubMed Central

    Emami, S; Mir, L; Gespach, C; Rosselin, G

    1989-01-01

    Intestinal epithelial cells from 19-day-old rat fetuses underwent electropermeabilization and were successfully transfected by three recombinant plasmids containing the cloned oncogenes from the human adenovirus type 2 early region E1A (SLC-11 cells) and polyoma virus and simian virus 40 large T tumor antigens (SLC-21 and SLC-41 cells). SLC-11 cells were propagated for 21 months in culture (current passage, 76; doubling time, 17 hr) and were immortalized by E1A, as shown by RNA transfer blot (Northern blot) analysis and indirect immunofluorescence of the nuclear oncoproteins. These cells were not tumorigenic in either athymic nude mice or syngeneic Wistar rats and showed a nearly normal karyotype with minimal chromosomal changes. The immortalized epithelial cell line SLC-11 retained several of the phenotypes observed in the parent cells of the intestinal mucosa, including cytoplasmic villin, cytokeratins, enkephalinase, and cell surface receptors sensitive to vasoactive intestinal peptide. It is concluded that immortal SLC-11 cells are a suitable model for studying the proliferation and differentiation of epithelial intestinal cells and analyzing cancer progression in the gastrointestinal tract. Images PMID:2470094

  5. Factors influencing the transfection efficiency and cellular uptake mechanisms of Pluronic P123-modified polypropyleneimine/pDNA polyplexes in multidrug resistant breast cancer cells.

    PubMed

    Gu, Jijin; Hao, Junguo; Fang, Xiaoling; Sha, Xianyi

    2016-04-01

    Generally, the major obstacles for efficient gene delivery are cellular internalization and endosomal escape of nucleic acid such as plasmid DNA (pDNA) or small interfering RNA (siRNA). We previously developed Pluronic P123 modified polypropyleneimine (PPI)/pDNA (P123-PPI/pDNA) polyplexes as a gene delivery system. The results showed that P123-PPI/pDNA polyplexes revealed higher transfection efficiency than PPI/pDNA polyplexes in multidrug resistant breast cancer cells. As a continued effort, the present investigation on the factors influencing the transfection efficiency, cellular uptake mechanisms, and intracellular fate of P123-PPI/pDNA polyplexes is reported. The presence of P123 was the main factor influencing the transfection efficiency of P123-PPI/pDNA polyplexes in MCF-7/ADR cells, but other parameters, such as N/P ratio, FBS concentration, incubation time and temperature were important as well. The endocytic inhibitors against clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis were involved in the internalization to investigate their effects on the cellular uptake and transfection efficiency of P123-PPI/pDNA polyplexes in vitro. The data showed that the internalization of P123-PPI/pDNA polyplexes was obtained from both CME and CvME. Colocalization experiments with TRITC-transferrin (CME indicator), Alexa Fluor 555-CTB (CvME indicator), monoclonal anti-α-tubulin (microtubule indicator), and LysoTracker Green (Endosome/lysosome indicator) were carried out to confirm the internalization routes. The results showed that both CME and CvME played vital roles in the effective transfection of P123-PPI/pDNA polyplexes. Endosome/lysosome system and skeleton, including actin filament and microtubule, were necessary for the transportation after internalization. PMID:26741268

  6. Transfection of BmCPV genomic dsRNA in silkmoth-derived Bm5 cells: stability and interactions with the core RNAi machinery.

    PubMed

    Swevers, Luc; Kolliopoulou, Anna; Li, Zheng; Daskalaki, Maria; Verret, Frederic; Kalantidis, Kriton; Smagghe, Guy; Sun, Jingchen

    2014-05-01

    While several studies have been conducted to investigate the stability of dsRNA in the extracellular medium (hemolymph, gut content, saliva), little is known regarding the persistence of dsRNA once it has been introduced into the cell. Here, we investigate the stability of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) genomic dsRNA fragments after transfection into Bombyx-derived Bm5 cells. Using RT-PCR as a detection method, we found that dsRNA could persist for long periods (up to 8 days) in the intracellular environment. While the BmCPV genomic dsRNA was processed by the RNAi machinery, its presence had no effects on other RNAi processes, such as the silencing of a luciferase reporter by dsLuc. We also found that transfection of BmCPV genomic dsRNA could not establish a viral infection in the Bm5 cells, even when co-transfections were carried out with dsRNAs targeting Dicer and Argonaute genes, suggesting that the neutralization by RNAi does not play a role in the establishment of an in vitro culture system. The mechanism of the dsRNA stability in Bm5 cells is discussed, as well as the implications for the establishment for an in vitro culture system for BmCPV. PMID:24636911

  7. High-throughput transfection and engineering of primary cells and cultured cell lines - an invaluable tool for research as well as drug development.

    PubMed

    Müller-Hartmann, Herbert; Faust, Nicole; Kazinski, Michael; Kretzschmar, Titus

    2007-11-01

    The manipulation of eukaryotic cells by introducing nucleic acids and other substrates using chemical, physical or viral methods is one of the ground-breaking tools in the life sciences. Changes in the molecular equipment of a cell induced by introducing different molecules not only enable the dissection of signal transduction and metabolic pathways, but also allow the exploitation of engineered cells as bio-factories for the production of proteins in the processes of target research and drug development. In addition to the application of engineered cells for modern cell-based assays, medically relevant engineered cells can be used in clinical settings for adoptive immunotherapy or gene therapy. With the advent of methods exploiting RNA interference (RNAi), gene identification and functional validation in eukaryotic cells have clearly become one of the most exciting methods in life sciences during the past few years. To accelerate research and development in these areas, high-quality, high-throughput approaches (i.e., using sample formats of at least 96 wells) for cell engineering are needed with increasing demand. Recent developments, especially in the field of electroporation, now allow the efficient, high-throughput engineering of virtually any cell type, including primary cells, many of which were previously considered difficult or even impossible to transfect. Primary cells freshly isolated from native tissues are gaining more and more interest, as data obtained with these cells are considered to be of higher physiological relevance than data obtained with immortalized cell lines that have been cultured for extensive periods. In this review, the various methods for cell engineering (with focus on higher eukaryotic cells) are summarized and their impact for high-throughput applications in research and drug development is discussed.

  8. Redirecting T cells to Ewing's sarcoma family of tumors by a chimeric NKG2D receptor expressed by lentiviral transduction or mRNA transfection.

    PubMed

    Lehner, Manfred; Götz, Gabriel; Proff, Julia; Schaft, Niels; Dörrie, Jan; Full, Florian; Ensser, Armin; Muller, Yves A; Cerwenka, Adelheid; Abken, Hinrich; Parolini, Ornella; Ambros, Peter F; Kovar, Heinrich; Holter, Wolfgang

    2012-01-01

    We explored the possibility to target Ewing's sarcoma family of tumors (ESFT) by redirecting T cells. To this aim, we considered NKG2D-ligands (NKG2D-Ls) as possible target antigens. Detailed analysis of the expression of MICA, MICB, ULBP-1, -2, and -3 in fourteen ESFT cell lines revealed consistent expression of at least one NKG2D-L. Thus, for redirecting T cells, we fused a CD3ζ/CD28-derived signaling domain to the ectodomain of NKG2D, however, opposite transmembrane orientation of this signaling domain and NKG2D required inverse orientation fusion of either of them. We hypothesized that the particularly located C-terminus of the NKG2D ectodomain should allow reengineering of the membrane anchoring from a native N-terminal to an artificial C-terminal linkage. Indeed, the resulting chimeric NKG2D receptor (chNKG2D) was functional and efficiently mediated ESFT cell death triggered by activated T cells. Notably, ESFT cells with even low NKG2D-L expression were killed by CD8(pos) and also CD4(pos) cells. Both, mRNA transfection and lentiviral transduction resulted in high level surface expression of chNKG2D. However, upon target-cell recognition receptor surface levels were maintained by tranfected RNA only during the first couple of hours after transfection. Later, target-cell contact resulted in strong and irreversible receptor down-modulation, whereas lentivirally mediated expression of chNKG2D remained constant under these conditions. Together, our study defines NKG2D-Ls as targets for a CAR-mediated T cell based immunotherapy of ESFT. A comparison of two different methods of gene transfer reveals strong differences in the susceptibility to ligand-induced receptor down-modulation with possible implications for the applicability of RNA transfection. PMID:22355347

  9. Chemical Oxidation of a Redox-Active, Ferrocene-Containing Cationic Lipid: Influence on Interactions with DNA and Characterization in the Context of Cell Transfection

    PubMed Central

    Aytar, Burcu S.; Muller, John P. E.; Golan, Sharon; Kondo, Yukishige; Talmon, Yeshayahu; Abbott, Nicholas L.; Lynn, David M.

    2012-01-01

    We report an approach to the chemical oxidation of a ferrocene-containing cationic lipid [bis(11-ferrocenylundecyl)dimethylammonium bromide, BFDMA] that provides redox-based control over the delivery of DNA to cells. We demonstrate that BFDMA can be oxidized rapidly and quantitatively by treatment with Fe(III)sulfate. This chemical approach, while offering practical advantages compared to electrochemical methods used in past studies, was found to yield BFDMA/DNA lipoplexes that behave differently in the context of cell transfection from lipoplexes formed using electrochemically oxidized BFDMA. Specifically, while lipoplexes of the latter do not transfect cells efficiently, lipoplexes of chemically oxidized BFDMA promoted high levels of transgene expression (similar to levels promoted by reduced BFDMA). Characterization by SANS and cryo-TEM revealed lipoplexes of chemically and electrochemically oxidized BFDMA to both have amorphous nanostructures, but these lipoplexes differed significantly in size and zeta potential. Our results suggest that differences in zeta potential arise from the presence of residual Fe2+ and Fe3+ ions in samples of chemically oxidized BFDMA. Addition of the iron chelating agent EDTA to solutions of chemically oxidized BFDMA produced samples functionally similar to electrochemically oxidized BFDMA. These EDTA-treated samples could also be chemically reduced by treatment with ascorbic acid to produce samples of reduced BFDMA that do promote transfection. Our results demonstrate that entirely chemical approaches to oxidation and reduction can be used to achieve redox-based ‘on/off’ control of cell transfection similar to that achieved using electrochemical methods. PMID:22980739

  10. Efficient gene transfection in the neurotypic cells by star-shaped polymer consisting of β-cyclodextrin core and poly(amidoamine) dendron arms.

    PubMed

    Liang, Bing; Deng, Jun Jie; Yuan, Fang; Yang, Ning; Li, Wei; Yin, Jian Rui; Pu, Shu Xiang; Xie, Long Chang; Gao, Cong; Zhang, Li Ming

    2013-04-15

    In order to develop the effective vectors that had high gene transfection capability and low cytotoxicity in the neuronal cells, we tested the star-shaped polymer consisting of β-cyclodextrin core and poly(amidoamine) (PAMAM) dendron arms [β-CD-(D3)7] as the vector to transfect the human neuroblastoma SH-SY5Y cells. The physicochemical properties of the β-CD-(D3)7/plasmid DNA (pDNA) complexes were characterized by using gel electrophoresis, dynamic light scattering, transmission electron microscopy and zeta-potential experiments. Among the human neuroblastoma SH-SY5Y cells, β-CD-(D3)7/pDNA complex demonstrated a lower toxicity compared to those of PAMAM (G=4, with an ethylenediamine core)/pDNA complex. When the N/P ratio was over 20, it was observed that PAMAM had a faster increment in toxicity compared to β-CD-(D3)7. Fluorescent image, confocal microscopy image and flow cytometry showed that β-CD-(D3)7/pDNA complexes had significantly higher transgene activity than that of PAMAM/pDNA complexes. For example, the transfection efficiency was 20% and 7.5% for β-CD-(D3)7/pDNA and PAMAM/pDNA complexes, respectively. These results indicated that β-CD-(D3)7 might be a promising candidate for neurotypic cells gene delivery with the characteristics of good biocompatibility, relatively high gene transfection capability and potential in vivo gene delivery ability. PMID:23544527

  11. Secreted or nonsecreted forms of acidic fibroblast growth factor produced by transfected epithelial cells influence cell morphology, motility, and invasive potential.

    PubMed Central

    Jouanneau, J; Gavrilovic, J; Caruelle, D; Jaye, M; Moens, G; Caruelle, J P; Thiery, J P

    1991-01-01

    Addition of exogenous acidic fibroblast growth factor (aFGF) to NBT-II epithelial carcinoma cells results in fibroblastic transformation and cell motility. We have generated aFGF-producing NBT-II cells by transfection with recombinant expression vectors containing human aFGF cDNA, or the human aFGF cDNA coupled to a signal peptide (SP) sequence. The effects of the nonsecreted and the secreted 16-kDa growth factor on the morphology, motility, and cell invasive potential (gelatinase activity) were compared. aFGF coupled to a SP was actively secreted out of the producing cells. The secretion of aFGF was not necessary for induction of gelatinase activity, as this was observed in NBT-II cells producing aFGF with or without SP. Production of aFGF, whether secreted or not secreted, resulted in increased in vitro motility of most isolated clones; however, there was no correlation between aFGF level and motility rate. The data suggest that expression of aFGF in NBT-II cells induces metastatic potential through an autocrine or intracrine mechanism. Images PMID:1707175

  12. A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: process development and product quality assessment.

    PubMed

    Rajendra, Yashas; Hougland, Maria D; Alam, Riazul; Morehead, Teresa A; Barnard, Gavin C

    2015-05-01

    Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS-KO host cell line. By creating a bi-allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS-KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)-mediated transfections were performed at high cell density (4 × 10(6) cells/mL) followed by immediate growth arrest at 32 °C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre-complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7-day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc-fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7-day process. Titers

  13. Stoichiometry of expressed alpha(4)beta(2)delta gamma-aminobutyric acid type A receptors depends on the ratio of subunit cDNA transfected.

    PubMed

    Wagoner, Kelly R; Czajkowski, Cynthia

    2010-05-01

    The gamma-aminobutyric acid type A receptor (GABA(A)R) is the target of many depressants, including benzodiazepines, anesthetics, and alcohol. Although the highly prevalent alphabetagamma GABA(A)R subtype mediates the majority of fast synaptic inhibition in the brain, receptors containing delta subunits also play a key role, mediating tonic inhibition and the actions of endogenous neurosteroids and alcohol. However, the fundamental properties of delta-containing GABA(A)Rs, such as subunit stoichiometry, are not well established. To determine subunit stoichiometry of expressed delta-containing GABA(A)Rs, we inserted the alpha-bungarotoxin binding site tag in the alpha(4), beta(2), and delta subunit N termini. An enhanced green fluorescent protein tag was also inserted into the beta(2) subunit to shift its molecular weight, allowing us to separate subunits using SDS-PAGE. Tagged alpha(4)beta(2)delta GABA(A)Rs were expressed in HEK293T cells using various ratios of subunit cDNA, and receptor subunit stoichiometry was determined by quantitating fluorescent alpha-bungarotoxin bound to each subunit on Western blots of surface immunopurified tagged GABA(A)Rs. The results demonstrate that the subunit stoichiometry of alpha(4)beta(2)delta GABA(A)Rs is regulated by the ratio of subunit cDNAs transfected. Increasing the ratio of delta subunit cDNA transfected increased delta subunit incorporation into surface receptors with a concomitant decrease in beta(2) subunit incorporation. Because receptor subunit stoichiometry can directly influence GABA(A)R pharmacological and functional properties, considering how the transfection protocols used affect subunit stoichiometry is essential when studying heterologously expressed alpha(4)beta(2)delta GABA(A)Rs. Successful bungarotoxin binding site tagging of GABA(A)R subunits is a novel tool with which to accurately quantitate subunit stoichiometry and will be useful for monitoring GABA(A)R trafficking in live cells.

  14. Formation of solid lipid nanoparticle (SLN)-gene vector complexes for transfection of mammalian cells in vitro.

    PubMed

    Rudolph, Carsten; Rosenecker, Joseph

    2012-03-01

    Solid lipid nanoparticles (SLNs) offer several technological advantages over standard DNA carriers such as cationic lipids or cationic polymers. However, in the absence of endosomolytic agents such as chloroquine, gene-transfer efficiency mediated by SLN-derived gene vectors consisting of optimized lipid composition remains lower compared to those achieved with standard transfection agents. This protocol describes the incorporation of a dimeric human immunodeficiency virus type-1 (HIV-1) TAT peptide into SLN gene vectors to increase gene-transfer efficiency. This results in higher transfection rates than for standard transfection agents in vitro; the ternary SLN-gene vector complexes usually result in transfection levels equal to or higher than those observed with gene vector complexes formulated with branched polyethylenimine (PEI) 25 kDa. One significant advantage of using this method is the low cytotoxicity of the SLN gene vectors. The application of the gene-transfer technique is limited to relatively low plasmid DNA (pDNA) concentrations of the resulting complexes (10 µg/mL). At higher concentrations, the particles tend to aggregate and precipitate. Therefore, their use for in vivo application, which generally requires high pDNA concentrations, is limited.

  15. A titration procedure of the Junonia coenia densovirus and quantitation of transfection by its cloned genomic DNA in four lepidopteran cell lines.

    PubMed

    Li, Y; Jousset, F X; Giraud, C; Rolling, F; Quiot, J M; Bergoin, M

    1996-03-01

    A sensitive and reproducible tissue culture biossay method was developed based on indirect immunofluorescence to titrate virus suspensions of the Junonia coenia densovirus (JcDNV) and to quantify transfections by its cloned genomic DNA. Four lepidopteran cell lines, the SPC-SL 52 from Spodoptera littoralis, the SPC-PL 40 and the SPC-PL 65 cells derived from Spodoptera litura ovaries and hemocytes, respectively, and the SC-LD 135 from Lymantria dispar were compared for their efficiency to support viral replication. The viral titres expressed as TCID50/ml averaged 10(5) for SPC-SL 52, SPC-PL 40 and SC-LD 135 cells, but were above 10(7) for SPC-PL 65 cells. Even with this most sensitive cell line, the rate of infected cells did not exceed 75% and decreased progressively by serial subcultures. Two transfection protocols were used to compare the sensitivity of the same four cell lines to a recombinant plasmid encompassing an infectious sequence of JcDNV genome. SPC-SL 52 cells were found to be the most sensitive, and the lipofection method resulted in about a 5-fold increase compared to the calcium phosphate precipitation protocol. The rescued virions proved to be infectious and the restriction profiles of their DNA were identical to that of wild type virions.

  16. The Production and Characteristics of a Mouse's Embryonic Stem Cell Lineage, Transfected by the Glia Neurotrophic Factor and Gene Fused with the Green Fluorescent Protein Gene

    PubMed Central

    Arsenieva, E. L.; Kuzmin, I. V.; Manuilova, E. S.; Novosadova, E. V.; Murkin, E. V.; Pavlova, G. V.; Tarantul, V. Z.

