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Vector System (vector + system)
Selected AbstractsProduction and characterization of a recombinant beta-1,4-endoglucanase (glycohydrolase family 9) from the termite Reticulitermes flavipesARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2010Xuguo Zhou Abstract Cell-1 is a host-derived beta-1,4-endoglucanase (Glycohydrolase Family 9 [GHF9]) from the lower termite Reticulitermes flavipes. Here, we report on the heterologous production of Cell-1 using eukaryotic (Baculovirus Expression Vector System; BEVS) and prokaryotic (E. coli) expression systems. The BEVS-expressed enzyme was more readily obtained in solubilized form and more active than the E. coli,expressed enzyme. Km and Vmax values for BEVS-expressed Cell-1 against the model substrate CMC were 0.993% w/v and 1.056,µmol/min/mg. Additional characterization studies on the BEVS-expressed enzyme revealed that it possesses activity comparable to the native enzyme, is optimally active around pH 6.5,7.5 and 50,60°C, is inhibited by EDTA, and displays enhanced activity up to 70°C in the presence of CaCl2. These findings provide a foundation on which to begin subsequent investigations of collaborative digestion by coevolved host and symbiont digestive enzymes from R. flavipes that include GHF7 exoglucanases, GHF1 beta glucosidases, phenol-oxidizing laccases, and others. © 2010 Wiley Periodicals, Inc. [source] An Approach for Enhancing Heterologous Production of Providencia Rettgeri Penicillin Acylase in Escherichia coliBIOTECHNOLOGY PROGRESS, Issue 3 2000C. Perry Chou Heterologous production of Providencia rettgeri penicillin acylase (PAC) was optimized inEscherichia coli. Several factors, including carbon, temperature, and host effects, were identified to be critical for the enzyme overproduction. The optimum culture conditions for the enzyme production vary for different host/vector systems. With the optimization, both volumetric and specific PAC activities could be significantly improved by more than 50-fold compared to the native expression in P. rettgeri. The heterologous production could be possibly limited by translation or posttranslational steps, depending on the culture temperature and host/vector system. To our knowledge, this is the first evidence demonstrating the limiting step for the production of P. rettgeri PAC and the existence of the P. rettgeri PAC precursor. [source] Genotoxicity of naturally occurring indole compounds: correlation between covalent DNA binding and other genotoxicity testsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2002M. Vijayaraj Reddy Abstract 3-Methylindole (3MI), melatonin (Mel), serotonin (Ser), and tryptamine (Tryp) were evaluated in vitro for their potential to induce DNA adducts, DNA strand breaks, chromosomal aberrations (Abs), inhibition of DNA synthesis, and mutations. All compounds produced DNA adducts in calf thymus DNA in the presence of rat liver S9. In cultured rat hepatocytes, all produced DNA adducts but none induced DNA strand breaks. In Chinese hamster ovary cells, 3MI and Mel produced DNA adducts, Abs, and inhibition of DNA synthesis with and without S9, except that Mel without S9 did not form adducts. Ser formed DNA adducts, was an equivocal Abs inducer, and suppressed DNA synthesis. Tryp induced neither adducts nor Abs, but did suppress DNA synthesis with S9. Ser and Tryp were less cytotoxic than 3MI and Mel. Mel, Ser, and Tryp failed to induce mutations in Salmonella and E. coli strains with or without S9. 3MI and Mel produced DNA adducts but not mutations in Salmonella TA100 with S9. 3MI and its metabolite indole 3-carbinol also did not induce mutations in a shuttle vector system in human cells. The lack of correlation between DNA adducts and other genotoxicity endpoints for these indole compounds may be due to the higher sensitivity of the 32P-postlabeling adduct assay or it may indicate that the indole-DNA adducts per se are not mutagenic and are not able to induce strand breaks or alkali-labile lesions. The indole-induced Abs may result from cytotoxicity and suppression of DNA synthesis with minimal if any contribution from DNA adducts. Environ. Mol. Mutagen. 