    2009-01-01

    The influence that the expression of the human (glial-derived neurotrophic factor (GDNF)) neurotrophic factor has on the morphology and proliferative activity of embryonic stem cells (SC) of a mouse with R1 lineage, as well as their ability to form embroid bodies (EB), has been studied. Before that, using a PCR (polymerase chain reaction) coupled with reverse transcription, it was shown that, in this very lineage of the embryonic SC, the expression of the receptors' genes is being fulfilled for the neurotropfic RET and GFRα1 glia factor. The mouse's embryonic SC lineage has been obtained, transfected by the human GDNF gene, and has been fused with the "green" fluorescent protein (GFP) gene. The presence of the expression of the human GDNF gene in the cells was shown by northern hybridization and the synthesis of its albuminous product by immunocitochemical coloration with the use of specific antibodies. The reliable slowing-down of the embriod-body formation by the embryonic SC transfected by the GDNF gene has been shown. No significant influence of the expression of the GDNF gene on the morphology and the proliferative activity of the transfected embryonic SCs has been found when compared with the control ones. PMID:22649595

  17. Preliminary study on non-viral transfection of F9 (factor IX) gene by nucleofection in human adipose-derived mesenchymal stem cells

    PubMed Central

    Olmedillas López, Susana; Garcia-Arranz, Mariano; Garcia-Olmo, Damian

    2016-01-01

    Background. Hemophilia is a rare recessive X-linked disease characterized by a deficiency of coagulation factor VIII or factor IX. Its current treatment is merely palliative. Advanced therapies are likely to become the treatment of choice for the disease as they could provide a curative treatment. Methods. The present study looks into the use of a safe non-viral transfection method based on nucleofection to express and secrete human clotting factor IX (hFIX) where human adipose tissue derived mesenchymal stem cells were used as target cells in vitro studies and NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice were used to analyze factor IX expression in vivo studies. Previously, acute liver injury was induced by an injected intraperitoneal dose of 500 mg/kg body weight of acetaminophen. Results. Nucleofection showed a percentage of positive cells ranging between 30.7% and 41.9% and a cell viability rate of 29.8%, and cells were shown to secrete amounts of hFIX between 36.8 and 71.9 ng/mL. hFIX levels in the blood of NSG mice injected with ASCs transfected with this vector, were 2.7 ng/mL 48 h after injection. Expression and secretion of hFIX were achieved both in vitro cell culture media and in vivo in the plasma of mice treated with the transfected ASCs. Such cells are capable of eventually migrating to a previously damaged target tissue (the liver) where they secrete hFIX, releasing it to the bloodstream over a period of at least five days from administration. Conclusions. The results obtained in the present study may form a preliminary basis for the establishment of a future ex vivo non-viral gene/cellular safe therapy protocol that may eventually contribute to advancing the treatment of hemophilia. PMID:27114871

  18. Effects of bone marrow-derived mesenchymal stem cells transfected with survivin on pulmonary fibrosis in mice

    PubMed Central

    ZHOU, MI; CHEN, DONG-LING; JIANG, TAO; FENG, YAN-MEI; HAN, XIAO-LI

    2015-01-01

    The aim of the present study was to investigate the effects of bone marrow-derived mesenchymal stem cells (BMSCs) transfected with survivin on lung fibrosis in mice. Mice with bleomycin-induced pulmonary fibrosis were allocated at random to group A, B or C, and injected with 1×106 survivin gene-expressing BMSCs, 1×106 BMSCs or normal saline, respectively. A total of 6 mice were sacrificed from each group on days 7, 14 and 28 after treatment. The extent of alveolitis and pulmonary fibrosis was assessed and the apoptotic rates of the BMSCs and survivin-expressing BMSCs were detected. The content of surfactant protein A (SP-A) in the lung and hydroxyproline (Hyp) in the serum was measured. The mRNA expression levels of transforming growth factor (TGF)-β1 and matrix metalloproteinase (MMP)-9 in the lung tissue of the mice was detected. Furthermore, the protein expression levels of caspase-3 and −9 were detected. The apoptotic rates of the BMSCs (group B) and survivin-expressing BMSCs (group A) were 14.466±1.953 and 7.718±0.493%, respectively. The degree of lung fibrosis in groups A and B was reduced compared with that in group C. The hydroxyproline content in groups A and B was reduced compared with that in group C, and the SP-A content in groups A and B was increased compared with that in group C. The mRNA expression levels of TGF-β1 in group A were reduced compared with those in group B, and the levels in group B were reduced compared with those in group C. By contrast, the mRNA expression levels of MMP-9 in group A were increased compared with those in groups B and C, and the levels in group B were increased compared with those in group A. The expression levels of caspase-3 and −9 in group A were elevated compared with those in groups B and C. In conclusion, BMSCs are effective in preventing bleomycin-induced lung fibrosis, and survivin may enhance the protective effects of BMSCs. PMID:26640562

  19. eNOS transfection of adipose-derived stem cells yields bioactive nitric oxide production and improved results in vascular tissue engineering.

    PubMed

    McIlhenny, Stephen; Zhang, Ping; Tulenko, Thomas; Comeau, Jason; Fernandez, Sarah; Policha, Aleksandra; Ferroni, Matthew; Faul, Elizabeth; Bagameri, Gabor; Shapiro, Irving; DiMuzio, Paul

    2015-11-01

    This study evaluates the durability of a novel tissue engineered blood vessel (TEBV) created by seeding a natural vascular tissue scaffold (decellularized human saphenous vein allograft) with autologous adipose-derived stem cells (ASC) differentiated into endothelial-like cells. Previous work with this model revealed the graft to be thrombogenic, likely due to inadequate endothelial differentiation as evidenced by minimal production of nitric oxide (NO). To evaluate the importance of NO expression by the seeded cells, we created TEBV using autologous ASC transfected with the endothelial nitric oxide synthase (eNOS) gene to produce NO. We found that transfected ASC produced NO at levels similar to endothelial cell (EC) controls in vitro which was capable of causing vasorelaxation of aortic specimens ex vivo. TEBV (n = 5) created with NO-producing ASC and implanted as interposition grafts within the aorta of rabbits remained patent for two months and demonstrated a non-thrombogenic surface compared to unseeded controls (n = 5). Despite the xenograft nature of the scaffold, the TEBV structure remained well preserved in seeded grafts. In sum, this study demonstrates that upregulation of NO expression within adult stem cells differentiated towards an endothelial-like lineage imparts a non-thrombogenic phenotype and highlights the importance of NO production by cells to be used as endothelial cell substitutes in vascular tissue engineering applications.

  20. Pancreatic Cancer Cell Exosome-Mediated Macrophage Reprogramming and the Role of MicroRNAs 155 and 125b2 Transfection using Nanoparticle Delivery Systems.

    PubMed

    Su, Mei-Ju; Aldawsari, Hibah; Amiji, Mansoor

    2016-01-01

    Exosomes are nano-sized endosome-derived small intraluminal vesicles, which are important facilitators of intercellular communication by transporting contents, such as protein, mRNA, and microRNAs, between neighboring cells, such as in the tumor microenvironment. The purpose of this study was to understand the mechanisms of exosomes-mediated cellular communication between human pancreatic cancer (Panc-1) cells and macrophages (J771.A1) using a Transwell co-culture system. Following characterization of exosome-mediated cellular communication and pro-tumoral baseline M2 macrophage polarization, the Panc-1 cells were transfected with microRNA-155 (miR-155) and microRNA-125b-2 (miR-125b2) expressing plasmid DNA using hyaluronic acid-poly(ethylene imine)/hyaluronic acid-poly(ethylene glycol) (HA-PEI/HA-PEG) self-assembling nanoparticle-based non-viral vectors. Our results show that upon successful transfection of Panc-1 cells, the exosome content was altered leading to differential communication and reprogramming of the J774.A1 cells to an M1 phenotype. Based on these results, genetic therapies targeted towards selective manipulation of tumor cell-derived exosome content may be very promising for cancer therapy. PMID:27443190

  1. Regulated expression of chimaeric genes containing the 5'-flanking regions of human growth hormone-related genes in transiently transfected rat anterior pituitary tumor cells.

    PubMed Central

    Cattini, P A; Eberhardt, N L

    1987-01-01

    The expression and hormonal regulation of chimaeric genes containing the 5'-flanking regions of the normal human growth hormone (hGH-1), the variant hGH (hGH-2) and chorionic somatomammotropin (hCS-1) genes fused to the chloramphenicol acetyl transferase (CAT) gene has been examined after transient transfection into cultured rat pituitary (GC), and non-pituitary (HeLa and Rat 2) tumor cells. As assessed by levels of CAT activity, the hGH-1 and hCS-1 gene hybrids were expressed at 5- to 25-fold higher levels in GC cells than in HeLa or Rat 2 cells. The hGH-2 gene hybrid was expressed at very low levels in all 3 cell types. Triiodothyronine treatment of transiently transfected GC cells had little effect on CAT activity from the hGH-1 gene hybrid but increased CAT activity from the hCS-1 gene hybrid. A slight but significant increase in CAT expression was detected with both genes after dexamethasone treatment. The data indicate that elements present on the hGH-1 and hCS-1 genes' 5'-flanking DNA are required for the efficient expression of these genes in GC cells. Images PMID:3644237

  2. Pancreatic Cancer Cell Exosome-Mediated Macrophage Reprogramming and the Role of MicroRNAs 155 and 125b2 Transfection using Nanoparticle Delivery Systems

    PubMed Central

    Su, Mei-Ju; Aldawsari, Hibah; Amiji, Mansoor

    2016-01-01

    Exosomes are nano-sized endosome-derived small intraluminal vesicles, which are important facilitators of intercellular communication by transporting contents, such as protein, mRNA, and microRNAs, between neighboring cells, such as in the tumor microenvironment. The purpose of this study was to understand the mechanisms of exosomes-mediated cellular communication between human pancreatic cancer (Panc-1) cells and macrophages (J771.A1) using a Transwell co-culture system. Following characterization of exosome-mediated cellular communication and pro-tumoral baseline M2 macrophage polarization, the Panc-1 cells were transfected with microRNA-155 (miR-155) and microRNA-125b-2 (miR-125b2) expressing plasmid DNA using hyaluronic acid-poly(ethylene imine)/hyaluronic acid-poly(ethylene glycol) (HA-PEI/HA-PEG) self-assembling nanoparticle-based non-viral vectors. Our results show that upon successful transfection of Panc-1 cells, the exosome content was altered leading to differential communication and reprogramming of the J774.A1 cells to an M1 phenotype. Based on these results, genetic therapies targeted towards selective manipulation of tumor cell-derived exosome content may be very promising for cancer therapy. PMID:27443190

  3. RNA-transfected CD40-activated B cells induce functional T-cell responses against viral and tumor antigen targets: implications for pediatric immunotherapy.

    PubMed

    Coughlin, Christina M; Vance, Barbara A; Grupp, Stephan A; Vonderheide, Robert H

    2004-03-15

    Vaccination with antigen-presenting cells (APCs) engineered to mimic mechanisms of immune stimulation represents a promising approach for cancer immunotherapy. Dendritic cell vaccines have entered phase 3 testing in adult malignancies, but such vaccines in children have been limited. We demonstrate that CD40-activated B cells (CD40-B) transfected with RNA may serve as an alternative vaccine that can be generated from small blood volumes regardless of patient age. CD40-B from pediatric patients are efficient APCs and can be loaded with RNA as an antigenic payload, permitting simultaneous targeting of multiple antigenic epitopes without the necessity of HLA matching. For viral and tumor antigens, CD40-B/RNA technology induced cytotoxic T lymphocytes (CTLs) from adults and children, which could be identified with peptide/major histocompatibility complex (MHC) tetramers. These CTLs secreted interferon-gamma (IFN-gamma) and killed targets in an MHC-restricted fashion. For pooled neuroblastoma RNA and autologous neuroblastoma RNA, CTLs that lysed neuroblastoma cell lines, including CTLs specific against the widely expressed tumor-antigen survivin, were generated. These findings support a novel platform for tumor-specific vaccine or adoptive immunotherapies in pediatric malignancies.

  4. A high-throughput functional assay for characterization of gamma-aminobutyric acid(A) channel modulators using cryopreserved transiently transfected cells.

    PubMed

    Liu, Jay; Chen, Tongming; Norris, Tyrell; Knappenberger, Kathy; Huston, Julie; Wood, Michael; Bostwick, Robert

    2008-12-01

    The ionotropic -aminobutyric acid (GABA)A receptors are an important family of drug targets for a variety of neurological and psychiatric disorders. Selective modulation of certain subtypes of the receptor could lead to novel or improved therapies. However, the discovery of subtype-selective compounds has been hampered by the lack of a high-throughput functional assay and the difficulty in establishing stable cell lines expressing GABAA receptor in a proper subunit composition. To meet drug discovery need we developed a fluorescent imaging plate reader(FLIPR)-based membrane potential assay with sufficient robustness and reproducibility for use in a high-throughput format. Two major subtypes of GABAA receptor were used: GABAA1 and GABAA2, which are composed of (alpha1)2(beta2)2gama2 and (alpha1)2(beta3)2gama2, respectively. We expressed the receptors by transiently co-transfecting cells with the three subunit DNAs in separate constructs, and by controlling the ratio of the DNA amount for each subunit transfected we forced the cells to express GABAA receptors in a pharmacologically relevant form. A large batch of transfected human embryonic kidney 293 cells were cryopreserved and used to screen and evaluate GABAA modulators.In these cells, agonist activation of GABAA receptor resulted in Cl- efflux and membrane depolarization, which was detected by FLIPR as an increase in fluorescence signal. Based on our characterization of several known GABAA modulators and a test set of compounds known to bind to the GABAA benzodiazepine site, we have demonstrated the validity and utility of this assay for discovery of novel pharmacological agents acting at GABAA receptors.

  5. Transcriptional and post-transcriptional limitations of high-yielding, PEI-mediated transient transfection with CHO and HEK-293E cells.

    PubMed

    Rajendra, Yashas; Kiseljak, Divor; Baldi, Lucia; Wurm, Florian M; Hacker, David L

    2015-01-01

    Transient gene expression (TGE) in human embryonic kidney (HEK-293) and Chinese hamster ovary (CHO) cells is a well-established technology for the rapid generation of recombinant proteins. Although the maximum TGE yields have reached 1 g/L or more, the amount of plasmid DNA (pDNA) required for transfection remains high. Although greater than 10(3) copies of pDNA are present per transfected cell, protein yields are still lower than those achieved in recombinant cell lines with only one or a few copies of the transgene. This indicates a clear limitation to TGE in terms of the maximum level of recombinant protein production. In this study, we investigated the limitations to high-yielding TGE processes with CHO and HEK-293E cells using a monoclonal antibody as a model protein. For either cell host, both the intracellular and intranuclear pDNA levels increased linearly with the amount of pDNA added to the culture. In contrast, transgene mRNA accumulation reached a plateau as the intranuclear pDNA amount increased, suggesting a limitation in pDNA transcription. A post-transcriptional limitation to TGE yields was revealed by calculating the amount of antibody produced per transgene mRNA (mRNA utilization). For both hosts the transgene mRNA utilization decreased dramatically when transfected pDNA amounts increased beyond the level giving the maximum protein yield. The post-transcriptional limitation did not appear to be due to bottlenecks in antibody assembly or secretion, suggesting that transgene mRNA translation may be limiting. The results show that TGE yields are not limited by pDNA delivery into the nuclei, but in pDNA and transgene mRNA utilization.

  6. Persistent human cardiac Na+ currents in stably transfected mammalian cells: Robust expression and distinct open-channel selectivity among Class 1 antiarrhythmics.

    PubMed

    Wang, Ging Kuo; Russell, Gabriella; Wang, Sho-Ya

    2013-01-01

    Miniature persistent late Na(+) currents in cardiomyocytes have been linked to arrhythmias and sudden death. The goals of this study are to establish a stable cell line expressing robust persistent cardiac Na(+) currents and to test Class 1 antiarrhythmic drugs for selective action against resting and open states. After transient transfection of an inactivation-deficient human cardiac Na(+) channel clone (hNav1.5-CW with L409C/A410W double mutations), transfected mammalian HEK293 cells were treated with 1 mg/ml G-418. Individual G-418-resistant colonies were isolated using glass cylinders. One colony with high expression of persistent Na(+) currents was subjected to a second colony selection. Cells from this colony remained stable in expressing robust peak Na(+) currents of 996 ± 173 pA/pF at +50 mV (n = 20). Persistent late Na(+) currents in these cells were clearly visible during a 4-second depolarizing pulse albeit decayed slowly. This slow decay is likely due to slow inactivation of Na(+) channels and could be largely eliminated by 5 μM batrachotoxin. Peak cardiac hNav1.5-CW Na(+) currents were blocked by tetrodotoxin with an IC(50) value of 2.27 ± 0.08 μM (n = 6). At clinic relevant concentrations, Class 1 antiarrhythmics are much more selective in blocking persistent late Na(+) currents than their peak counterparts, with a selectivity ratio ranging from 80.6 (flecainide) to 3 (disopyramide). We conclude that (1) Class 1 antiarrhythmics differ widely in their resting- vs. open-channel selectivity, and (2) stably transfected HEK293 cells expressing large persistent hNav1.5-CW Na(+) currents are suitable for studying as well as screening potent open-channel blockers. PMID:23695971

  7. Combined effect of ultrasound/SonoVue microbubble on CD4(+)CD25(+) regulatory T cells viability and optimized parameters for its transfection.

    PubMed

    Shi, Chunying; Zhang, Yu; Yang, Haichao; Dong, Tianxiu; Chen, Yaodong; Xu, Yutong; Yang, Xiuhua

    2015-09-01

    The purpose of this study was to investigate the combined effect of ultrasound and SonoVue microbubble on CD4(+)CD25(+) regulatory T cells (Tregs) viability and to explore the appropriate parameters for Tregs transfection. Tregs were separated from peripheral venous blood of patients with hepatocellular carcinoma and seeded in 96-well plates. The optimal ultrasound exposure time and optimal SonoVue microbubble concentration for Tregs were measured by mechanical index (MI) of 1.2 or 1.4, exposure time of 0, 30, 60, 90, 120, 150, 180s, and 0, 10, 20, 30, 40, 50μL/100μL microbubble per well, respectively. In addition, the combined effect of ultrasound and microbubble on Tregs viability was evaluated according to the following parameters: MI 1.2/1.4+exposure time of 120, 150, 180s+0, 10, 20, 30, 40, 50μL/100μL microbubble per well. Tregs viability investigations were performed in order to explore the optimal transfection condition. The efficiency of plasmid transfer was determined by detection of luciferase activity on the microscopic examinations. The proliferation of Tregs could be promoted by ultrasound exposures, while being decreased with the increasing concentration of microbubbles. Under the current experimental conditions, the optimal ultrasound parameters were MI=1.4 and exposure time=150/180s. The optimal microbubble concentration was 10μL/100μL. Compared with treatment with ultrasound or microbubbles alone, the transfection efficiency of Tregs improved 50% by combining ultrasound and microbubble. The results indicate that both ultrasound and microbubble could affect the Tregs proliferation and the optimal Treg transfection rate was obtained by treating with 10% microbubbles and ultrasound exposure for 150/180s under ultrasound MI of 1.4.

  8. Modifications in the head group and in the spacer of cholesterol-based cationic lipids promote transfection in melanoma B16-F10 cells and tumours.

    PubMed

    Reynier, P; Briane, D; Coudert, R; Fadda, G; Bouchemal, N; Bissieres, P; Taillandier, E; Cao, A

    2004-01-01

    A series of four cationic lipids derived from cholesterol was synthesised and their efficiencies to vectorise nucleic acids were compared. The investigation concerns the effects of systematic chemical modifications in the polar head and in the spacer. The cationic lipid molecules used are in the same family of 3beta[N-(N',N',N'-trimethylaminoethane)-carbamoyl] cholesterol iodide (TMAEC-Chol), presenting a spacer of two or three carbons and a quaternary ammonium polar head ramified with methyl or ethyl groups. These lipids formed stable liposomes sizing from 100 to 200 nm when prepared with the colipid dioleoyl phosphatidylethanolamine (DOPE). The goal of this work was to investigate the effect of the chemical structure of these cationic lipids on lipofection. Their ability to form complexes with DNA, their cytotoxicity and their transfection efficiency in vitro and in vivo were studied. Results were compared with those obtained from the well known cholesterol-based cationic lipid DC-Chol. In a melanoma cell line (B16-F10), results showed that either the polar head or the spacer affected the cytotoxicity. Cationic lipids with three ethyl groups in the head are more toxic than those with three methyl groups while cationic lipids with three carbons in the spacer are less toxic than those with two carbons in the spacer. The best transfection level was obtained in vitro and in vivo with cationic lipids having 3C in the spacer. Data indicated that among these lipids, in vivo gene transfer is advantaged by the methylated polar head while in vitro the best level was obtained with the ethylated one. Finally, it was observed that the chemical structure influences the transfection in the presence of serum while the complex charge and the DOPE ratios in liposomes preferentially affect the interaction with erythrocytes. Argumentations are proposed to explain the discrepancies between in vitro and in vivo transfection results concerning the optimal charge ratio and the chemical

  9. Involvement of claudin-7 in HIV infection of CD4(-) cells

    PubMed Central

    Zheng, Junying; Xie, Yiming; Campbell, Richard; Song, Jun; Massachi, Samira; Razi, Miriam; Chiu, Robert; Berenson, James; Yang, Otto O; Chen, Irvin SY; Pang, Shen

    2005-01-01

    Background Human immunodeficiency virus (HIV) infection of CD4(-) cells has been demonstrated, and this may be an important mechanism for HIV transmission. Results We demonstrated that a membrane protein, claudin-7 (CLDN-7), is involved in HIV infection of CD4(-) cells. A significant increase in HIV susceptibility (2- to 100-fold) was demonstrated when CLDN-7 was transfected into a CD4(-) cell line, 293T. In addition, antibodies against CLDN-7 significantly decreased HIV infection of CD4(-) cells. Furthermore, HIV virions expressing CLDN-7 on their envelopes had a much higher infectivity for 293T CD4(-) cells than the parental HIV with no CLDN-7. RT-PCR results demonstrated that CLDN-7 is expressed in both macrophages and stimulated peripheral blood leukocytes, suggesting that most HIV virions generated in infected individuals have CLDN-7 on their envelopes. We also found that CLDN-7 is highly expressed in urogenital and gastrointestinal tissues. Conclusion Together these results suggest that CLDN-7 may play an important role in HIV infection of CD4(-) cells. PMID:16368003

  10. Tissue-specific expression of cell-surface Qa-2 antigen from a transfected Q7b gene of C57BL/10 mice

    PubMed Central

    1987-01-01

    We screened a cDNA library prepared from a BALB.B10 CTL clone that expresses Qa-2 antigen, and isolated four clones derived from Q7b, a Qa region gene of C57BL/10. One of these Q7b cDNAs and the Q7b chromosomal gene were subcloned into expression vectors and transfected into L cells and R1.1 thymoma cells. We found that the chromosomal Q7b gene expresses Qa-2 on the surface of R1.1 cells, but not on L cells while the Q7b cDNA expresses protein on the surface of both cell types. The levels of Qa-2 expression do not correlate with the total levels of Q7b mRNA in these transfectants. Our results suggest that the tissue- specific expression of Qa-2 may be controlled, in part, by mechanisms of alternate RNA splicing. By using hybrid gene constructs, we have mapped the tissue-specific element to the 3' part of the gene, downstream of a site near the middle of exon 4. The hybrid polypeptides differ significantly in their transmembrane and cytoplasmic regions. These portions of the protein also may play a role in the tissue- specific expression of Qa-2. PMID:3502706

  11. Successful transfer of plasmid DNA into in vitro cells transfected with an inorganic plasmid-Mg/Al-LDH nanobiocomposite material as a vector for gene expression

    NASA Astrophysics Data System (ADS)

    Jaffri Masarudin, Mas; Yusoff, Khatijah; Rahim, Raha Abdul; Zobir Hussein, Mohd

    2009-01-01

    The delivery of a full plasmid, encoding the green fluorescent protein gene into African monkey kidney (Vero3) cells, was successfully achieved using nanobiocomposites based on layered double hydroxides. This demonstrated the potential of using the system as an alternative DNA delivery vector. Intercalation of the circular plasmid DNA, pEGFP-N2, into Mg/Al-NO3- layered double hydroxides (LDH) was accomplished through anion exchange routes to form the nanobiocomposite material. The host was previously synthesized at the Mg2+ to Al3+ molar ratio Ri = 2 and subsequently intercalated with plasmid DNA. Size expansion of the interlamellae host from 8.8 Å in LDH to 42 Å was observed in the resulting nanobiocomposite, indicating stable hybridization of the plasmid DNA. The powder x-ray diffraction (PXRD) results, supplemented with Fourier-transform infrared (FTIR) spectroscopy, compositional and electrophoresis studies confirmed the encapsulation episode of the biomaterial. In order to elucidate the use of this resulting nanobiocomposite as a delivery vector, an MTT assay was performed to determine any cytotoxic effects of the host towards cells. The intercalated pEGFP-N2 anion was later successfully recovered through acidification with HNO3 after treatment with DNA-degrading enzymes, thus also showing the ability of the LDH host to protect the intercalated biomaterial from degradation. Cell transfection studies on Vero3 cells were then performed, where cells transfected with the nanobiocomposite exhibited fluorescence as early as 12 h post-treatment compared to naked delivery of the plasmid itself.

  12. Modulation of metallothionein-III mRNA content and growth rate of rat C6-glial cells by transfection with human 5-HT1D receptor genes.

    PubMed

    Amoureux, M C; Wurch, T; Pauwels, P J

    1995-09-14

    The mRNA content of the brain-specific metallothionein-III (MT-III) protein was measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR) in two transformed glial cell lines: rat C6-glial and human U-373 MG cells. Low levels of MT-III mRNA were detected compared to a high expression of this mRNA in primary cultures of rat astrocytes. C6-glial cell lines stably transfected with a human 5-HT1D alpha or 5-HT1D beta receptor gene showed a decrease (87 to 93%) in basal [3H]thymidine incorporation, whereas their MT-III mRNA content was more than 30-fold increased compared to plasmid transfected C6-glial cells. The inverse proportion between mitogenic activity and MT-III mRNA content suggests that MT-III may act as a growth inhibitory factor in rat C6-glial cells. PMID:7677777

  13. Novel stably transfected human reporter cell line AIZ-AR as a tool for an assessment of human androgen receptor transcriptional activity.

    PubMed

    Bartonkova, Iveta; Novotna, Aneta; Dvorak, Zdenek

    2015-01-01

    Androgen receptor plays multiple physiological and pathological roles in human organism. In the current paper, we describe construction and characterization of a novel stably transfected human reporter cell line AIZ-AR for assessment of transcriptional activity of human androgen receptor. Cell line AIZ-AR is derived from human prostate carcinoma epithelial cell line 22Rv1 that was transfected with reporter plasmid containing 3 copies of androgen response regions (ARRs) followed by a single copy of androgen response element (ARE) from the promoter region of human prostate specific antigen (PSA) gene. AIZ-AR cells remained fully functional for more than 60 days and over 25 passages in the culture and even after cryopreservation. Time-course analyses showed that AIZ-AR cells allow detection of AR ligands as soon as after 8 hours of the treatment. We performed dose-response analyses with 23 steroids in 96-well plate format. We observed activation of AR by androgens, but not by estrogens and mineralocorticoids. Some glucocorticoids and progesterone also induced luciferase, but their potencies were 2-3 orders of magnitude weaker as compared to androgens. Taken together, we have developed a rapid, sensitive, selective, high-throughput and reproducible tool for detection of human AR ligands, with potential use in pharmacological and environmental applications.

  14. Using intron splicing trick for preferential gene expression in transduced cells: an approach for suicide gene therapy.

    PubMed

    Pourzadegan, F; Shariati, L; Taghizadeh, R; Khanahmad, H; Mohammadi, Z; Tabatabaiefar, M A

    2016-01-01

    Suicide gene therapy is one of the most innovative approaches in which a potential toxic gene is delivered to the targeted cancer cell by different target delivery methods. We constructed a transfer vector to express green fluorescent protein (GFP) in transduced cells but not in packaging cells. We placed gfp under the control of the cytomegalovirus (CMV) promoter, which is positioned between the two long-terminal repeats in reverse direction. The intron-2 sequence of the human beta globin gene with two poly-A signals and several stop codons on the antisense strand was placed on the leading strand between the CMV promoter and gfp. For lentiviral production, the HEK293T and line were co-transfected with the PMD2G, psPAX2 and pLentiGFP-Ins2 plasmids. The HEK293T and line were transduced with this virus. PCR was performed for evaluation of intron splicing in transduced cells. The GFP expression was seen in 65% of the cells transduced. The PCR amplification of the genomic DNA of transduced cells confirmed the splicing of intron 2. The strategy is significant to accomplish our goal for preserving the packaging cells from the toxic gene expression during viral assembly and the resultant reduction in viral titration. Also it serves to address several other issues in the gene therapy.

  15. Use of a transfected and amplified Drosophila heat shock promoter construction for inducible production of toxic mouse c-myc proteins in CHO cells

    SciTech Connect

    Wurm, F.M.; Gwinn, K.A.; Papoulas, O.; Pallavicini, M.; Kingston, R.E.

    1987-07-24

    After transfection and selection with methotrexate, CHO cell lines were established which contained up to 2000 copies of an expression vector for c-myc protein. The vector contained the Drosophila heat shock protein 70 (hsp70) promoter fused with the coding region of the mouse c-myc gene. Incubation of cells for up to 3 hours at 43/sup 0/C resulted in at least a 100-fold induction of recombinant c-myc mRNA. When cells were shifted back to 37/sup 0/C, within 1 to 4 hours, this RNA was translated into protein to yield about 250 ..mu..g per 10/sup 9/ cells. Cells died a few hours later, suggesting that high concentrations of intracellular c-myc are cytotoxic. 47 refs., 5 figs.

  16. Enhanced transfection efficiency and targeted delivery of self-assembling h-R3-dendriplexes in EGFR-overexpressing tumor cells.

    PubMed

    Li, Jun; Li, Shengnan; Xia, Songyun; Feng, Jinfeng; Zhang, Xuedi; Hao, Yanli; Chen, Lei; Zhang, Xiaoning

    2015-09-22

    The efficient gene transfection, cellular uptake and targeted delivery in vivo are key issues for non-viral gene delivery vectors in cancer therapy. To solve these issues, we designed a new targeted gene delivery system based on epidermal growth factor receptor (EGFR) targeting strategy. An anti-EGFR monoclonal antibody h-R3 was introduced to dendriplexes of PAMAM and DNA via electrostatic interactions to form self-assembled h-R3-PAMAM-DNA complexes (h-R3-dendriplexes). Dendriplexes h-R3-dendriplexes represented excellent DNA encapsulation ability and formed unique nanostructures. Compared to dendriplexes, h-R3-dendriplexes presented lower cytotoxicity, higher gene transfection efficiency, excellent endosome escape ability and high nuclear accumulation in the EGFR-overexpressing HepG2 cells. Both ex vivo fluorescence imaging and confocal results of frozen section revealed that h-R3-dendriplexes showed higher targeted delivery and much better gene expression in the tumors than dendriplexes at the same N/P ratio, and h-R3-dendriplexes had accumulation primarily in the tumor and kidney. Moreover, h-R3-dendriplexes for p53 delivery indicated efficient cell growth inhibition and potentiated paclitaxel-induced cell death. These results indicate that the h-R3-dendriplexes represent a great potential to be used as efficient targeted gene delivery carriers in EGFR-overexpressing tumor cells. PMID:26309162

  17. [Construction of venus vector carrying IGFBP7 gene and its expression in K562 cells].

    PubMed

    Wu, Shui-Yan; Hu, Shao-Yan; Cen, Jian-Nong; Chen, Zi-Xing

    2012-02-01

    The aim of this study was to construct venus vector carrying the gene encoding insulin-like growth factor binding protein 7 (IGFBP7), which provides an effective platform for exploring the function of this gene in leukemia. After digestion by restriction endonuclease, the IGFBP7 gene was recombined with the transfer plasmid. The venus particles were packaged using 293T cells to transfect K562 cells, and identification was performed by means of flow cytometry, RT-PCR and Western blot. The results showed that the sequence of cloned IGFBP7 gene was the same as that in GenBank. The size of product restricted by BamHI was same as the predicted one. GFP expression was observed in 293T and K562 cells with the fluorescent microscopy and flow cytometry. The expression level of mRNA and protein of IGFBP7 was confirmed by RT-PCR and Western blotting in K562 cells. It is concluded that venus vector carrying IGFBP7 gene has been successfully constructed and provides basis for exploring function of IGFBP7 in K562 cells.

  18. Induction of CML28-specific cytotoxic T cell responses using co-transfected dendritic cells with CML28 DNA vaccine and SOCS1 small interfering RNA expression vector

    SciTech Connect

    Zhou Hongsheng; Zhang Donghua . E-mail: hanson2008@gmail.com; Wang Yaya; Dai Ming; Zhang Lu; Liu Wenli; Liu Dan; Tan Huo; Huang Zhenqian

    2006-08-18

    CML28 is an attractive target for antigen-specific immunotherapy. SOCS1 represents an inhibitory control mechanism for DC antigen presentation and the magnitude of adaptive immunity. In this study, we evaluated the potential for inducing CML28-specific cytotoxic T lymphocytes (CTL) responses by dendritic cells (DCs)-based vaccination. We constructed a CML28 DNA vaccine and a SOCS1 siRNA vector and then cotransfect monocyte-derived DCs. Flow cytometry analysis showed gene silencing of SOCS1 resulted in higher expressions of costimulative moleculars in DCs. Mixed lymphocyte reaction (MLR) indicated downregulation of SOCS1 stronger capability to stimulate proliferation of responder cell in DCs. The CTL assay revealed transfected DCs effectively induced autologous CML28-specific CTL responses and the lytic activities induced by SOCS1-silenced DCs were significantly higher compared with those induced by SOCS1-expressing DCs. These results in our study indicates gene silencing of SOCS1 remarkably enhanced the cytotoxicity efficiency of CML28 DNA vaccine in DCs.

  19. Coupling of a transfected human brain A1 adenosine receptor in CHO-K1 cells to calcium mobilisation via a pertussis toxin-sensitive mechanism.

    PubMed Central

    Iredale, P. A.; Alexander, S. P.; Hill, S. J.

    1994-01-01

    1. The presence of A1 adenosine receptors in CHO-K1 cells transfected with the human brain A1 sequence was confirmed by ligand binding studies using 8-cyclopentyl-[3H] 1,3-dipropylxanthine ([3H]-DPCPX). 2. Alterations in intracellular calcium ([Ca2+]i) were measured with the calcium-sensitive dye, fura-2. 3. N6-cyclopentyladenosine (CPA), the selective A1 agonist, and 5'-N-ethylcarboxaminoadenosine (NECA), a relatively non-selective adenosine receptor agonist, elicited rapid, biphasic increases in [Ca2+]i which involved both mobilisation from intracellular stores and calcium entry. 4. The calcium response to CPA was significantly inhibited by the selective A1 antagonist DPCPX. The non-selective adenosine receptor, xanthine amino congener (XAC), was less potent. 5. The calcium response to CPA was completely prevented by pretreatment of the cells with pertussis toxin implicating the involvement of Gi in the receptor-mediated response. 6. In summary, we present evidence for the coupling of transfected human brain A1 adenosine receptors in CHO-K1 cells to mobilisation of [Ca2+]i via a pertussis toxin-sensitive G protein. PMID:8032613

  20. Transfection of CXCR-4 using microbubble-mediated ultrasound irradiation and liposomes improves the migratory ability of bone marrow stromal cells.

    PubMed

    Wang, Gong; Zhuo, Zhongxiong; Zhang, Qian; Xu, Yali; Wu, Shengzheng; Li, Lu; Xia, Hongmei; Gao, Yunhua

    2015-01-01

    Bone marrow stromal cells (BMSCs) have proven useful for the treatment of various human diseases and injuries. However, their reparative capacity is limited by their poor migration and homing ability, which are primarily dependent on the SDF-1/CXCR4 axis. Most subcultured BMSCs lack CXCR4 receptor expression on the cell surface and exhibit impaired migratory capacity. To increase responsiveness to SDF-1 and promote cell migration and survival of cultured BMSCs, we used a combination of ultrasound-targeted microbubble destruction (UTMD) and liposomes to increase CXCR4 expression in vitro. We isolated and cultured rat BMSCs to their third passage and transduced them with recombinant plasmid pDsRed-CXCR4 using microbubble-mediated ultrasound irradiation and liposomes. Compared to some viral vectors, the method we employed here resulted in significantly better transfection efficiency, CXCR4 expression, and technical reproducibility. The benefits of this approach are likely due to the combination of "sonoporation" caused by shockwaves and microjet flow resulting from UTMD-generated cavitation. Following transfection, we performed a transwell migration assay and found that the migration ability of CXCR4-modified BMSCs was 9-fold higher than controls. The methods we describe here provide an effective, safe, non-viral means to achieve high levels of CXCR4 expression. This is associated with enhanced migration of subcultured BMSCs and may be useful for clinical application as well.

  1. Generation and usage of aequorin lentiviral vectors for Ca(2+) measurement in sub-cellular compartments of hard-to-transfect cells.

    PubMed

    Lim, Dmitry; Bertoli, Alessandro; Sorgato, M Catia; Moccia, Francesco

    2016-05-01

    Targeted aequorin-based Ca(2+) probes represent an unprecedented tool for the reliable measurement of Ca(2+) concentration and dynamics in different sub-cellular compartments. The main advantages of aequorin are its proteinaceous nature, which allows attachment of a signal peptide for targeting aequorin to virtually any sub-cellular compartment; its low Ca(2+)-binding capacity; the wide range of Ca(2+) concentrations that can be measured, ranging from sub-micromolar to millimolar; its robust performance in aggressive environments, e.g., the strong acidic pH of the lysosomal lumen. Lentiviral vectors represent a popular tool to transduce post-mitotic or hard-to-transfect cells both in vitro and in vivo. Furthermore, it has great potential for gene therapy. Last generation lentiviral vectors represent a perfect compromise for combining large insert size, ease of production and handling, and high degree of biosafety. Here, we describe strategies for cloning aequorin probes - targeted to the cytosol, sub-plasma membrane cytosolic domains, the mitochondrial matrix, and the endoplasmic reticulum lumen - into lentiviral vectors. We describe methods for the production of lentiviral particles, and provide examples of measuring Ca(2+) dynamics by such aequorin-encoding lentiviral vectors in sub-cellular compartments of hard-to-transfect cells, including immortalized striatal neurons, primary cerebellar granule neurons and endothelial progenitor cells, which provide suitable in vitro models for the study of different human diseases. PMID:26992273

  2. An in vitro study on the genotoxic effect of substituted furans in cells transfected with human metabolizing enzymes: 2,5-dimethylfuran and furfuryl alcohol

    PubMed Central

    Huffman, Minor P.; Høie, Anja H.; Svendsen, Camilla; Brunborg, Gunnar; Murkovic, Michael; Glatt, Hansruedi; Husøy, Trine

    2016-01-01

    2,5-Dimethylfuran (DMF) and furfuryl alcohol (FFA) are two substituted furans that are formed during the processing of foods and have also been used as food flavorings. DMF and FFA are proposed to be bioactivated by human sulfotransferases (SULTs) which are not expressed in conventional cell lines used for genotoxicity testing. Therefore, in addition to the standard V79 cell line, we used a transfected V79 derived cell line co-expressing human cytochrome P450 (CYP) 2E1 and human SULT1A1 to assess the genotoxicity of DMF and FFA. The alkaline single cell gel electrophoresis (SCGE) assay was used to detect DNA damage in the form of single strand breaks and alkali-labile sites after exposure to DMF (0.5h; 0.5, 1, 1.5 or 2mM) or FFA (3h; 1, 3, 6 or 15mM). DMF induced DNA damage in V79 cells in a concentration-dependent manner irrespective of the expression of human CYP2E1 and SULT1A1. Almost no increase in the level of DNA damage was detected after exposure to FFA, except for a weak effect at the highest concentration in the transfected cell line. The results suggest that DNA damage in V79 cells from exposure to DMF detected by the alkaline SCGE assay is independent of human CYP2E1 and SULT1A1, and the genotoxic effect of FFA, as assessed by SCGE, is minimal in V79 cells. PMID:27226491

  3. An in vitro study on the genotoxic effect of substituted furans in cells transfected with human metabolizing enzymes: 2,5-dimethylfuran and furfuryl alcohol.