40:1,17, 2002. © 2002 Wiley-Liss, Inc. [source] Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivoEXPERIMENTAL PHYSIOLOGY, Issue 1 2005Z. Shevtsova The brain parenchyma consists of several different cell types, such as neurones, astrocytes, microglia, oligodendroglia and epithelial cells, which are morphologically and functionally intermingled in highly complex three-dimensional structures. These different cell types are also present in cultures of brain cells prepared to serve as model systems of CNS physiology. Gene transfer, either in a therapeutic attempt or in basic research, is a fascinating and promising tool to manipulate both the complex physiology of the brain and that of isolated neuronal cells. Viral vectors based on the parvovirus, adeno-associated virus (AAV), have emerged as powerful transgene delivery vehicles. Here we describe highly efficient targeting of AAV vectors to either neurones or astrocytes in cultured primary brain cell cultures. We also show that transcriptional targeting can be achieved by the use of small promoters, significantly boosting the transgene capacity of the recombinant viral genome. However, we also demonstrate that successful targeting of a vector in vitro does not necessarily imply that the same targeting works in the adult brain. Cross-packaging the AAV-2 genome in capsids of other serotypes adds additional benefits to this vector system. In the brain, the serotype-5 capsid allows for drastically increased spread of the recombinant vector as compared to the serotype-2 capsid. Finally, we emphasize the optimal targeting approach, in which the natural tropism of a vector for a specific cell type is employed. Taken together, these data demonstrate the flexibility which AAV-based vector systems offer in physiological research. [source] Kinetic study of sn -glycerol-1-phosphate dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix K1FEBS JOURNAL, Issue 3 2002Jin-Suk Han A gene having high sequence homology (45,49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94,96 °C at near neutral pH. This enzyme was subjected to two-substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)H- dependent dihydroxyacetone phosphate reduction and NAD+ -dependent,glycerol-1-phosphate (Gro1P) oxidation. NADP+ -dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P)+ acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol- 1-phosphate dehydrogenase from A. pernix follows a ordered bi,bi mechanism. [source] Heterologous expression of nonribosomal peptide synthetases in B. subtilis: construction of a bi-functional B. subtilis/E. coli shuttle vector systemFEMS MICROBIOLOGY LETTERS, Issue 2 2002Sascha Doekel Abstract A major obstacle in investigating the biosynthesis of pharmacologically important peptide antibiotics is the heterologous expression of the giant biosynthetic genes. Recently, the genetically engineered strain Bacillus subtilis KE30 has been reported as an excellent surrogate host for the heterologous expression of an entire nonribosomal peptide synthetase (NRPS) gene cluster. In this study, we expand the applicability of this strain, by the development of four Escherichia coli/B. subtilis shuttle expression vectors. Comparative overproduction of hybrid NRPS proteins derived from both organisms revealed a significant beneficial effect of overproducing proteins in B. subtilis KE30 as underlined by the production of stable nondegradative proteins, as well as the formation of active phosphopantetheinylated holo-proteins. [source] Reprogramming Hansenula polymorpha for penicillin production: expression of the Penicillium chrysogenum pcl geneFEMS YEAST RESEARCH, Issue 7 2007Loknath Gidijala Abstract We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host,vector system based on methionine auxotrophy and the H. polymorpha MET6 gene, which encodes a putative cystathionine ,-lyase. With this new host,vector system, the Penicillium chrysogenum pcl gene, encoding peroxisomal phenylacetyl-CoA ligase (PCL), was expressed in H. polymorpha. PCL has a potential C-terminal peroxisomal targeting signal type 1 (PTS1). Our data demonstrate that a green fluorescent protein,PCL fusion protein has a dual location in the heterologous host in the cytosol and in peroxisomes. Mutation of the PTS1 of PCL (SKI-COOH) to SKL-COOH restored sorting of the fusion protein to peroxisomes only. Additionally, we demonstrate that peroxisomal PCL,SKL produced in H. polymorpha displays normal enzymatic activities. [source] Applications of Sleeping Beauty transposons for nonviral gene therapyIUBMB LIFE, Issue 6 2007Hanzhong Liu Abstract Virus-based gene therapy has advanced to clinical trials; however, this approach may result in serious adverse events including oncogenesis and the possibility of triggering fatal immune responses. Nonviral gene delivery approaches have a better safety profile, but their in vivo application has been largely limited in the past due to their inefficient delivery into cells and lack of stable chromosomal integration that is necessary for long-term therapeutic benefit. However, recent advances suggest that the use of Sleeping Beauty transposons, a novel integrating nonviral vector system, are capable of achieving