    PubMed

    Huffman, Minor P; Høie, Anja H; Svendsen, Camilla; Brunborg, Gunnar; Murkovic, Michael; Glatt, Hansruedi; Husøy, Trine

    2016-09-01

    2,5-Dimethylfuran (DMF) and furfuryl alcohol (FFA) are two substituted furans that are formed during the processing of foods and have also been used as food flavorings. DMF and FFA are proposed to be bioactivated by human sulfotransferases (SULTs) which are not expressed in conventional cell lines used for genotoxicity testing. Therefore, in addition to the standard V79 cell line, we used a transfected V79 derived cell line co-expressing human cytochrome P450 (CYP) 2E1 and human SULT1A1 to assess the genotoxicity of DMF and FFA. The alkaline single cell gel electrophoresis (SCGE) assay was used to detect DNA damage in the form of single strand breaks and alkali-labile sites after exposure to DMF (0.5h; 0.5, 1, 1.5 or 2mM) or FFA (3h; 1, 3, 6 or 15mM). DMF induced DNA damage in V79 cells in a concentration-dependent manner irrespective of the expression of human CYP2E1 and SULT1A1. Almost no increase in the level of DNA damage was detected after exposure to FFA, except for a weak effect at the highest concentration in the transfected cell line. The results suggest that DNA damage in V79 cells from exposure to DMF detected by the alkaline SCGE assay is independent of human CYP2E1 and SULT1A1, and the genotoxic effect of FFA, as assessed by SCGE, is minimal in V79 cells. PMID:27226491

  4. Transfection of Platyhelminthes

    PubMed Central

    Moguel, Bárbara; Bobes, Raúl J.; Carrero, Julio C.; Laclette, Juan P.

    2015-01-01

    Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species. PMID:26090388

  5. Transfection of Platyhelminthes.

    PubMed

    Moguel, Bárbara; Bobes, Raúl J; Carrero, Julio C; Laclette, Juan P

    2015-01-01

    Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species.

  6. Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions

    PubMed Central

    Backliwal, Gaurav; Hildinger, Markus; Chenuet, Sebastien; Wulhfard, Sarah; De Jesus, Maria; Wurm, Florian M.

    2008-01-01

    Transient transfection allows for fast production of recombinant proteins. However, the current bottlenecks in transient transfection are low titers and low specific productivity compared to stable cell lines. Here, we report an improved transient transfection protocol that yields titers exceeding 1 g/l in HEK293E cells. This was achieved by combining a new highly efficient polyethyleneimine (PEI)-based transfection protocol, optimized gene expression vectors, use of cell cycle regulators p18 and p21, acidic Fibroblast Growth Factor, exposure of cells to valproic acid and consequently the maintenance of cells at high cell densities (4 million cells/ml). This protocol was reproducibly scaled-up to a working volume of 2 l, thus delivering >1 g of purified protein just 2 weeks after transfection. This is the fastest approach to gram quantities of protein ever reported from cultivated mammalian cells and could initiate, upon further scale-up, a paradigm shift in industrial production of such proteins for any application in biotechnology. PMID:18617574

  7. Establishment of human endothelial cell lines in a serum-free culture and its application for expression of transfected prepro endothelin gene.

    PubMed

    Kobayashi, M; Kondo, M; Mitsui, Y

    1991-12-01

    Endothelial cells cover the inner layer of vascular tube and are involved in the pathogenesis of some diseases in heart and brain. A development of endothelial cell lines in a serum-free culture lead to our discovery of endothelin (ET) and is expected to be breakthrough for other new fields of medical science. In this paper, we report establishment of vascular endothelial cell lines in serum-free and protein-free culture, transfection of prepro ET cDNA into them, and it's expression and processing to mature ET. A human endothelial cell line, ECV304, has been subcultivated in medium with 10% fetal bovine serum. We tested serum-free culture of this cell line and a several months later, established a cell line, t-HUE2 which would show stable growth in serum-free medium ASF301 containing EGF, insulin, transferrin and albumin as protein components. After additional 12-months' struggle for obtaining a new cell line which can grow without any protein components, we established t-HUE4 cell line which can be subcultivated in basal medium of HamF-12. Although the cell line was derived from human umbilical vein endothelial cells, production and secretion of ET by the cell line into culture medium was not detected with radioimmunoassay method. By transfecting prepro ET cDNA into t-HUE2 and Raji (a human B cell line), we examined the regulation of gene expression in the transformed t-HUE2 and Raji cell lines. The results indicated that all the transformants expressed high level of prepro ET mRNA but that Raji transformant did not produce detectable amounts of ET nor ER precursor protein (big ET). In addition, conversion ratio of ET to big ET in t-HUE2 transformants was much higher than those in normal endothelial cells. Thus, post-transcriptional regulation of prepro ET gene in human B lymphocyte cell line and induction of ET converting enzyme in human vascular endothelial cell line were suggested. In addition to the present study, usefulness of t-HUE2 cell line in serum

  8. Optimal conditions for freezing CHO-S and HEK293-EBNA cell lines: influence of Me2SO, freeze density, and PEI-mediated transfection on revitalization and growth of cells, and expression of recombinant protein.

    PubMed

    Kleman, Marika I; Oellers, Kerstin; Lullau, Elke

    2008-08-01

    To avoid the time consuming, labor intensive seed-train expansion and to improve production reliability and consistency, portions of bulk cryopreserved cells from the same cultivation can be utilized as inocula or alternatively may be used to undertake transient transfections for large-scale bioreactor production. In this study, the conditions for large-scale freezing in cryobags were optimized utilizing a design of experiment approach. We showed that relatively high density of 30-40 x 10(6) cells/mL and relatively low Me(2)SO concentrations of 5-6% in the freezing media are optimal to freeze HEK293-EBNA and CHO-S cells in a controlled manner in order to achieve high viable cell recovery and growth post-thawing. The immediate transfer of freshly thawed cells into culture medium resulted in better cell growth compared to cells that were centrifuged in order to remove Me(2)SO. This was the case as long as the residual Me(2)SO did not exceed 0.2-0.3%. The best time to perform transient 25 kDa polyethylenimine-mediated transfection of pCEP4-EGFP plasmid into freshly thawed, one-step inoculated cells is after 72-96 h in culture. At this time point, the numbers of EGFP-positive cells in the freshly thawed culture mimic perfectly that of cells grown continuously. Finally, our data showed that it is possible to freeze transiently polyethyleneimine-transfected HEK293-EBNA cells and maintain growth rate and expression of recombinant protein following thawing. The optimal time point for freezing cells was 4 h after transfection.

  9. L-FABP T94A decreased fatty acid uptake and altered hepatic triglyceride and cholesterol accumulation in Chang liver cells stably transfected with L-FABP.

    PubMed

    Gao, Na; Qu, Xia; Yan, Jin; Huang, Qi; Yuan, Hao-Yong; Ouyang, Dong-Sheng

    2010-12-01

    Liver fatty acid-binding protein (L-FABP, FABP1) is a highly conserved key factor in lipid metabolism. This study was undertaken to verify whether the T94A mutation in the L-FABP gene affects fatty acid uptake and intracellular esterification into specific lipid pools. Candidate SNPs were recreated using site-directed mutagenesis and tested for physical function in stably transfected Chang liver cell lines. We found that the T94A mutant of L-FABP lowered FFA uptake but had no effect on FFA efflux. L-FABP T94A-expressing cells showed decreased triglyceride content and increased cholesterol accumulation compared to the wild-type control for cells incubated with an FFA mixture (oleate: palmitate, 2:1 ratio). In conclusion, our study provided additional indications of the functional relevance of the L-FABP T94A SNP in hepatic fatty acid and lipid metabolism in humans.

  10. Estrogen response in the hFOB 1.19 human fetal osteoblastic cell line stably transfected with the human estrogen receptor gene.

    PubMed

    Harris, S A; Tau, K R; Enger, R J; Toft, D O; Riggs, B L; Spelsberg, T C

    1995-10-01

    The gene coding for the human wild-type estrogen receptor (ER) was stably transfected into the human fetal osteoblastic cell line hFOB 1.19, a clonal cell line which is conditionally immortilized with a temperature sensitive mutant of SV40 large T antigen (tsA58). Five subclones were obtained which express various levels of ER mRNA and protein. The subclone with the highest level of functional (nuclear bound) ER, hFOB/ER9, contained 3,931 (+/- 1,341) 17beta-estradiol molecules bound/nucleus as determined by the nuclear binding (NB) assay. Using the dextran coated charcoal (DCC) method, the level of total cytosolic ER measured was 204 (+/- 2) fmol/mg protein. This subclone was examined further for estradiol (E2) responsiveness. The ER expressed in hFOB/ER9 cells was shown to be functional using a transiently transfected ERE-TK-luciferase construct. Expression of luciferase from this construct increased approximately 25-fold in hFOB/ER9 cells following 10(-9)M E2 treatment. This effect on ERE-TK-luciferase expression was both dose and steroid dependant. Further, treatment of hFOB/ER9 cells with 10(-9)M E2 resulted in a 2.5-4.0-fold increase in endogenous progesterone receptor (PR) levels detected by steroid binding assays, and a noticeable increase in both the A and B forms of PR by western blot assay. The establishment of this estrogen responsive human osteoblastic cell line should provide an excellent model system for the study of estrogen action on osteoblast function.

  11. Regression of intracerebral rat glioma isografts by therapeutic subcutaneous immunization with interferon-gamma, interleukin-7, or B7-1-transfected tumor cells.

    PubMed

    Visse, E; Siesjö, P; Widegren, B; Sjögren, H O

    1999-01-01

    Progress in the definition of the roles of various costimulators and cytokines in determining the type and height of immune responses has made it important to explore genetically altered tumor cells expressing such molecules for therapeutic immunizations. We have studied the effect of therapeutic subcutaneous (s.c.) immunizations on the growth of preexisting intracerebral brain tumor isografts in the rat. Transfectant glioma cell clones expressing either rat interferon-gamma (IFN-gamma), rat interleukin-7 (IL-7), or rat B7-1 were selected. After irradiation (80 Gy) the clones were used for immunization (administered in up to four s.c. doses in a hind leg over 14-day intervals starting 1 day after the intracranial isografting of the parental tumor). Significant growth inhibition of the intracerebral parental tumors was induced by transfectants expressing IFN-gamma and IL-7, respectively. The strongest effect was observed with IFN-gamma-expressing cells, resulting in cures in 37% of the males and in 100% of the females. Immunization with IL-7 had a similar, strong initial effect, with significantly prolonged survival in the majority of the rats but a lower final cure rate (survival for >150 days). The B7-1-expressing tumor clones induced cures in seven of eight female rats; however, no cures were seen in the male rats. It was also shown that the B7-1-expressing cells were themselves strongly immunogenic in female rats, requiring high cell numbers to result in a progressively growing tumor upon s.c. isografting; this was not the case in male rats. As a whole, the results imply that despite the unfavorable location of intracerebral tumors, therapeutic s.c. immunizations with certain types of genetically altered tumor cells can induce complete regressions with permanent survival and without gross neurological or other apparent signs of brain damage. The present results demonstrate complete regressions when immunizations are initiated shortly after intracranial

  12. Processing of two homologous precursors, pro-neuropeptide Y and pro-pancreatic polypeptide, in transfected cell lines expressing different precursor convertases.

    PubMed

    Wulff, B S; Johansen, T E; Dalbøge, H; O'Hare, M M; Schwartz, T W

    1993-06-25

    The processing of two homologous precursors, pro-neuropeptide Y (pro-NPY) and pro-pancreatic poly-peptide (pro-PP), was studied in four neuroendocrine cell lines after transfection: CA-77 medullary thyroid carcinoma cells, AtT-20 corticotrope pituitary cells, RIN2A-19 pancreatic endocrine cells, and NB1 neuroblastoma cells. Northern blot analysis indicated that the AtT-20 cells only expressed precursor convertase 3; in contrast, NB1 cells only expressed precursor convertase 2, whereas the RIN2A-19 and CA-77 cells expressed both enzymes. Despite these differences in expression pattern of precursor convertases the four cell lines were, surprisingly, indistinguishable in respect to their processing of pro-PP and pro-NPY. In all four cell lines, pro-NPY was almost completely converted to NPY, and, in all four cell lines, only around 50% of the PP precursor was converted to PP. The relatively poor processing efficiency of pro-PP was rather similar to the processing efficiency of the endogenously produced precursors in the respective cell lines, pro-calcitonin (CA-77), proopiomelanocortin (AtT-20), proinsulin (RIN2A-19), and pro-vasoactive intestinal polypeptide (NB1). At least in the CA-77 cells, NPY and PP were apparently sorted to the regulated secretory pathway, as upon stimulation with secretagogue the release of the transfected peptides increased in parallel with the endogenously expressed peptide, calcitonin gene-related peptide. Mutagenesis studies showed that on the N-terminal side of the di-basic processing site, the otherwise important difference in structure between PP and NPY, a proline for glutamine in position 34, was not responsible for the difference in processing efficiency. On the C-terminal side of the processing site, the efficient processing of pro-NPY could not be transferred to pro-PP by exchanging the whole C-terminal domains of the precursors. It is concluded that pro-NPY is processed more efficiently than pro-PP in all neuroendocrine cell lines

  13. Modification of tumor cell exosome content by transfection with wt-p53 and microRNA-125b expressing plasmid DNA and its effect on macrophage polarization.

    PubMed

    Trivedi, M; Talekar, M; Shah, P; Ouyang, Q; Amiji, M

    2016-01-01

    Exosomes are responsible for intercellular communication between tumor cells and others in the tumor microenvironment. These microvesicles promote oncogensis and can support towards metastasis by promoting a pro-tumorogenic environment. Modifying the exosomal content and exosome delivery are emerging novel cancer therapies. However, the clinical translation is limited due to feasibility of isolating and delivery of treated exosomes as well as an associated immune response in patients. In this study, we provide proof-of-concept for a novel treatment approach for manipulating exosomal content by genetic transfection of tumor cells using dual-targeted hyaluronic acid-based nanoparticles. Following transfection with plasmid DNA encoding for wild-type p53 (wt-p53) and microRNA-125b (miR-125b), we evaluate the transgene expression in the SK-LU-1 cells and in the secreted exosomes. Furthermore, along with modulation of wt-p53 and miR-125b expression, we also show that the exosomes (i.e., wt-p53/exo, miR-125b/exo and combination/exo) have a reprogramed global miRNA profile. The miRNAs in the exosomes were mainly related to the activation of genes associated with apoptosis as well as p53 signaling. More importantly, these altered miRNA levels in the exosomes could mediate macrophage repolarization towards a more pro-inflammatory/antitumor M1 phenotype. However, further studies, especially in vivo studies, are warranted to assess the direct influence of such macrophage reprogramming on cancer cells and oncogenesis post-treatment. The current study provides a novel platform enabling the development of therapeutic strategies affecting not only the cancer cells but also the tumor microenvironment by utilizing the 'bystander effect' through genetic transfer with secreted exosomes. Such modification could also support antitumor environment leading to decreased oncogenesis. PMID:27500388

  14. Modification of tumor cell exosome content by transfection with wt-p53 and microRNA-125b expressing plasmid DNA and its effect on macrophage polarization

    PubMed Central

    Trivedi, M; Talekar, M; Shah, P; Ouyang, Q; Amiji, M

    2016-01-01

    Exosomes are responsible for intercellular communication between tumor cells and others in the tumor microenvironment. These microvesicles promote oncogensis and can support towards metastasis by promoting a pro-tumorogenic environment. Modifying the exosomal content and exosome delivery are emerging novel cancer therapies. However, the clinical translation is limited due to feasibility of isolating and delivery of treated exosomes as well as an associated immune response in patients. In this study, we provide proof-of-concept for a novel treatment approach for manipulating exosomal content by genetic transfection of tumor cells using dual-targeted hyaluronic acid-based nanoparticles. Following transfection with plasmid DNA encoding for wild-type p53 (wt-p53) and microRNA-125b (miR-125b), we evaluate the transgene expression in the SK-LU-1 cells and in the secreted exosomes. Furthermore, along with modulation of wt-p53 and miR-125b expression, we also show that the exosomes (i.e., wt-p53/exo, miR-125b/exo and combination/exo) have a reprogramed global miRNA profile. The miRNAs in the exosomes were mainly related to the activation of genes associated with apoptosis as well as p53 signaling. More importantly, these altered miRNA levels in the exosomes could mediate macrophage repolarization towards a more pro-inflammatory/antitumor M1 phenotype. However, further studies, especially in vivo studies, are warranted to assess the direct influence of such macrophage reprogramming on cancer cells and oncogenesis post-treatment. The current study provides a novel platform enabling the development of therapeutic strategies affecting not only the cancer cells but also the tumor microenvironment by utilizing the ‘bystander effect' through genetic transfer with secreted exosomes. Such modification could also support antitumor environment leading to decreased oncogenesis. PMID:27500388

  15. Development of stably transfected human and rat hepatoma cell lines for the species-specific assessment of xenobiotic response enhancer module (XREM)-dependent induction of drug metabolism.

    PubMed

    Fery, Yvonne; Mueller, Stefan O; Schrenk, Dieter

    2010-11-01

    Based on our current knowledge, PXR holds a key position in the induction of a selective battery of enzymes and transporters of drug metabolism. In order to prevent serious adverse drug effects or unpredicted drug-drug interactions (DDI), it is compulsory to investigate the possible inducing potency of drugs under development. Furthermore, analysis of the inducing potency of environmental pollutants and new or manufactured chemicals is part of toxicological risk assessment. In non-transfected human HepG2 and rat H4IIE hepatoma cells, we examined the characteristics of expression of 45 genes involved in drug metabolism. A few gene products such as CYP2B6 or CYP3A4 mRNA were prominent in HepG2 cells while their major rat counterparts were, e.g., CYP2B3 or CYP3A1/3A3. Furthermore, a number of xenobiotic receptors including PXR were expressed in both cell lines. A number of genes were regulated in a cell type and species-specific manner after incubation with the prototypical PXR agonists rifampicin or dexamethasone, respectively. Then, we established cell-based reporter gene assays for screening for PXR-dependent induction of drug metabolism. HepG2 and H4IIE cells were stably transfected with a reporter gene containing PXR responsive elements (XREMs) which mediate the induction of PXR target genes such as CYP3A enzymes. With both stable cell lines the CYP inducers clotrimazole, dexamethasone, omeprazole, phenobarbital, rifampicin, as well as the drug candidate EMD 392949 and the brominated flame retardants hexabromocylododecane (HBCD) and a pentabromodiphenyl ether (pentaBDE) mixture were screened. In the human HepG2-XREM3 and rat H4IIE-XREM3 cells, clotrimazole and HBCD were found as common activators of the human and rat PXR whereas pentaBDE was more effective with the human cell system. Omeprazole and phenobarbital did not induce the rat PXR-dependent reporter gene expression in H4IIE-XREM3 cells, while a moderate increase was found in HepG2-XREM3 cells. EMD 392949

  16. 1,25-dihydroxyvitamin D inhibits vitamin E succinate-induced apoptosis in C3H10T1/2 cells but not Harvey ras-transfected cells.

    PubMed

    Stedman, Lynn; Nickel, Kwangok P; Castillo, S Sianna; Andrade, Juan; Burgess, John R; Teegarden, Dorothy

    2003-01-01

    In this study, the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on regulation of apoptosis was compared in control C3H10T1/2 mouse fibroblast cells and those transfected with the Harvey ras oncogene. A known apoptotic stimulator, vitamin E succinate (VES), reduced cell number in a time- and dose-dependent manner in both cell types. In an assay for viable cells, there were significantly more C3H10T1/2 cells cotreated with VES and 1,25(OH)2D3 (-5.0 +/- 10.5% of vehicle-treated controls) compared to VES alone treated cells (-60.8 +/- 5.6%). In contrast, 1,25(OH)2D3 did not change the percentage of viable cells following treatment by VES in ras-transfected cells [-67.3 +/- 7.5%, VES alone compared to 57.3 +/-v 15.7% with VES and 1,25(OH)2D3 ]. Further studies confirmed that 1,25(OH)2D3 inhibited VES-mediated apoptosis (1.27 +/- 0.34-fold over vehicle control) compared to VES treatment alone (2.29 +/- 0.56-fold increase) in C3H10T1/2 cells, but not in ras-transfected cells [3.07 +/- 0.43-fold increase, VES treatment alone; 3.64 +/- 0.42-fold increase, VES and 1,25(OH)2D3]. Both C3H10T1/2 and ras-transfected cells treated with VES had increased concentrations of cellular VES with very little change in a-tocopherol, indicating that the cells took up VES intact. In addition, both cell lines contained similar levels of nuclear vitamin D receptor (VDR); however, the ras-transfected cells had reduced VDRE transcriptional activity. In conclusion, VES exerts its effect intact and 1,25(OH)2D3 preferentially protects C3H10T1/2 cells, whereas ras-transformed cells were not protected from VES-mediated apoptosis.

  17. Conversion of MDCK cell line to suspension culture by transfecting with human siat7e gene and its application for influenza virus production.

    PubMed

    Chu, Chia; Lugovtsev, Vladimir; Golding, Hana; Betenbaugh, Michael; Shiloach, Joseph

    2009-09-01

    MDCK cells are currently being considered as an alternative to embryonated eggs for influenza virus propagation and hemagglutinin (HA) production intended for vaccine manufacturing. MDCK cells were found suitable for the virus production but their inability to grow in suspension burdens the process of scale up and hence their production capability. Anchorage-dependent MDCK cells were converted to anchorage-independent cells, capable of growing in suspension as a result of transfection with the human siat7e gene (ST6GalNac V). This gene was previously identified as having an important role in cellular adhesion when the transcriptions of genes from anchorage-dependent and anchorage-independent HeLa cells were compared. Unlike the parental MDCK cells, the siat7e-expressing cells were capable of growing in shake flasks as suspension cultures, achieving maximum concentration of 7 x 10(5) cells/mL while keeping close to 100% viability throughout the growth phase. In production experiments, the siat7e-expressing cells were infected with the Influenza B/Victoria/504/2000 strain. It was determined that the cell-derived viruses retained similar antigenic properties as those obtained from egg-derived viruses and their nucleotide sequences were identical. The specific production of hemagglutinin (expressed in hemagglutination units per 10(6) cells) from the siat7e-expressing cells was approximately 20 times higher than the specific production from the parental MDCK cells. If this suspension process scales up, the production potential of HA from 10 L of siat7e-expressing cells at a concentration of 10(6) cells/mL would be equivalent to the amount of HA obtained from 10,000 embryonated eggs. PMID:19706449

  18. Conversion of MDCK cell line to suspension culture by transfecting with human siat7e gene and its application for influenza virus production

    PubMed Central

    Chu, Chia; Lugovtsev, Vladimir; Golding, Hana; Betenbaugh, Michael; Shiloach, Joseph

    2009-01-01

    MDCK cells are currently being considered as an alternative to embryonated eggs for influenza virus propagation and hemagglutinin (HA) production intended for vaccine manufacturing. MDCK cells were found suitable for the virus production but their inability to grow in suspension burdens the process of scale up and hence their production capability. Anchorage-dependent MDCK cells were converted to anchorage-independent cells, capable of growing in suspension as a result of transfection with the human siat7e gene (ST6GalNac V). This gene was previously identified as having an important role in cellular adhesion when the transcriptions of genes from anchorage-dependent and anchorage-independent HeLa cells were compared. Unlike the parental MDCK cells, the siat7e-expressing cells were capable of growing in shake flasks as suspension cultures, achieving maximum concentration of 7 × 105 cells/mL while keeping close to 100% viability throughout the growth phase. In production experiments, the siat7e-expressing cells were infected with the Influenza B/Victoria/504/2000 strain. It was determined that the cell-derived viruses retained similar antigenic properties as those obtained from egg-derived viruses and their nucleotide sequences were identical. The specific production of hemagglutinin (expressed in hemagglutination units per 106 cells) from the siat7e-expressing cells was approximately 20 times higher than the specific production from the parental MDCK cells. If this suspension process scales up, the production potential of HA from 10 L of siat7e-expressing cells at a concentration of 106 cells/mL would be equivalent to the amount of HA obtained from 10,000 embryonated eggs. PMID:19706449

  19. Cytotoxic activity of Bacillus thuringiensis Cry proteins on mammalian cells transfected with cadherin-like Cry receptor gene of Bombyx mori (silkworm).

    PubMed Central

    Tsuda, Yoko; Nakatani, Fumiki; Hashimoto, Keiko; Ikawa, Satoshi; Matsuura, Chikako; Fukada, Takashi; Sugimoto, Kenji; Himeno, Michio

    2003-01-01

    Cry1Aa, an insecticidal protein produced by Bacillus thuringiensis, has been shown to bind to cadherin-like protein, BtR175, in Bombyx mori (silkworm) midgut. We previously reported three variant alleles of BtR175 (BtR175a, b and c). When transiently expressed in COS7 cells, all the three BtR175 variants bound to Cry1Aa. We stably expressed BtR175b in HEK293 cells. These BtR175b-expressing cells swelled and died in the presence of activated Cry1Aa in a dose- and time-dependent manner, showing that BtR175b itself can impart Cry1Aa-susceptibility to mammalian cells. These cells were more susceptible to Cry1Aa than to Cry1Ab and Cry1Ac. Since dispersed B. mori midgut cells were reported to be highly susceptible to Cry1Ac, this result suggested that other Cry1Ac-specific receptor(s) were simultaneously working with BtR175 in the midgut cells. Advantages are also discussed of applying these transfected mammalian cells to toxicity assays of mutant Cry proteins. PMID:12403648

  20. CYP3A4-transfected Caco-2 cells as a tool for understanding biochemical absorption barriers: studies with sirolimus and midazolam.

    PubMed

    Cummins, Carolyn L; Jacobsen, Wolfgang; Christians, Uwe; Benet, Leslie Z

    2004-01-01

    CYP3A4-transfected Caco-2 cells were used as an in vitro system to predict the importance of drug metabolism and transport on overall drug absorption. We examined the transport and metabolism of two drugs; midazolam, an anesthetic agent and CYP3A4 substrate, and sirolimus, an immunosuppressant and a dual CYP3A4/P-glycoprotein (P-gp) substrate, in the presence of cyclosporine (CsA, a CYP3A4/P-gp inhibitor) or N-[4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl]-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine (GG918) (an inhibitor of P-gp and not CYP3A4). All major CYP3A4 metabolites were formed in the cells (1-OH > 4-OH midazolam and 39-O-desmethyl > 12-OH > 11-OH sirolimus), consistent with results from human liver microsomes. There was no bidirectional transport of midazolam across CYP3A4-transfected Caco-2 cells, whereas there was a 2.5-fold net efflux of sirolimus (1 microM) that disappeared in the presence of CsA or GG918. No change in the absorption rate or extraction ratio (ER) for midazolam was observed when P-gp was inhibited with GG918. Addition of GG918 had a modest impact on the absorption rate and ER for sirolimus (increased 58% and decreased 25%, respectively), whereas a 6.1-fold increase in the absorption rate and a 75% decrease in the ER were found when sirolimus was combined with CsA. Although both midazolam and sirolimus metabolites were preferentially excreted to the apical compartment, only sirolimus metabolites were transported by P-gp as determined from inhibition studies with GG918. Using CYP3A4-transfected Caco-2 cells we determined that, in contrast to P-gp, CYP3A4 is the major factor limiting sirolimus absorption. The integration of CYP3A4 and P-gp into a combined in vitro system was critical to unveil the relative importance of each biochemical barrier. PMID:14569063

  1. Construction of a tissue engineered intervertebral disc with high biological activity using an allogeneic intervertebral disc supplemented with transfected nucleus pulposus cells expressing exogenous dopamine beta-hydroxylase.

    PubMed

    Bai, M; Wang, Y H; Yin, H P; Li, S W

    2015-09-09

    This study addressed the in vitro construction and biological activity of tissue engineered intervertebral discs with exogenous human dopamine beta-hydroxylase (DBH) nucleus pulposus cells. pSNAV2.0-DBH expression plasmids were utilized to enhance the survival rates of intervertebral disc tissue cells. Various concentrations of transfected nucleus pulposus cells were injected into the discs, and DBH mRNA expression was determined using polymerase chain reaction amplification. Polysaccharide content and total collagen protein content in the engineered disc nucleus pulposus tissue were determined. The visible fluorescence intensities of the 1 x 10(5) and 1 x 10(6) groups vs the 1 x 10(4) group were significantly increased (P < 0.05); no significant difference was observed between the 1 x 10(5) and 1 x 10(6) groups (P > 0.05) at 7 days after injection. DBH mRNA expression could be detected in the all but the EGFP control group at 14 days culture. No significant difference was observed in the protein content between the 1 x 10(4) and the control groups at various times, while the protein content was significantly higher in the 1 x 10(5) vs the control and the 1 x 10(4) groups at 7-, 14-, and 21-day cultures. These results demonstrate that a tissue engineered intervertebral disc with high biological activity can be constructed by utilizing allogeneic intervertebral discs stored in liquid nitrogen and a 1 x 10(5) transfected nucleus pulposus cell complex with in vitro culture for 14 days. This model can be used in animal experiments to study the biological activity of the engineered discs.

  2. Construction of a tissue engineered intervertebral disc with high biological activity using an allogeneic intervertebral disc supplemented with transfected nucleus pulposus cells expressing exogenous dopamine beta-hydroxylase.

    PubMed

    Bai, M; Wang, Y H; Yin, H P; Li, S W

    2015-01-01

    This study addressed the in vitro construction and biological activity of tissue engineered intervertebral discs with exogenous human dopamine beta-hydroxylase (DBH) nucleus pulposus cells. pSNAV2.0-DBH expression plasmids were utilized to enhance the survival rates of intervertebral disc tissue cells. Various concentrations of transfected nucleus pulposus cells were injected into the discs, and DBH mRNA expression was determined using polymerase chain reaction amplification. Polysaccharide content and total collagen protein content in the engineered disc nucleus pulposus tissue were determined. The visible fluorescence intensities of the 1 x 10(5) and 1 x 10(6) groups vs the 1 x 10(4) group were significantly increased (P < 0.05); no significant difference was observed between the 1 x 10(5) and 1 x 10(6) groups (P > 0.05) at 7 days after injection. DBH mRNA expression could be detected in the all but the EGFP control group at 14 days culture. No significant difference was observed in the protein content between the 1 x 10(4) and the control groups at various times, while the protein content was significantly higher in the 1 x 10(5) vs the control and the 1 x 10(4) groups at 7-, 14-, and 21-day cultures. These results demonstrate that a tissue engineered intervertebral disc with high biological activity can be constructed by utilizing allogeneic intervertebral discs stored in liquid nitrogen and a 1 x 10(5) transfected nucleus pulposus cell complex with in vitro culture for 14 days. This model can be used in animal experiments to study the biological activity of the engineered discs. PMID:26400296

  3. Liposome-mediated transfection of wild-type P53 DNA into human prostate cancer cells is improved by low-frequency ultrasound combined with microbubbles

    PubMed Central

    BAI, WEN-KUN; ZHANG, WEI; HU, BING; YING, TAO

    2016-01-01

    Prostate cancer is a common type of cancer in elderly men. The aim of the present study was to evaluate the effects of ultrasound exposure in combination with SonoVue microbubbles on liposome-mediated transfection of wild-type P53 genes into human prostate cancer cells. PC-3 human prostate cancer cells were exposed to ultrasound; duty cycle was controlled at 20% (2 sec on, 8 sec off) for 5 min with and without SonoVue microbubble echo-contrast agent using a digital sonifier (frequency, 21 kHz; intensity, 46 mW/cm2). The cells were divided into eight groups, as follows: Group A (SonoVue + wild-type P53), group B (ultrasound + wild-type P53), group C (SonoVue + ultrasound + wild-type P53), group D (liposome + wild-type P53), group E (liposome + SonoVue + wild-type P53), group F (liposome + wild-type P53 + ultrasound), group G (liposome + wild-type P53 + ultrasound + SonoVue) and the control group (wild-type P53). Following treatment, a hemocytometer was used to measure cell lysis, reverse transcription-quantitative polymerase chain reaction and western blotting were performed to detect P53 gene transfection efficiency, Cell Counting Kit-8 was employed to reveal cell proliferation and Annexin V/propidium iodide staining was used to determine cell apoptosis. Cell lysis was minimal in each group. Wild-type P53 gene and protein expression were significantly increased in the PC-3 cells in group G compared with the control and all other groups (P<0.01). Cell proliferation was significantly suppressed in group G compared with the control group and all other groups (P<0.01). Cell apoptosis levels in group G were significantly improved compared with the control group and all other groups (P<0.01). Thus, the results of the present study indicate that the use of low-frequency and low-energy ultrasound in combination with SonoVue microbubbles may be a potent physical method for increasing liposome gene delivery efficiency. PMID:27313702

  4. Epstein-Barr virus infection of CR2-transfected epithelial cells reveals the presence of MHC class II on the virion.

    PubMed

    Knox, P G; Young, L S

    1995-10-20

    Epithelial cell lines transfected with the Epstein-Barr virus (EBV) receptor CR2 are susceptible to infection by EBV. Following infection with certain EBV strains we found that these cells became positive for MHC class II. The class II was confirmed as being of viral and not target cell origin by immunostaining with HLA-specific monoclonal antibodies. Electron microscopic immunogold staining confirmed the presence of MHC class II on the surface of the virion. While some MHC class I was also found on the EB virion, other cell surface molecules were absent. Dual color immunofluorescence and confocal microscopy analysis demonstrated colocalization of class II with EBV-encoded structural proteins (MA and VCA) in infected epithelial cells. However, preincubation of EBV with antibodies against either MHC class I or MHC class II failed to affect either EBV binding or EBV infection. The presence of MHC on the surface of the EB virion may be a consequence of the intracellular pathways through which productive virus exits from the cell and may influence the target cell tropism of EBV. PMID:7483258

  5. The 5' flanking region of the gene for the Epstein-Barr virus-encoded nuclear antigen 2 contains a cell type specific cis-acting regulatory element that activates transcription in transfected B-cells.

    PubMed Central

    Ricksten, A; Olsson, A; Andersson, T; Rymo, L

    1988-01-01

    We have recently identified the promoter that positions the initiation (cap) site for RNA encoding the Epstein-Barr virus (EBV) determined nuclear antigen 2 (EBNA2) in transfected COS-1 cells. The cells were transfected with recombinant vectors that contained the BamHI WYH region of the EBV genome. In order to delineate regulatory DNA sequences required for the expression of EBNA2 the 5' flanking region of the gene was linked to reporter genes in expression vectors and transfected into EBV genome-negative lymphoid DG75 cells. We demonstrate that several cis-acting elements contribute to a transcriptional enhancer activity found in the region between nucleotides-553 and -86 relative to the cap site. The enhancer was active in lymphoid DG75 cells but not in HeLa cells and stimulated transcription also from the heterologous thymidine kinase (TK) and beta-globin promoters. Nuclear extracts of lymphoid cells contained protein factors that bound to the enhancer. The in vitro introduction of a mutation in the enhancer sequence that substantially reduced the transcription stimulatory activity concurrently blocked the binding of one of the factors. Images PMID:2843816

  6. Improving the osteogenesis of human bone marrow mesenchymal stem cell sheets by microRNA-21-loaded chitosan/hyaluronic acid nanoparticles via reverse transfection.

    PubMed

    Wang, Zhongshan; Wu, Guangsheng; Wei, Mengying; Liu, Qian; Zhou, Jian; Qin, Tian; Feng, Xiaoke; Liu, Huan; Feng, Zhihong; Zhao, Yimin

    2016-01-01

    Cell sheet engineering has emerged as a novel approach to effectively deliver seeding cells for tissue regeneration, and developing human bone marrow mesenchymal stem cell (hBMMSC) sheets with high osteogenic ability is a constant requirement from clinics for faster and higher-quality bone formation. In this work, we fabricated biocompatible and safe chitosan (CS)/hyaluronic acid (HA) nanoparticles (NPs) to deliver microRNA-21 (miR-21), which has been proved to accelerate osteogenesis in hBMMSCs; then, the CS/HA/miR-21 NPs were cross-linked onto the surfaces of culture plates with 0.2% gel solution to fabricate miR-21-functionalized culture plates for reverse transfection. hBMMSC sheets were induced continuously for 14 days using a vitamin C-rich method on the miR-21-functionalized culture plates. For the characterization of CS/HA/miR-21 NPs, the particle size, zeta potential, surface morphology, and gel retardation were sequentially investigated. Then, the biological effects of hBMMSC sheets on the miR-21-functionalized culture plates were evaluated. The assay results demonstrated that the hBMMSC sheets could be successfully induced via the novel reverse transfection approach, and miR-21 delivery significantly enhanced the in vitro osteogenic differentiation of hBMMSC sheets in terms of upregulating calcification-related gene expression and enhancing alkaline phosphatase production, collagen secretion, and mineralized nodule formation. The enhanced osteogenic activity of hBMMSC sheets might promisingly lead to more rapid and more robust bone regeneration for clinical use. PMID:27274237

  7. Improving the osteogenesis of human bone marrow mesenchymal stem cell sheets by microRNA-21-loaded chitosan/hyaluronic acid nanoparticles via reverse transfection

    PubMed Central

    Wang, Zhongshan; Wu, Guangsheng; Wei, Mengying; Liu, Qian; Zhou, Jian; Qin, Tian; Feng, Xiaoke; Liu, Huan; Feng, Zhihong; Zhao, Yimin

    2016-01-01

    Cell sheet engineering has emerged as a novel approach to effectively deliver seeding cells for tissue regeneration, and developing human bone marrow mesenchymal stem cell (hBMMSC) sheets with high osteogenic ability is a constant requirement from clinics for faster and higher-quality bone formation. In this work, we fabricated biocompatible and safe chitosan (CS)/hyaluronic acid (HA) nanoparticles (NPs) to deliver microRNA-21 (miR-21), which has been proved to accelerate osteogenesis in hBMMSCs; then, the CS/HA/miR-21 NPs were cross-linked onto the surfaces of culture plates with 0.2% gel solution to fabricate miR-21-functionalized culture plates for reverse transfection. hBMMSC sheets were induced continuously for 14 days using a vitamin C-rich method on the miR-21-functionalized culture plates. For the characterization of CS/HA/miR-21 NPs, the particle size, zeta potential, surface morphology, and gel retardation were sequentially investigated. Then, the biological effects of hBMMSC sheets on the miR-21-functionalized culture plates were evaluated. The assay results demonstrated that the hBMMSC sheets could be successfully induced via the novel reverse transfection approach, and miR-21 delivery significantly enhanced the in vitro osteogenic differentiation of hBMMSC sheets in terms of upregulating calcification-related gene expression and enhancing alkaline phosphatase production, collagen secretion, and mineralized nodule formation. The enhanced osteogenic activity of hBMMSC sheets might promisingly lead to more rapid and more robust bone regeneration for clinical use. PMID:27274237

  8. Connexin 32 and its derived homotypic gap junctional intercellular communication inhibit the migration and invasion of transfected HeLa cells via enhancement of intercellular adhesion.