long-lasting therapeutic levels of transgene expression in preclinical settings. These observations and other ongoing relevant studies may unlock the therapeutic potential of nonviral gene therapy for human diseases. iubmb Life, 59: 1 - 6, 2007 [source] Phenotypic analysis of the sensitivity of HIV-1 to inhibitors of the reverse transcriptase, protease, and integrase using a self-inactivating virus vector systemJOURNAL OF MEDICAL VIROLOGY, Issue 3 2001Gergely Jármy Abstract Conventional phenotypic analysis of resistance of the human immunodeficiency virus (HIV) to antiviral therapy is time-consuming and requires culture of infectious virus. Although phenotypic analyses may be desirable, rapid generation of test results and decentralized availability of the test system will be important to achieve utility in the clinical practice. This study describes the design of an alternative phenotypic resistance test using replication incompetent viral vectors. Chimeric HIV vectors containing a marker gene were generated. The env and most of the regulatory and accessory genes of HIV were removed. In addition, the 3,U3 region was deleted to obtain a self-inactivating construct. Cotransfection of the plasmid with a plasmid that provided the vesicular stomatitis virus glycoprotein resulted in the production of replication-incompetent virus vectors. Infection of susceptible cells with the vectors led to marker gene expression. Vector production in the presence of protease (PR) inhibitors, or infection in the presence of reverse transcriptase (RT) or integrase (IN) inhibitors reduced marker gene expression in a dose-dependent manner. Marker gene activity was preserved at higher drug levels if vectors contained RT and PR genes from resistant virus isolates. Sensitivity to nucleoside and non-nucleoside RT inhibitors, protease and integrase inhibitors could be determined in 10 working days. The phenotypic drug resistance test using replication-incompetent HIV vectors significantly speeds up drug resistance measurements and allows testing at reduced biosafety levels. This will make clinical use of phenotypic assessment of antiviral resistance more feasible. J. Med. Virol. 64:223,231, 2001. © 2001 Wiley-Liss, Inc. [source] A novel synthetic peptide vector system for optimal gene delivery to bone marrow stromal cellsJOURNAL OF PEPTIDE SCIENCE, Issue 3 2007Pan Haitao Abstract A 23-amino acid, bifunctional, integrin-targeted synthetic peptide was evaluated for ex vivo gene delivery to rabbit bone marrow stromal cells (BMSCs). The peptide (K)16GRGDSPC consists of an amino terminal domain of 16 lysines for electrostatic binding of DNA, and a 7-amino acid integrin-binding domain at the carboxyl terminal. PcDNA3-EGFP plasmids were transfected into BMSCs by (K)16GRGDSPC and the positive cells gave out a bright green fluorescence. High levels of gene delivery of pcDNA3-TGF-,1 plasmids were obtained with 2 to 4 µg/ml DNA concentration, with (K)16GRGDSPC at an optimal peptide: DNA w/w ratio of 3:1, with a required exposure time of more than 4 h but shorter than 24 h for BMSC exposure to the peptide/DNA complexes with completely absent serum in the initial stage; with 100 µM chloroquine and at least 8 h exposure for BMSC exposure to chloroquine; with a fusogenic peptide at an optimal (K)16GRGDSPC/DNA/fusogenic peptide w/w ratio of 3:1:5; and with Lipofectamine 2000 at an optimal (K)16GRGDSPC/DNA/Lipofectamine 2000 w/w ratio of 3:1:2 at a constant DNA concentration of 2 µg/ml. Chloroquine, the fusogenic peptide and Lipofectamine 2000 all significantly promoted gene delivery, but chloroquine was more effective than the fusogenic peptide and had obvious synergistic effects with Lipofectamine 2000. Under optimal conditions, TGF-,1 gene was transfected into BMSCs without observable toxicity, and the stable expression was examined by RT-PCR and Western blot analysis. The stable transgenic cells showed obvious bands. This novel synthetic peptide, providing a new way for the use of polylysine and RGD motif in DNA vector system, is potentially well suited to ex vivo gene delivery to BMSCs for experimental and clinical applications in the field of bone tissue engineering. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd. [source] One-plasmid tunable coexpression for mycobacterial protein,protein interaction studiesPROTEIN SCIENCE, Issue 11 2009Yong Chang Abstract A single plasmid that allows controlled coexpression has been developed for use in mycobacteria. The tetracycline inducible promoter, PtetO, was used to provide tetracycline-dependent induction of one gene, while the Psmyc, Pimyc, or Phsp promoters were used to provide three different levels of constitutive expression of a second gene. The functions of these four individual promoters were established using green fluorescent protein (GFP) and a newly identified red fluorescence inducible protein from Geobacillus sterothermophilus strain G1.13 (RFIP) as reporters. The tandem use of GFP and RFIP as reporter genes allowed optimization of the tunable coexpression in Mycobacterium smegmatis; either time at a fixed inducer concentration or changes in inducer concentration could be used to control the protein:protein ratio. This single vector system was used to coexpress the two-protein Mycobacterium tuberculosis stearoyl-CoA ,9 desaturase complex (integral membrane desaturase Rv3229c and NADPH oxidoreductase Rv3230c) in M. smegmatis. The catalytic activity was found to increase in a manner corresponding to increasing the level of Rv3230c relative to a fixed level of Rv3229c. This system, which can yield finely tuned coexpression of the fatty acid desaturase complex in mycobacteria, may be useful for study of other multicomponent complexes. Furthermore, the tunable coexpression strategy used herein should also be applicable in other species with minor modifications. [source] Lentiviral vectors that carry anti-HIV shRNAs: problems and solutionsTHE JOURNAL OF GENE MEDICINE, Issue 9 2007Olivier ter Brake Abstract Background HIV-1 replication can be inhibited with RNA interference (RNAi) by expression of short hairpin RNA (shRNA) from a lentiviral vector. Because lentiviral vectors are based on HIV-1, viral sequences in the vector system are potential targets for the antiviral shRNAs. Here, we investigated all possible routes by which shRNAs can target the lentiviral vector system. Methods Expression cassettes for validated shRNAs with targets within HIV-1 Leader, Gag-Pol, Tat/Rev and Nef sequences were inserted in the lentiviral vector genome. Third-generation self-inactivating HIV-1-based lentiviral vectors were produced and lentiviral vector capsid production and transduction titer determined. Results RNAi against HIV-1 sequences within the vector backbone results in a reduced transduction titer while capsid production was unaffected. The notable exception is self-targeting of the shRNA encoding sequence, which does not affect transduction titer. This is due to folding of the stable shRNA hairpin structure, which masks the target for the RNAi machinery. Targeting of Gag-Pol mRNA reduces both capsid production and transduction titer, which was improved with a human codon-optimized Gag-Pol construct. When Rev mRNA was targeted, no reduction in capsid production and transduction titer was observed. Conclusions Lentiviral vector titers can be negatively affected when shRNAs against the vector backbone and the Gag-Pol mRNA are expressed during lentiviral vector production. Titer reductions due to targeting of the Gag-Pol mRNA can be avoided with a human codon-optimized Gag-Pol packaging plasmid. The remaining targets in the vector backbone may be modified by point mutations to resist RNAi-mediated degradation during vector production. Copyright © 2007 John Wiley & Sons, Ltd. [source] Generation of stable retrovirus packaging cell lines after transduction with herpes simplex virus hybrid amplicon vectors,THE JOURNAL OF GENE MEDICINE, Issue 3 2002Miguel Sena-Esteves Abstract Background A number of properties have relegated the use of Moloney murine leukemia virus (Mo-MLV)-based retrovirus vectors primarily to ex vivo protocols. Direct implantation of retrovirus producer cells can bypass some of the limitations, and in situ vector production may result in a large number of gene transfer events. However, the fibroblast nature of most retrovirus packaging cells does not provide for an effective distribution of vector producing foci in vivo, especially in the brain. Effective development of new retrovirus producer cells with enhanced biologic properties may require the testing of a large number of different cell types, and a quick and efficient method to generate them is needed. Methods Moloney murine leukemia virus (Mo-MLV) gag-pol and env genes and retrovirus vector sequences carrying lacZ were cloned into different minimal HSV/AAV hybrid amplicons. Helper virus-free amplicon vectors were used to co-infect glioma cells in culture. Titers and stability of retrovirus vector production were assessed. Results Simultaneous infection of two glioma lines, Gli-36 (human) and J3T (dog), with both types of amplicon vectors, generated stable packaging populations that produced retrovirus titers of 0.5,1.2×105 and 3.1,7.1×103 tu/ml, respectively. Alternatively, when cells were first infected with retrovirus vectors followed by infection with HyRMOVAmpho amplicon vector, stable retrovirus