    PubMed

    Yang, Jie; Liu, Bing; Wang, Qin; Yuan, Dongdong; Hong, Xiaoting; Yang, Yan; Tao, Liang

    2011-01-01

    The effects of connexin (Cx) and its derived homotypic gap junctional intercellular communication (GJIC) between tumor cells on the invasion of metastatic cancers and the underlying mechanisms remain unclear. In this study, we investigated the influence of Cx32 and the homotypic GJIC mediated by this Cx on the migration, invasion and intercellular adhesion of transfected HeLa cells. The expression of Cx32 significantly increased cell adhesion and inhibited migration and invasion. The inhibition of GJIC by oleamide, a widely used GJIC inhibitor, reduced the enhanced adhesion and partly reversed the decreased migration and invasion that had been induced by Cx32 expression. Blockage of the p38 and extracellular signal-regulated kinase 1 and 2 mitogen-activated protein kinase (ERK1/2 MAPKs) pathways using their specific inhibitors attenuated the effects of Cx32, but not those of GJIC, on cell adhesion, migration and invasion. These results indicate that the homotypic GJIC mediated by Cx32, as well as the Cx itself, inhibit cell migration and invasion, most likely through the elevation of intercellular adhesion. The suppressive effect of Cx32 on the migration and invasion of cancer cells, but not that of its derived homotypic GJIC, partly depends on the activation of the p38 and the ERK1/2 MAPKs pathways.

  9. Accelerated Repair and Reduced Mutagenicity of DNA Damage Induced by Cigarette Smoke in Human Bronchial Cells Transfected with E.coli Formamidopyrimidine DNA Glycosylase

    PubMed Central

    Foresta, Mara; Izzotti, Alberto; La Maestra, Sebastiano; Micale, Rosanna; Poggi, Alessandro; Vecchio, Donatella; Frosina, Guido

    2014-01-01

    Cigarette smoke (CS) is associated to a number of pathologies including lung cancer. Its mutagenic and carcinogenic effects are partially linked to the presence of reactive oxygen species and polycyclic aromatic hydrocarbons (PAH) inducing DNA damage. The bacterial DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG) repairs both oxidized bases and different types of bulky DNA adducts. We investigated in vitro whether FPG expression may enhance DNA repair of CS-damaged DNA and counteract the mutagenic effects of CS in human lung cells. NCI-H727 non small cell lung carcinoma cells were transfected with a plasmid vector expressing FPG fused to the Enhanced Green Fluorescent Protein (EGFP). Cells expressing the fusion protein EGFP-FPG displayed accelerated repair of adducts and DNA breaks induced by CS condensate. The mutant frequencies induced by low concentrations of CS condensate to the Na+K+-ATPase locus (ouar) were significantly reduced in cells expressing EGFP-FPG. Hence, expression of the bacterial DNA repair protein FPG stably protects human lung cells from the mutagenic effects of CS by improving cells’ capacity to repair damaged DNA. PMID:24498234

  10. Expression and purification of soluble and stable ectodomain of natural killer cell receptor LLT1 through high-density transfection of suspension adapted HEK293S GnTI(-) cells.

    PubMed

    Bláha, Jan; Pachl, Petr; Novák, Petr; Vaněk, Ondřej

    2015-05-01

    Lectin-like transcript 1 (LLT1, gene clec2d) was identified to be a ligand for the single human NKR-P1 receptor present on NK and NK-T lymphocytes. Naturally, LLT1 is expressed on the surface of NK cells, stimulating IFN-γ production, and is up-regulated upon activation of other immune cells, e.g. TLR-stimulated dendritic cells and B cells or T cell receptor-activated T cells. While in normal tissues LLT1:NKR-P1 interaction (representing an alternative "missing-self" recognition system) play an immunomodulatory role in regulation of crosstalk between NK and antigen presenting cells, LLT1 is upregulated in glioblastoma cells, one of the most lethal tumors, where it acts as a mediator of immune escape of glioma cells. Here we report transient expression and characterization of soluble His176Cys mutant of LLT1 ectodomain in an eukaryotic expression system of human suspension-adapted HEK293S GnTI(-) cell line with uniform N-glycans. The His176Cys mutation is critical for C-type lectin-like domain stability, leading to the reconstruction of third canonical disulfide bridge in LLT1, as shown by mass spectrometry. Purified soluble LLT1 is homogeneous, deglycosylatable and forms a non-covalent homodimer whose dimerization is not dependent on presence of its N-glycans. As a part of production of soluble LLT1, we have adapted HEK293S GnTI(-) cell line to growth in suspension in media facilitating transient transfection and optimized novel high cell density transfection protocol, greatly enhancing protein yields. This transfection protocol is generally applicable for protein production within this cell line, especially for protein crystallography. PMID:25623399

  11. Inclusion of S-sepharose beads in the culture medium significantly improves recovery of secreted rBPI(21) from transfected CHO-K1 cells.

    PubMed

    Horwitz, A H; Carroll, S F; Williams, R E; Liu, P S

    2000-02-01

    rBPI(23), a recombinant N-terminal fragment of human bactericidal/permeability-increasing protein (BPI), kills gram-negative bacteria and binds endotoxin. rBPI(21), a variant, in which cysteine 132 is changed to alanine, retains the activities of rBPI(23). Initial attempts using conventional ion-exchange chromatography to purify rBPI(23) from culture supernatants of transfected CHO-K1 cells resulted in lower than expected yields. Also, ELISA of supernatants from CHO-K1 transfectants expressing rBPI(23) or rBPI(21) yielded variable signals. Results from pulse-chase experiments using [(35)S]methionine had indicated that rBPI(23) could not be detected in the culture medium by 7 h of chase, suggesting that these proteins were degraded and/or bound to cells, media components, or vessel surfaces. To address these issues, we developed a novel process whereby sterile S-Sepharose beads were added directly to the cell culture medium. For attached cells, the beads were added to confluent cultures with serum-free medium for the expression phase, while for suspension-adapted cells, beads were added at the beginning of culture growth. The S-Sepharose was then separated from cells and media and washed, and BPI was eluted with high-salt buffer. This approach yielded up to a 50-fold improvement in recovery of rBPI(23) and rBPI(21) from roller bottles, shake flasks, and 2-liter fermenters. It also resulted in improved detection and quantitation of secreted rBPI(23) and rBPI(21) by ELISA. Results of competition binding studies with iodinated rBPI(21) in conjunction with unlabeled rBPI(21) and rBPI(23) or with heparin demonstrated that these proteins bound specifically and with high affinity to heparan-containing sites on the surface of the CHO-K1 cells. We conclude that the S-Sepharose included in the culture medium captures the BPI protein products as they are secreted and protects them from degradation and/or irreversible binding to cell surfaces. This method has been scaled up to

  12. Transfection with extracellularly UV-damaged DNA induces human and rat cells to express a mutator phenotype towards parvovirus H-1

    SciTech Connect

    Dinsart, C.; Cornelis, J.J.; Klein, B.; van der Eb, A.J.; Rommelaere, J.

    1984-02-01

    Human and rat cells transfected with UV-irradiated linear double-stranded DNA from calf thymus displayed a mutator activity. This phenotype was identified by growing a lytic thermosensitive single-stranded DNA virus (parvovirus H-1) in those cells and determining viral reversion frequencies. Likewise, exogenous UV-irradiated closed circular DNAs, either double-stranded (simian virus 40) or single-stranded (phi X174), enhanced the ability of recipient cells to mutate parvovirus H-1. The magnitude of mutator activity expression increased along with the number of UV lesions present in the inoculated DNA up to a saturation level. Unirradiated DNA displayed little inducing capacity, irrespective of whether it was single or double stranded. Deprivation of a functional replication origin did not impede UV-irradiated simian virus 40 DNA from providing rat and human cells with a mutator function. Our data suggest that in mammalian cells a trans-acting mutagenic signal might be generated from UV-irradiated DNA without the necessity for damaged DNA to replicate.

  13. Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line

    SciTech Connect

    Jeang, K.T.; Cho, M.S.; Hayward, G.S.

    1984-10-01

    A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The authors isolated a clonal Ltk/sup +/ cell line which expressed the /sup 35/methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH/sub 2/p198-3 cells was phosphorylated (presumably by a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH/sub 2/l198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors.

  14. Transfected adenosine A1 receptor-mediated modulation of thrombin-stimulated phospholipase C and phospholipase A2 activity in CHO cells.

    PubMed

    Dickenson, J M; Hill, S J

    1997-02-19

    Thrombin receptor activation in Chinese hamster ovary (CHO) cells stimulates the hydrolysis of inositol phospholipids and the release of arachidonic acid. Our previous studies have shown that activation of the human transfected adenosine A1 receptor in CHO cells (CHO-A1) potentiates the accumulation of inositol phosphates elicited by endogenous P2U purinoceptors and CCKA receptors. In this study we have investigated whether adenosine A1 receptor activation can modulate thrombin-stimulated arachidonic acid release and/or inositol phospholipid hydrolysis in CHO-A1 cells. Thrombin stimulated [3H]arachidonic acid release and total [3H]inositol phosphate accumulation in CHO-A1 cells. Both these responses to thrombin were were insensitive to pertussis toxin. The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), potentiated thrombin-stimulated [3H]arachidonic acid. In marked contrast, PMA inhibited thrombin-stimulated [3H]inositol phosphate accumulation. The selective protein kinase C inhibitor Ro 31-8220 (3-¿1-[3-(2-isothioureido)propyl] indol-3-yl¿-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dione) had no effect on thrombin-stimulated [3H]arachidonic acid release but reversed the potentiation of thrombin-stimulated [3H]arachidonic acid release elicited by PMA. The selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) augmented the release of [3H]arachidonic acid produced by thrombin. Co-activation of the adenosine A1 receptor also potentiated thrombin-stimulated [3H]inositol phosphate accumulation. The synergistic interactions between the adenosine A1 receptor and thrombin were abolished in pertussis-toxin-treated cells. The potentiation of [3H]arachidonic acid release by CPA was blocked by the protein kinase C inhibitors Ro 31-8220 and GF 109203X (3-[1-[3-(dimethylamino)propyl]-1 H-indol-3-yl]-4-(1 H-indol-3-yl)- 1H-pyrrole-2,5-dione). In conclusion, thrombin receptor activation in CHO-A1 cells stimulates the accumulation of [3H

  15. Transfection in the third dimension

    PubMed Central

    Dhaliwal, Anandika; Oshita, Victor; Segura, Tatiana

    2013-01-01

    An understanding of parameters that modulate gene transfer in 3-D will assist in the formation of gene delivery systems and scaffolds, which can mediate efficient non-viral delivery for guiding in-vivo tissue regeneration and therapy. We have previously demonstrated the cell area and length, integrin expression, and RhoGTPases mediated signalling to be pivotal parameters that guide gene transfer to mouse mesenchymal stem cells (mMSCs) cultured in 2-D and are modulated by ECM proteins. In this study, we were interested in determining if cationic polymer mediated gene transfer to cells seeded in 3-D would occur through different mechanisms as compared to 2-D. In particular, we examined the endocytosis pathways used to internalize polyplexes, and the role of cytoskeletal dynamics and RhoGTPases on non-viral gene transfer for cells seeded in 2-D and 3-D. Inhibition of clathrin- and caveolae- mediated endocytosis resulted in more drastic decrease in overall transgene expression for cells seeded in 3-D than those in 2-D. In addition, polyplex internalization was only significantly decreased in 3-D when clathrin-mediated endocytosis was inhibited, while caveolae-mediated endocytosis inhibition for cells seeded in 2-D resulted in the strongest polyplex internalization inhibition. Actin and microtubule polymerization affected 2-D and 3-D transfection differently. Microtubule depolymerization enhanced transgene expression in 2-D, but inhibited transgene expression in 3-D. Last, inhibition of RhoGTPases also affected 2-D and 3-D transfection differently. The inhibition of ROCK effector resulted in a decrease of transgene expression and internalization for cells seeded in 3-D, but not 2-D and the inhibition of effector PAK1 resulted in an increase of transgene expression for both 2-D and 3-D. Overall, our study suggests that the process of gene transfer occurs through different mechanisms for cells seeded in 2-D compared to those seeded in 3-D. PMID:23929354

  16. Use of optogenetic technology in cell culture models

    NASA Astrophysics Data System (ADS)

    Erofeev, A. I.; Zakharova, O. A.; Matveev, M. V.; Terekhin, S. G.; Bezprozvanny, I. B.; Vlasova, O. L.

    2016-08-01

    Optogenetic is a powerful method that allows to modulate cellular physiological properties. In our article, we demonstrate changes of electrical properties of cellular membranes on HEK-293T and hippocampal neurons transfected with channelrhodopsins and halorhodopsins induced by blue and orange light stimulation.

  17. HD-03/ES: A Herbal Medicine Inhibits Hepatitis B Surface Antigen Secretion in Transfected Human Hepatocarcinoma PLC/PRF/5 Cells.

    PubMed

    Varma, Sandeep R; Sundaram, R; Gopumadhavan, S; Vidyashankar, Satyakumar; Patki, Pralhad S

    2013-01-01

    HD-03/ES is a herbal formulation used for the treatment of hepatitis B. However, the molecular mechanism involved in the antihepatitis B (HBV) activity of this drug has not been studied using in vitro models. The effect of HD-03/ES on hepatitis B surface antigen (HBsAg) secretion and its gene expression was studied in transfected human hepatocarcinoma PLC/PRF/5 cells. The anti-HBV activity was tested based on the inhibition of HBsAg secretion into the culture media, as detected by HBsAg-specific antibody-mediated enzyme assay (ELISA) at concentrations ranging from 125 to 1000  μ g/mL. The effect of HD-03/ES on HBsAg gene expression was analyzed using semiquantitative multiplex RT-PCR by employing specific primers. The results showed that HD-03/ES suppressed HBsAg production with an IC50 of 380  μ g/mL in PLC/PRF/5 cells for a period of 24 h. HD-03/ES downregulated HBsAg gene expression in PLC/PRF/5 cells. In conclusion, HD-03/ES exhibits strong anti-HBV properties by inhibiting the secretion of hepatitis B surface antigen in PLC/PRF/5 cells, and this action is targeted at the transcription level. Thus, HD-03/ES could be beneficial in the treatment of acute and chronic hepatitis B infections.

  18. The induction of serine/threonine protein phosphorylations by a PDGFR/TrkA chimera in stably transfected PC12 cells.

    PubMed

    Biarc, Jordane; Chalkley, Robert J; Burlingame, A L; Bradshaw, Ralph A

    2012-05-01

    Stably transfected PC12 cells expressing a chimeric receptor composed of the extracellular domain of the platelet-derived growth factor receptor BB and the transmembrane and intracellular domains of TrkA, the nerve growth factor receptor, were stimulated for 20 min with platelet-derived growth factor and the resulting phosphoproteome was determined from affinity purified tryptic peptides identified by tandem MS (MS/MS) analyses. The changes in the levels of individual phosphorylation sites in stimulated cells versus control were ascertained by the stable isotope labeling of amino acids in cell culture technique. A total of 2035 peptides (806 proteins) were indentified and quantified in both data sets. Of these, 424 phosphopeptides on 259 proteins were found to be up-regulated and 392 sites on 206 proteins were down-regulated (1.8-fold or more). Protein kinases and phosphatases, as well as sites in many proteins involved in G-protein signaling, were prominently represented in the up-regulated group and more than half of the kinase up-regulated phosphosites could be clustered into three sequence motifs; a similar distribution was also found for the down-regulated sites. A comparison of the up-regulated motif profile observed to that calculated from a previous study of the EGFR-induced phosphoproteome in human HeLa cells at the same time point showed a considerable amount of similarity, supporting the view that RTK signal transduction pathways and downstream modifications are likely to be extensively overlapping.

  19. HD-03/ES: A Herbal Medicine Inhibits Hepatitis B Surface Antigen Secretion in Transfected Human Hepatocarcinoma PLC/PRF/5 Cells

    PubMed Central

    Varma, Sandeep R.; Sundaram, R.; Gopumadhavan, S.; Vidyashankar, Satyakumar; Patki, Pralhad S.

    2013-01-01

    HD-03/ES is a herbal formulation used for the treatment of hepatitis B. However, the molecular mechanism involved in the antihepatitis B (HBV) activity of this drug has not been studied using in vitro models. The effect of HD-03/ES on hepatitis B surface antigen (HBsAg) secretion and its gene expression was studied in transfected human hepatocarcinoma PLC/PRF/5 cells. The anti-HBV activity was tested based on the inhibition of HBsAg secretion into the culture media, as detected by HBsAg-specific antibody-mediated enzyme assay (ELISA) at concentrations ranging from 125 to 1000 μg/mL. The effect of HD-03/ES on HBsAg gene expression was analyzed using semiquantitative multiplex RT-PCR by employing specific primers. The results showed that HD-03/ES suppressed HBsAg production with an IC50 of 380 μg/mL in PLC/PRF/5 cells for a period of 24 h. HD-03/ES downregulated HBsAg gene expression in PLC/PRF/5 cells. In conclusion, HD-03/ES exhibits strong anti-HBV properties by inhibiting the secretion of hepatitis B surface antigen in PLC/PRF/5 cells, and this action is targeted at the transcription level. Thus, HD-03/ES could be beneficial in the treatment of acute and chronic hepatitis B infections. PMID:23691296

  20. The use of transfected fibroblasts and transgenic mice establishes that stimulation of T cells by the Mycoplasma arthritidis mitogen is mediated by E alpha.

    PubMed

    Cole, B C; David, C S; Lynch, D H; Kartchner, D R

    1990-01-15

    Mycoplasma arthritidis produces a soluble protein which is active for murine and human lymphocytes when presented by Ia-bearing accessory cells. By using fibroblasts transfected in vitro with various class II Ag, we demonstrated that presentation of the M. arthritidis mitogen (MAM) to T cells was mediated by E alpha-containing molecules. We also showed that splenocytes from transgenic mice expressing E alpha heterozygously (B10.TRG E alpha+) or homozygously (B10.E alpha TG +/+) underwent a similar proliferation in response to MAM as compared with the failure of control B10.TRG E alpha- splenocytes to respond to MAM. Although splenocytes from inbred C3H and CBA mice exhibited much higher proliferative responses to MAM than did those from B10.TRG.E alpha+ or B10.E alpha TG +/+ mice, flow cytometry showed similar levels of E alpha expression. Furthermore, gamma-irradiated splenocytes from B10.TRG E alpha + mice presented MAM to T hybridoma cells with a similar efficacy as did splenocytes from C3H mice. The lesser response to MAM of lymphocytes from the E alpha transgenic mice as compared with those from C3H and B10.K mice was likewise not due to differential expression of their V beta TCR. We conclude that presentation of MAM to T cells is accomplished by E alpha-containing molecules. The studies also suggest that the conserved, nonpolymorphic regions of class II molecules may play an important role in host immune response to microbial products.

  1. Sepiapterin attenuates 1-methyl-4-phenylpyridinium-induced apoptosis in neuroblastoma cells transfected with neuronal NOS: role of tetrahydrobiopterin, nitric oxide, and proteasome activation.

    PubMed

    Shang, Tiesong; Kotamraju, Srigiridhar; Zhao, Hongtao; Kalivendi, Shasi V; Hillard, Cecilia J; Kalyanaraman, B

    2005-10-15

    In this study, we investigated the molecular mechanism of toxicity of 1-methyl-4-phenylpyridinium (MPP+), an ultimate toxic metabolite of a mitochondrial neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, that causes parkinsonism in experimental animals and humans. Using wild-type and human neuronal nitric oxide synthase (nNOS) stably transfected neuroblastoma cells (SH-SY5Y), we showed that nNOS overexpression in SH-SY5Y cells greatly enhanced proteasome activity and mitigated MPP+-induced apoptosis. During MPP+-induced oxidative stress, intracellular BH4 levels decreased, resulting in nNOS "uncoupling" (i.e., switching from nitric oxide to superoxide generation). Increasing the intracellular BH4 levels by sepiapterin supplementation restored the nNOS activity, inhibited superoxide formation, increased proteasome activity, decreased protein ubiquitination, and attenuated apoptosis in MPP+-treated cells. Implications of BH4 depletion in dopaminergic cells and sepiapterin supplementation to augment the striatal nNOS activity in the pathogenesis mechanism and treatment of Parkinson disease are discussed. PMID:16198233

  2. Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells.

    PubMed Central

    Cawley, K C; Akita, C G; Walsh, D A

    1989-01-01

    Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme. Images Fig. 1. Fig. 2. Fig. 3. PMID:2481439

  3. Optimization of Transfection Conditions for siRNA Screening.

    PubMed

    Montoya, Justin J; Azorsa, David O

    2016-01-01

    RNAi screening of mammalian cells is often performed using siRNAs and cationic lipids as transfection reagents. Efficiency of transfection depends on growth characteristics of the cells and the cationic lipid used. With a large selection of cationic lipids available, it can often be difficult to select the optimal lipid and lipid:siRNA (vol:wt) ratio. Here, we describe the process of optimizing siRNA transfection conditions for efficient reverse transfection of mammalian cells using specific positive and negative siRNA controls. PMID:27581281

  4. Cationic phospholipids: structure transfection activity relationships

    SciTech Connect

    Koynova, Rumiana; Tenchov, Boris

    2010-01-18

    Synthetic cationic lipids are presently the most widely used non-viral gene carriers. Examined here is a particularly attractive cationic lipid class, triester phosphatidylcholines (PCs) exhibiting low toxicities and good transfection efficiency. Similarly to other cationic lipids, they form stable complexes (lipoplexes) with the polyanionic nucleic acids. A summary of studies on a set of {approx}30 cationic PCs reveals the existence of a strong, systematic dependence of their transfection efficiency on the lipid hydrocarbon chain structure: transfection activity increases with increase of chain unsaturation from 0 to 2 double bonds per lipid and decreases with increase of chain length in the range {approx}30-50 total number of chain carbon atoms. Maximum transfection was observed for ethyl phosphate PCs (EPCs) with monounsaturated 14:1 chains (total of 2 double bonds and 30 chain carbon atoms). Lipid phase behavior is known to depend strongly on the chain molecular structure and the above relationships thus substantiate a view that cationic PC phase propensities are an important determinant of their activity. Indeed, X-ray structural studies show that the rate of DNA release from lipoplexes as well as transfection activity well correlate with non-lamellar phase progressions observed in cationic PC mixtures with membrane lipids. These findings appear to be of considerable interest because, according to current views, key processes in lipid-mediated transfection such as lipoplex disassembly and DNA release within the cells are believed to take place upon cationic lipid mixing with cellular lipids.

  5. Miglustat (NB-DNJ) works as a chaperone for mutated acid beta-glucosidase in cells transfected with several Gaucher disease mutations.

    PubMed

    Alfonso, Pilar; Pampín, Sandra; Estrada, Jorge; Rodríguez-Rey, José Carlos; Giraldo, Pilar; Sancho, Javier; Pocoví, Miguel

    2005-01-01

    Gaucher disease (GD) is a disorder of glycosphinglipid metabolism caused by deficiency of lysosomal acid beta-glucosidase (GC), resulting in progressive deposition of glucosylceramide in macrophages. The glucose analogue, N-butyl-deoxynojirimycin (NB-DNJ, Miglustat), is an inhibitor of the ceramide-specific glucosyltransferase (CSG) which catalyzes the first step of glycosphingolipids biosynthesis and is currently approved for the oral treatment of type 1 GD. Using site-directed mutagenesis, we constructed plasmids containing wild-type and several mutations in glucocerebrosidase (GBA) gene. The plasmids were transfected into COS-7 cells and stable transfected cell lines were obtained by geneticin (G418) selection. Cells were cultured during 6 days with medium with or without 10 microM NB-DNJ. The addition of NB-DNJ to COS-7 cell medium leads to 1.3-, 2.1-, 2.3-, 3.6-, and 9.9-fold increase in the activity of S364R, wild-type, N370S, V15M, and M123T GC, respectively. However, no significant changes were observed in the activity of the L444P, L336P, and S465del mutated proteins, but a small decrease in the rare P266L variant was observed. These results suggest that NB-DNJ, in addition to the inhibitory effect on CSG, also works as a "chemical chaperone", increasing the activity of acid beta-glucosidase of wild-type and several GC mutated proteins, including the most frequent N370S mutation. The specific location of the Miglustat binding site in GC is unknown. Potential binding sites in the enzyme have been searched for using computational molecular docking. The searching strategy identified three potential GC binding sites for Miglustat, one being the substrate-binding site of the enzyme, which was the best-ranked site by AutoDock program. Therefore, it is possible that Miglustat exerts its chaperoning activity on acid beta-glucosidase by acting as an inhibitor bound at the active site. This increase on the activity of the acid beta-glucosidase would imply that

  6. Bioassay for follicle stimulating activity of equine gonadotropic hormone in mare serum using frozen/thawed transiently transfected reporter cells.

    PubMed

    Sahmi, F; Nicola, E; Price, C A

    2012-09-01

    The objective was to establish a cell line-based bioassay for FSH in horse serum for screening samples with high eCG bioactivity. A cell line (HEK293) was transiently cotransfected with an FSH reporter expression plasmid and a cAMP-responsive β-galactosidase reporter plasmid. Cells were bulk frozen, and thawed for assay purposes. This assay was specific for FSH, with no cross-reaction with LH or insulin-like growth factor-1. Standard curves (eCG) and serum samples from pregnant mares passed parallel line bioassay validity tests (linearity and parallelism). Estimates of bioactivity with this bioassay were highly correlated with estimates obtained with the Steelman-Pohley hCG augmentation assay. The colorimetric end point permitted the use of this assay as a rapid screen for FSH bioactivity without the need for animal use or complex cell culture facilities. PMID:22578627

  7. Differential expression of cellular microRNAs in HPV-11 transfected cells. An analysis by three different array platforms and qRT-PCR.

    PubMed

    Dreher, Anita; Rossing, Maria; Kaczkowski, Bogumil; Nielsen, Finn Cilius; Norrild, Bodil

    2010-12-17

    Human papillomavirus type 11 (HPV-11) infects the genital and the respiratory tract leading to condylomas and respiratory papillomatosis. HPV infections are restricted to epithelial tissue and the progression through the virus lifecycle is tightly coordinated to the differentiation of the host cell. The changes of cellular microRNAs by HPV-11 gene expression were investigated in a cell culture model of HaCaT cells transfected with HPV-11, with the goal of understanding which cellular processes were affected by the virus. Human microRNA profiling was conducted on three different array platform systems and because very few microRNAs (miR-663, -638, -149* and -92b*) were consistently found in all three array data sets we performed extensive statistical analyses of the array data and the qRT-PCR validation. We assume that the most reliable differentially expressed microRNAs are the ones identified by more than one array platform. We also show that TaqMan® qRT-PCR validation is of limited use for less abundant microRNAs.

  8. Characterization of subtype-specific antibodies to the human D5 dopamine receptor: studies in primate brain and transfected mammalian cells.

    PubMed Central

    Bergson, C; Mrzljak, L; Lidow, M S; Goldman-Rakic, P S; Levenson, R

    1995-01-01

    To achieve a better understanding of how D5 dopamine receptors mediate the actions of dopamine in brain, we have developed antibodies specific for the D5 receptor. D5 antibodies reacted with recombinant baculovirus-infected Sf9 cells expressing the D5 receptor but not with the D1 receptor or a variety of other catecholaminergic and muscarinic receptors. Epitope-tagged D5 receptors expressed in mammalian cells were reactive with both D5 antibodies and an epitope-specific probe. A mixture of N-linked glycosylated polypeptides and higher molecular-mass species was detected on immunoblots of membrane fractions of D5-transfected cells and also of primate brain. D5 receptor antibodies intensely labeled pyramidal neurons in the prefrontal cortex, whereas spiny medium-sized neurons and aspiny large interneurons of the caudate nucleus were relatively lightly labeled. Antibodies to the D5 dopamine receptor should prove important in experimentally determining specific roles for the D5 and D1 receptors in cortical processes and diseases. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7536933

  9. Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts.

    PubMed

    Williamson, Chad D; Wong, Daniel S; Bozidis, Petros; Zhang, Aiping; Colberg-Poley, Anamaris M

    2015-09-01

    Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub-domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria-associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing substantial protein yields from HCMV-infected primary fibroblasts and from transfected HeLa cells. Furthermore, this unit includes protocols for isolation of detergent resistant membranes from subcellular fractions as well as techniques that allow visualization of the mitochondrial network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients.

  10. Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts

    PubMed Central

    Williamson, Chad D.; Wong, Daniel S.; Bozidis, Petros; Zhang, Aiping; Colberg-Poley, Anamaris M.

    2015-01-01

    Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub-domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria-associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing substantial protein yields from HCMV-infected primary fibroblasts and from transfected HeLa cells. Furthermore, this unit includes protocols for isolation of detergent resistant membranes from subcellular fractions as well as techniques that allow visualization of the mitochondria network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients. PMID:26331984

  11. Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA

    SciTech Connect

    Tsurimoto, T.; Fujiyama, A.; Matsubara, K.

    1987-01-01

    A human hepatocellular carcinoma cell line (Huh6-c15) was transfected with a recombinant DNA molecule that consists of tandemly arranged hepatitis B virus (HBV) genome and a neomycin-resistant gene. One clone resistant to G-418 produces and releases surface antigen and e antigen into medium at a high level and accumulates core particles intracellularly. This clone has a chromosomally integrated set of the original recombinant DNA and produces a 3.5-kilobase transcript corresponding to the pregenome RNA as well as HBV DNAs in an extrachromosomal form. Most of these DNAs were in single-stranded or partially double-stranded form and were packaged in the intracellular core particles. In the medium, particles were detected that contained HBV DNA and were morphologically indistinguishable from Dane particles. These results demonstrate that the HBV genome in an integrated state acted as a template for viral gene expression and replication. The cells were maintained for more than 6 months without losing the ability to produce the extrachromosomal HBV DNA and Dane-like particles. Thus, the cells can be used as a model system for analyses of gene expression and DNA replication of HBV in human hepatocytes.

  12. Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line

    NASA Astrophysics Data System (ADS)

    Yang, Chengbin; Panwar, Nishtha; Wang, Yucheng; Zhang, Butian; Liu, Maixian; Toh, Huiting; Yoon, Ho Sup; Tjin, Swee Chuan; Chong, Peter Han Joo; Law, Wing-Cheung; Chen, Chih-Kuang; Yong, Ken-Tye

    2016-04-01

    First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro

  13. Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line.

    PubMed

    Yang, Chengbin; Panwar, Nishtha; Wang, Yucheng; Zhang, Butian; Liu, Maixian; Toh, Huiting; Yoon, Ho Sup; Tjin, Swee Chuan; Chong, Peter Han Joo; Law, Wing-Cheung; Chen, Chih-Kuang; Yong, Ken-Tye

    2016-04-28

    First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro

  14. Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line.

    PubMed

    Yang, Chengbin; Panwar, Nishtha; Wang, Yucheng; Zhang, Butian; Liu, Maixian; Toh, Huiting; Yoon, Ho Sup; Tjin, Swee Chuan; Chong, Peter Han Joo; Law, Wing-Cheung; Chen, Chih-Kuang; Yong, Ken-Tye

    2016-04-28

    First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro

  15. Altered Arginine Metabolism in Cells Transfected with Human Wild-Type Beta Amyloid Precursor Protein (βAPP).

    PubMed

    Jęśko, Henryk; Wilkaniec, Anna; Cieślik, Magdalena; Hilgier, Wojciech; Gąssowska, Magdalena; Lukiw, Walter J; Adamczyk, Agata

    2016-01-01

    Alterations of enzymes linked to arginine metabolism have been recently implicated in Alzheimer's disease (AD). Despite strong association of arginine changes with nitric oxide (NO) pathway, the impact of amyloid β (Aβ) peptides on arginine degradation and re-synthesis is unknown. In the present study we compared expression levels of arginases (ARG1, ARG2), neuronal, endothelial and inducible NO synthase isoforms (NNOS, ENOS, INOS), enzymes that metabolize arginine or resynthesize it from citrulline and the levels of corresponding amino acids in rat pheochromocytoma (PC12) cells overexpressing human Aβ precursor protein (APPwt cells). Moreover, we investigated the changes in miRNAs responsible for modulation of arginine metabolism in AD brains. Real-time PCR analysis revealed in APPwt cells significant decreases of ARG1 and ARG2 which are responsible for lysing arginine into ornithine and urea; this reduction was followed by significantly lower enzyme activity. NNOS and ENOS mRNAs were elevated in APPwt cells while iNOS was undetectable in both cell lines. The expression of argininosuccinate synthase (ASS) that metabolizes citrulline was down-regulated without changes in argininosuccinate lyase (ASL). Ornithine decarboxylase (ODC), which decarboxylates ornithine to form putrescine was also reduced. Arginine, the substrate for both arginases and NOS, was unchanged in APPwt cells. However, citrulline concentration was significantly higher. Elevated miRNA-9 and miRNA-128a found in AD brain tissues might modulate the expression of ASS and NOS, respectively. Our results indicate that Aβ affects arginine metabolism and this influence might have important role in the pathomechanism of AD.

  16. Altered Arginine Metabolism in Cells Transfected with Human Wild-Type Beta Amyloid Precursor Protein (βAPP).

    PubMed

    Jęśko, Henryk; Wilkaniec, Anna; Cieślik, Magdalena; Hilgier, Wojciech; Gąssowska, Magdalena; Lukiw, Walter J; Adamczyk, Agata

    2016-01-01

    Alterations of enzymes linked to arginine metabolism have been recently implicated in Alzheimer's disease (AD). Despite strong association of arginine changes with nitric oxide (NO) pathway, the impact of amyloid β (Aβ) peptides on arginine degradation and re-synthesis is unknown. In the present study we compared expression levels of arginases (ARG1, ARG2), neuronal, endothelial and inducible NO synthase isoforms (NNOS, ENOS, INOS), enzymes that metabolize arginine or resynthesize it from citrulline and the levels of corresponding amino acids in rat pheochromocytoma (PC12) cells overexpressing human Aβ precursor protein (APPwt cells). Moreover, we investigated the changes in miRNAs responsible for modulation of arginine metabolism in AD brains. Real-time PCR analysis revealed in APPwt cells significant decreases of ARG1 and ARG2 which are responsible for lysing arginine into ornithine and urea; this reduction was followed by significantly lower enzyme activity. NNOS and ENOS mRNAs were elevated in APPwt cells while iNOS was undetectable in both cell lines. The expression of argininosuccinate synthase (ASS) that metabolizes citrulline was down-regulated without changes in argininosuccinate lyase (ASL). Ornithine decarboxylase (ODC), which decarboxylates ornithine to form putrescine was also reduced. Arginine, the substrate for both arginases and NOS, was unchanged in APPwt cells. However, citrulline concentration was significantly higher. Elevated miRNA-9 and miRNA-128a found in AD brain tissues might modulate the expression of ASS and NOS, respectively. Our results indicate that Aβ affects arginine metabolism and this influence might have important role in the pathomechanism of AD. PMID:26971935

  17. Granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by cDNA-transfected tumor cells induces a more potent antitumor response than exogenous GM-CSF.

    PubMed

    Shi, F S; Weber, S; Gan, J; Rakhmilevich, A L; Mahvi, D M

    1999-01-01

    Clinical cancer gene therapy trials have generally focused on the transfer of cytokine cDNA to tumor cells ex vivo and with the subsequent vaccination of the patient with these genetically altered tumor cells. This approach results in high local cytokine concentrations that may account for the efficacy of this technique in animal models. We hypothesized that the expression of certain cytokines by tumor cells would be a superior immune stimulant when compared with local delivery of exogenous cytokines. Granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA in a nonviral expression vector was inserted into MDA-MB-231 (human breast cancer), M21 (human melanoma), B16 (murine melanoma), and P815 (mastocytoma) cells by particle-mediated gene transfer. The ability of transfected tumor cells to generate a tumor-specific immune response was evaluated in an in vitro mixed lymphocyte-tumor cell assay and in an in vivo murine tumor protection model. Peripheral blood lymphocytes cocultured with human GM-CSF-transfected tumor cells were 3- to 5-fold more effective at lysis of the parental tumor cells than were peripheral blood lymphocytes incubated with irradiated tumor cells and exogenous human GM-CSF. Mice immunized with murine GM-CSF-transfected irradiated B16 murine melanoma cells or P815 mastocytoma cells were protected from subsequent tumor challenge, whereas mice immunized with the nontransfected tumors and cutaneous transfection of murine GM-CSF cDNA at the vaccination site developed tumors more frequently. The results indicate that GM-CSF protein expressed in human and murine tumor cells is a superior antitumor immune stimulant compared with exogenous GM-CSF in the tumor microenvironment. PMID:10078967

  18. Enhanced expression of multidrug resistance-associated protein 2 and reduced expression of aquaglyceroporin 3 in an arsenic-resistant human cell line.

    PubMed

    Lee, Te-Chang; Ho, I-Ching; Lu, Wen-Jen; Huang, Jin-ding

    2006-07-01

    Arsenic-resistant cells (R15), derived from a human lung adenocarcinoma cell line (CL3), were 10-fold more resistant to sodium arsenite (As(III)). Because R15 cells accumulated less arsenic than parental CL3 cells, this arsenic resistance may be due to higher efflux and/or lower uptake of As(III). We therefore compared expression of the multidrug resistance-associated proteins MRP1, MRP2, and MRP3 in these two cell lines. MRP2 expression was 5-fold higher in R15 cells than in CL3 cells, whereas MRP1 and MRP3 expression levels were similar. Furthermore, verapamil and cyclosporin A, inhibitors of multidrug resistance transporters, significantly reduced the efflux of arsenic from R15. Thus, increased arsenic extrusion by MRP2 may contribute to arsenic resistance in R15 cells. We also examined the expression of several aquaglyceroporins (AQPs), which mediate As(III) uptake by cells. Little AQP7 or AQP9 mRNA was detected by reverse transcription-PCR in either cell line, whereas AQP3 mRNA expression was 2-fold lower in R15 cells than in CL3 cells. When AQP3 expression in CL3 cells was knocked down by RNA interference, CL3 cells accumulated less arsenic and became more resistant to As(III). Conversely, overexpression of AQP3 in human embryonic kidney 293T cells increased arsenic accumulation, and the cells were more susceptible to As(III) than 293T cells transfected with vector alone. These results suggest that AQP3 is involved in As(III) accumulation. Taken together, our results suggest that enhanced expression of MRP2 and lower expression of AQP3 are responsible for lower arsenic accumulation in arsenic-resistant R15 cells.

  19. Hydrodynamics-based transfection of rat interleukin-10 gene attenuates porcine serum-induced liver fibrosis in rats by inhibiting the activation of hepatic stellate cells.

    PubMed

    Huang, Yue-Hong; Chen, Yun-Xin; Zhang, Li-Juan; Chen, Zhi-Xin; Wang, Xiao-Zhong

    2014-09-01

    Liver fibrosis is the common pathological outcome for the majority of chronic liver diseases. Interleukin-10 (IL-10) is a cytokine that downregulates proinflammatory responses and has a modulatory effect on liver fibrogenesis. However, little is known regarding the effect of rat interleukin‑10 (rIL‑10) gene by hydrodynamics-based transfection (HBT) on liver fibrosis in rats. The aim of this study was to investigate the effect of the rIL-10 gene by HBT on the progression of liver fibrosis induced by porcine serum (PS) in rats and explore its possible mechanism. Plasmid‑expressing rIL-10 was transferred into rats by HBT and immunohistochemistry and RT-PCR were used to detect the major organ expressing rIL-10. Liver fibrosis was induced in rats by intraperitoneal administration of PS for 8 weeks. Plasmid pcDNA3-rIL-10 solution was administered weekly by HBT starting at the 5th week. Liver function and hepatic histology were examined. The possible molecular mechanisms of rIL-10 gene therapy were assessed in liver tissue and hepatic stellate cells (HSCs) co-cultured with BRL cells (a hepatocyte line) in vitro. The results showed rIL-10 expression occurred mainly in the liver following rIL-10 gene transfer by HBT. Maintaining a stable expression of rIL-10 in serum was assessed by repeated administration. The rIL-10 gene treatment attenuated liver inflammation and fibrosis in PS-induced fibrotic rats, reduced the deposition of collagen and the expression of α-smooth muscle actin (α-SMA) in fibrotic rats. The in vitro experiment showed that the expression of a-SMA and procollagen type I in HSCs co-cultured with the BRL‑transfected rIL-10 gene were significantly decreased. These findings indicate that rIL-10 gene therapy by HBT attenuates PS-induced liver fibrosis in rats and that its mechanism is associated with rIL-10 inhibiting the activation of HSCs and promoting the degeneration of collagen.

  20. Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency

    PubMed Central

    Sork, Helena; Nordin, Joel Z; Turunen, Janne J; Wiklander, Oscar PB; Bestas, Burcu; Zaghloul, Eman M; Margus, Helerin; Padari, Kärt; Duru, Adil D; Corso, Giulia; Bost, Jeremy; Vader, Pieter; Pooga, Margus; Smith, CI Edvard; Wood, Matthew JA; Schiffelers, Raymond M; Hällbrink, Mattias; Andaloussi, Samir EL

    2016-01-01

    The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents. PMID:27111416

  1. Use of rhenium-188 for in vivo imaging and treatment of human cervical cancer cells transfected with lentivirus expressing sodium iodide symporter.

    PubMed

    Zhang, Min; Shi, Shuo; Guo, Rui; Miao, Yin; Li, Biao

    2016-10-01

    Although survival rates for cervical cancer have improved, they need further improvement in patients with distant metastases. The sodium iodine symporter (NIS) gene has often been used in cancer therapy and imaging. We examined the therapeutic effects of rhenium-188 (188Re) in a cervical cancer xenograft model expressing the NIS gene under the control of the tumor-specific human telomerase reverse transcriptase (hTERT) promoter. We constructed two recombinant lentiviral vectors expressing enhanced green fluorescent protein (eGFP) or the NIS gene driven by the hTERT promoter. To determine the tumor-specific transcriptional activity of the hTERT promoter, the eGFP-expressing vector was stably transfected into tumor cells and normal cells. A cervical cancer HeLa cell line stably expressing NIS (HeLa-TERTNIS) was created and examined in a similar way. HeLa and HeLa-TERTNIS tumor xenografts were transplanted in nude mice, and in vivo 188Re distribution was measured using micro-SPECT/CT imaging. The therapeutic effects of 188Re were assessed over 21 days on the basis of tumor volume and the immunohistochemical findings of excised tumors. eGFP expression controlled by the hTERT promoter was substantially higher in the tumor cells than normal cells. Quantitative PCR and western blotting confirmed that HeLa-TERTNIS cells expressed high levels of NIS mRNA and protein, respectively. Further, 188Re uptake and accumulation were significantly higher in HeLa-TERTNIS cells and xenografts than HeLa cells and xenografts. In vitro and in vivo, 188Re significantly reduced the survival of HeLa-TERTNIS cells and inhibited the growth of HeLa-TERTNIS xenografts, respectively. Immunohistochemical staining showed that HeLa-TERTNIS xenograft tumors expressed higher levels of NIS and caspase-3 and lower levels of Ki-67 than HeLa xenograft tumors. Our findings indicated that hTERT promoter-driven expression of the NIS gene in HeLa cells led to 188Re uptake and therapeutic effects. Thus, NIS

  2. Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

    PubMed Central

    Maeß, Marten B.; Wittig, Berith; Lorkowski, Stefan

    2014-01-01

    Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings. PMID:25226503

  3. Biotinylation of the Fcγ receptor ectodomains by mammalian cell co-transfection: application to the development of a surface plasmon resonance-based assay.

    PubMed

    Dorion-Thibaudeau, July; St-Laurent, Gilles; Raymond, Céline; De Crescenzo, Gregory; Durocher, Yves

    2016-02-01

    We here report the production of four biotinylated Fcγ receptor (FcγR) ectodomains and their subsequent stable capture on streptavidin-biosensor surfaces. For receptor biotinylation, we first describe an in-cell protocol based on the co-transfection of two plasmids corresponding to one of the FcγR ectodomains and the BirA enzyme in mammalian cells. This strategy is compared with a standard sequential in vitro enzymatic biotinylation with respect to biotinylation level and yield. Biotinylated FcγR ectodomains that have been prepared with both strategies are then compared by analytical ultracentrifugation and surface plasmon resonance (SPR) analyses. Overall, we demonstrate that in-cell biotinylation is an interesting alternative to standard biotinylation protocol, as it requires less purification steps while yielding higher titers. Finally, biotin-tagged FcγRs produced with the in-cell approach are successfully applied to the development of SPR-based assays to evaluate the impact of the glycosylation pattern of monoclonal antibodies on their interaction with CD16a and CD64. In that endeavor, we unambiguously observe that highly galactosylated trastuzumab (TZM-gal), non-glycosylated trastuzumab (TZM-NG), and reference trastuzumab are characterized by different kinetic profiles upon binding to CD16a and CD64 that had been captured at the biosensor surface via their biotin tag. More precisely, while TZM-NG binding to CD16a was not detected, TZM-gal formed a more stable complex with CD16a than our reference TZM. In contrast, both glycosylated TZM bound to captured CD64 in a stable and similar fashion, whereas the interaction of their non-glycosylated form with CD64 was characterized by a higher dissociation rate. PMID:26762306

  4. Transforming growth factor beta 1 (TGF beta 1) is an autocrine positive regulator of colon carcinoma U9 cells in vivo as shown by transfection of a TGF beta 1 antisense expression plasmid.

    PubMed

    Huang, F; Newman, E; Theodorescu, D; Kerbel, R S; Friedman, E

    1995-12-01

    A transforming growth factor beta1 (TGF beta1) antisense expression plasmid under constitutive control of the Rous sarcoma virus promoter was introduced into the highly tumorigenic and invasive colon carcinoma U9A cell line, which uses its autocrine TGF beta1 as a growth-stimulating factor. Stable transfectants were infrequent, and only the K6 transfectant exhibited 39 and 33%, respectively, of the levels of TGF beta1 mRNA and active, secreted TGF beta1 protein of the parental line. K6 exhibited no change in TGF beta2 expression, and TGF beta3 expression was not detected in either parental or transfectant cells. Compared to the parental line, the K6 antisense transfectant exhibited a 3-fold increase in lag time in anchorage-dependent colony formation. The parental line was 44 times as invasive through a collagen l-coated polycarbonate membrane in vitro as K6 cells and, after s.c. injection at low-cell inocula, U9A cells induced tumors 75 times as large in vivo as did the K6 antisense transfectant. The decreases in in vitro invasion and anchorage-dependent colony formation seen in K6 cells were largely reversed by the addition of TGF beta1. Tumors that did arise from the K6 antisense transfectant cells had lost antisense TGF beta1 expression and expressed the same TGF beta1 mRNA levels as controls. U9A cells were more metastatic to the liver after intrasplenic injection than K6 cells. These findings demonstrate a role for autocrine TGE beta1 in colon cancer tumorigenicity and invasion. They also show that a relatively small decrease in TGF beta1 levels was enough to markedly decrease colon carcinoma cell aggressiveness. This is not unprecedented, as we had found in an earlier study that a small, 2-4-fold increase in TGF beta1 protein levels in human colon cancers correlated with disease progression to metastases (E. Friedman et al., Cancer Epidemiol, Biomarkers & Prev., 4:549-554, 1995).

  5. In vivo Molecular Imaging and Radionuclide (131I) Therapy of Human Nasopharyngeal Carcinoma Cells Transfected with a Lentivirus Expressing Sodium Iodide Symporter

    PubMed Central

    Shi, Shuo; Zhang, Min; Guo, Rui; Miao, Ying; Hu, Jiajia; Xi, Yun; Li, Biao

    2015-01-01

    Introduction Despite recent improvements in the survival rates for nasopharyngeal carcinoma (NPC), novel treatment strategies are required to improve distant metastasis-free survival. The sodium iodine symporter (NIS) gene has been applied for in vivo imaging and cancer therapy. In this study, we examined the potential of NIS gene therapy as a therapeutic approach in NPC by performing non-invasive imaging using 125I and 131I therapy in vivo. Methods We constructed a lentiviral vector expressing NIS and enhanced green fluorescent protein (EGFP) under the control of the human elongation factor-1α (EF1α) promoter, and stably transfected the vector into CNE-2Z NPC cells to create CNE-2Z-NIS cells. CNE-2Z and CNE-2Z-NIS tumor xenografts were established in nude mice; 125I uptake, accumulation and efflux were measured using micro-SPECT/CT imaging; the therapeutic effects of treatment with 131I were assessed over 25 days by measuring tumor volume and immunohistochemical staining of the excised tumors. Results qPCR, immunofluorescence and Western blotting confirmed that CNE-2Z-NIS cells expressed high levels of NIS mRNA and protein. CNE-2Z-NIS cells and xenografts took up and accumulated significantly more 125I than CNE-2Z cells and xenografts. In vitro, 131I significantly reduced the clonogenic survival of CNE-2Z-NIS cells. In vivo, 131I effectively inhibited the growth of CNE-2Z-NIS xenografts. At the end of 131I therapy, CNE-2Z-NIS xenograft tumor cells expressed higher levels of NIS and caspase-3 and lower levels of Ki-67. Conclusion Lentiviruses effectively delivered and mediated long-lasting expression of NIS in CNE-2Z cells which enabled uptake and accumulation of radioisotopes and provided a significant therapeutic effect in an in vivo model of NPC. NIS-mediated radioiodine treatment merits further investigation as a potentially effective, low toxicity therapeutic strategy for NPC. PMID:25621996

  6. Rapid Method To Determine Intracellular Drug Concentrations in Cellular Uptake Assays: Application to Metformin in Organic Cation Transporter 1-Transfected Human Embryonic Kidney 293 Cells.

    PubMed

    Chien, Huan-Chieh; Zur, Arik A; Maurer, Tristan S; Yee, Sook Wah; Tolsma, John; Jasper, Paul; Scott, Dennis O; Giacomini, Kathleen M

    2016-03-01

    Because of the importance of intracellular unbound drug concentrations in the prediction of in vivo concentrations that are determinants of drug efficacy and toxicity, a number of assays have been developed to assess in vitro unbound concentrations of drugs. Here we present a rapid method to determine the intracellular unbound drug concentrations in cultured cells, and we apply the method along with a mechanistic model to predict concentrations of metformin in subcellular compartments of stably transfected human embryonic kidney 293 (HEK293) cells. Intracellular space (ICS) was calculated by subtracting the [(3)H]-inulin distribution volume (extracellular space, ECS) from the [(14)C]-urea distribution volume (total water space, TWS). Values obtained for intracellular space (mean ± S.E.M.; μl/10(6) cells) of monolayers of HEK cells (HEK-empty vector [EV]) and cells overexpressing human organic cation transporter 1 (HEK-OCT1), 1.21± 0.07 and 1.25±0.06, respectively, were used to determine the intracellular metformin concentrations. After incubation of the cells with 5 µM metformin, the intracellular concentrations were 26.4 ± 7.8 μM and 268 ± 11.0 μM, respectively, in HEK-EV and HEK-OCT1. In addition, intracellular metformin concentrations were lower in high K(+) buffer (140 mM KCl) compared with normal K(+) buffer (5.4 mM KCl) in HEK-OCT1 cells (54.8 ± 3.8 μM and 198.1 ± 11.2 μM, respectively; P < 0.05). Our mechanistic model suggests that, depending on the credible range of assumed physiologic values, the positively charged metformin accumulates to particularly high levels in endoplasmic reticulum and/or mitochondria. This method together with the computational model can be used to determine intracellular unbound concentrations and to predict subcellular accumulation of drugs in other complex systems such as primary cells. PMID:26700958

  7. Role of transfection and clonal selection in mediating radioresistance.

    PubMed Central

    Pardo, F S; Bristow, R G; Taghian, A; Ong, A; Borek, C

    1991-01-01

    Transfected oncogenes have been reported to increase the radioresistance of rodent cells. Whether transfected nononcogenic DNA sequences and subsequent clonal selection can result in radioresistant cell populations is unknown. The present set of experiments describe the in vitro radiosensitivity and tumorigenicity of selected clones of primary rat embryo cells and human glioblastoma cells, after transfection with a neomycin-resistance marker (pSV2neo or pCMVneo) and clonal selection. Radiobiological data comparing the surviving fraction at 2 Gy (SF2) and the mean inactivation dose show the induction of radioresistance in two rat embryo cell clones and one glioblastoma clone, as compared to untransfected cells. Wild-type and transfectant clones were injected into three strains of immune-deficient mice (scid, NIH, and nu/nu) to assay for tumorigenicity and metastatic potential. Only the glioblastoma parent line and its transfectant clones were tumorigenic. None of the cells produced spontaneous or experimentally induced metastases. Flow cytometric analyses indicated that the induction of radioresistance could not be attributed to changes in cell kinetics at the time of irradiation. Our results show that transfection of a neomycin-resistance marker and clonal selection can impart radioresistance on both normal and tumor cells. The work also indicates that altered radiation sensitivity does not necessarily correlate with changes in cell-cycle kinetics at the time of irradiation, tumorigenicity, or altered metastatic potential. Our findings have critical implications for transfection studies investigating determinants of cellular radiosensitivity. PMID:1961732

  8. Direct demonstration of Ca2+ binding defects in sarco-endoplasmic reticulum Ca2+ ATPase mutants overexpressed in COS-1 cells transfected with adenovirus vectors.

    PubMed

    Strock, C; Cavagna, M; Peiffer, W E; Sumbilla, C; Lewis, D; Inesi, G

    1998-06-12

    Single mutations of specific amino acids within the membrane-bound region of the sarco-endoplasmic reticulum Ca2+ (SERCA)-1 ATPase interfere with Ca2+ inhibition of ATPase phosphorylation by Pi (1), suggesting that these residues may be involved in complexation of two Ca2+ that are known to bind to the enzyme. However, direct measurements of Ca2+ binding in the absence of ATP have been limited by the low quantities of available mutant protein. We have improved the transfection efficiency by means of recombinant adenovirus vectors, yielding sufficient expression of wild type and mutant SERCA-1 ATPase for measurements of Ca2+ binding to the microsomal fraction of the transfected cells. We find that in the presence of 20 microM Ca2+ and in the absence of ATP, the Glu771 --> Gln, Thr799 --> Ala, Asp800 --> Asn, and Glu908 --> Ala mutants exhibit negligible binding, indicating that the oxygen functions of Glu771, Thr799, Asp800, and Glu908 are involved in interactions whose single disruption causes major changes in the highly cooperative "duplex" binding. Total loss of Ca2+ binding is accompanied by loss of Ca2+ inhibition of the Pi reaction. We also find that, at pH 7.0, the Glu309 --> Gln and the Asn796 --> Ala mutants bind approximately half as much Ca2+ as the wild type ATPase and do not interfere with Ca2+ inhibition of the Pi reaction. At pH 6.2, the Glu309 --> Gln mutant does not bind any Ca2+, and its phosphorylation by Pi is not inhibited by Ca2+. On the contrary, the Asn796 --> Ala mutant retains the behavior displayed at pH 7.0. This suggests that in the Glu309 --> Gln mutant, ionization of acidic functions in other amino acids (e.g. Glu771 and Asp800) occurs as the pH is shifted, thereby rendering Ca2+ binding possible. In the Asn796 --> Ala mutant, on the other hand, the Glu309 carboxylic function allows binding of inhibitory Ca2+ even at pH 6.2. In all cases mutational interference with the inhibition of the Pi reaction by Ca2+ can be overcome by raising

  9. The assessment of estrogenic or anti-estrogenic activity of chemicals by the human stably transfected estrogen sensitive MELN cell line: results of test performance and transferability.

    PubMed

    Witters, H; Freyberger, A; Smits, K; Vangenechten, C; Lofink, W; Weimer, M; Bremer, S; Ahr, P H-J; Berckmans, P

    2010-08-01

    The need for development and validation of in vitro hormone receptor transactivation assays as important alternative tools to study interactions with sex hormone receptors is outlined by international organisations, as such assays should be included in the OECD conceptual framework for the testing and assessment of endocrine active chemicals. Therefore as part of the European Union (EU)-sponsored 6th framework project ReProTect, the validation study with MELN cells, MCF-7 cells (ER+, estrogen receptor positive) which were stably transfected with the estrogen responsive gene ERE-betaGlob-Luc-SVNeo was set up. Standard operating procedures including a prescreen assay for unknown chemicals, an ER-agonist assay and an ER-antagonist assay were developed at the Flemish Institute for Technological Research, Belgium, and successfully transferred to Bayer Schering Pharma AG, Germany. Test results were obtained for 16 chemicals, and it was demonstrated that the MELN assay is transferable, robust and reproducible which allowed to rank chemical compounds according to their strong to weak affinity for the estrogen-alpha receptor, or identify negative chemicals within the test range up to 10(-5)M. Besides the screening for agonism, we demonstrated the suitability of MELN cells to test for antagonistic activity, which is of added value compared to current validated assays. As the MELN assay successfully passed the first modules of the ECVAM validation procedure, it now should be considered for further steps including the definition of a prediction model and application domain to get it accepted as an alternative screening assay, contributing to the 3R's with a reduction of animal experiments.

  10. Generation and preclinical immunogenicity study of dengue type 2 virus-like particles derived from stably transfected mosquito cells.

    PubMed

    Suphatrakul, Amporn; Yasanga, Thippawan; Keelapang, Poonsook; Sriburi, Rungtawan; Roytrakul, Thaneeya; Pulmanausahakul, Rojjanaporn; Utaipat, Utaiwan; Kawilapan, Yanee; Puttikhunt, Chunya; Kasinrerk, Watchara; Yoksan, Sutee; Auewarakul, Prasert; Malasit, Prida; Charoensri, Nicha; Sittisombut, Nopporn

    2015-10-13

    Recent phase IIb/III trials of a tetravalent live attenuated vaccine candidate revealed a need for improvement in the stimulation of protective immunity against diseases caused by dengue type 2 virus (DENV-2). Our attempts to develop particulate antigens for possibly supplementing live attenuated virus preparation involve generation and purification of recombinant DENV-2 virus-like particles (VLPs) derived from stably (prM+E)-expressing mosquito cells. Two VLP preparations generated with either negligible or enhanced prM cleavage exhibited different proportions of spherical particles and tubular particles of variable lengths. In BALB/c mice, VLPs were moderately immunogenic, requiring adjuvants for the induction of strong virus neutralizing antibody responses. VLPs with enhanced prM cleavage induced higher levels of neutralizing antibody than those without, but the stimulatory activity of both VLPs was similar in the presence of adjuvants. Comparison of EDIII-binding antibodies in mice following two adjuvanted doses of these VLPs revealed subtle differences in the stimulation of anti-EDIII binding antibodies. In cynomolgus macaques, VLPs with enhanced prM cleavage augmented strongly neutralizing antibody and EDIII-binding antibody responses in live attenuated virus-primed recipients, suggesting that these DENV-2 VLPs may be useful as the boosting antigen in prime-boost immunization. As the levels of neutralizing antibody induced in macaques with the prime-boost immunization were comparable to those infected with wild type virus, this virus-prime VLP-bo