packaging populations were obtained from Gli-36 and J3T cells producing retrovirus titers comparable to those obtained with a traditional retrovirus packaging cell line, ,CRIPlacZ. Conclusions This amplicon vector system should facilitate generation of new types of retrovirus producer cells. Conversion of cells with migratory or tumor/tissue homing properties could result in expansion of the spatial distribution or targeting capacity, respectively, of gene delivery by retrovirus vectors in vivo. Copyright © 2002 John Wiley & Sons, Ltd. [source] Virus-induced gene silencing in tomatoTHE PLANT JOURNAL, Issue 6 2002Yule Liu Abstract We have previously demonstrated that a tobacco rattle virus (TRV)-based vector can be used in virus-induced gene silencing (VIGS) to study gene function in Nicotiana benthamiana. Here we show that recombinant TRV infects tomato plants and induces efficient gene silencing. Using this system, we suppressed the PDS, CTR1 and CTR2 genes in tomato. Suppression of CTR1 led to a constitutive ethylene response phenotype and up-regulation of an ethylene response gene, CHITINASE B. This phenotype is similar to Arabidopsis ctr1 mutant plants. We have constructed a modified TRV vector based on the GATEWAY recombination system, allowing restriction- and ligation-free cloning. Our results show that tomato expressed sequence tags (ESTs) can easily be cloned into this modified vector using a single set of primers. Using this vector, we have silenced RbcS and an endogenous gene homologous to the tomato EST cLED3L14. In the future, this modified vector system will facilitate large-scale functional analysis of tomato ESTs. [source] Feline Immunodeficiency Virus-Mediated Viral Interleukin-10 Gene Transfer Prolongs Non-Vascularized Cardiac Allograft SurvivalAMERICAN JOURNAL OF TRANSPLANTATION, Issue 5 2003Shuang Fu Previous experiments demonstrated plasmid-, retroviral-, or adenoviral-mediated vIL-10 gene transfer could prolong allograft survival, but transgene expression was rapidly extinguished. Feline immunodeficiency virus (FIV) can integrate into genomic DNA of nondividing cells, resulting in indefinite transgene expression. We hypothesized FIV-mediated gene transfer could provide long-term gene expression, and improved allograft survival. FIV-vIL-10 and FIV-,-gal were produced using the FELIX vector system. With vector transfer to syngeneic cardiac grafts, ,-galactosidase reporter gene expression was noted as early as day 5, was strongly expressed at days 10 and 20, and persisted for 50 days after transplantation. For allografts, FIV-vIL-10 gene transfer more than doubled mean survival from 10 ± 1.6 to 22.3 ± 3 days. When combined with other immunosuppressants, such as anti-CD40L mAb, FTY720, or anti-CD3 mAb, the mean survival times were prolonged to 27 ± 4.6 days, 27.8 ± 4.6 days, and 45.5 ± 4.9 days, respectively. Multiple chemokine and chemokine receptor genes were induced by ischemia-reperfusion injury in syngeneic grafts, and in allogeneic grafts more genes were induced and to a greater degree. In allogeneic grafts transduced with FIV-IL-10, a number of the chemokine genes were suppressed. Therefore, FIV virus-mediated vIL-10 gene transfer prolongs allograft survival and, in combination with other agents, produces an additive effect. [source] Use of recombinant rotavirus VP6 nanotubes as a multifunctional template for the synthesis of nanobiomaterials functionalized with metalsBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2009Germán Plascencia-Villa Abstract The structural characteristics and predefined constant size and shape of viral assemblies make them useful tools for nanobiotechnology, in particular as scaffolds for constructing highly organized novel nanomaterials. In this work it is shown for the first time that nanotubes formed by recombinant rotavirus VP6 protein can be used as scaffolds for the synthesis of hybrid nanocomposites. Rotavirus VP6 was produced by the insect cell-baculovirus expression vector system. Nanotubes of several micrometers in length and various diameters in the nanometer range were functionalized with Ag, Au, Pt, and Pd through strong (sodium borohydride) or mild (sodium citrate) chemical reduction. The nanocomposites obtained were characterized by transmission electron microscopy (TEM), high-resolution TEM (HRTEM) with energy dispersive spectroscopy (EDS), dynamic light scattering, and their characteristic plasmon resonance. The outer surface of VP6 nanotubes had intrinsic affinity to metal deposition that allowed in situ synthesis of nanoparticles. Furthermore, the use of preassembled recombinant protein structures resulted in highly ordered integrated materials. It was possible to obtain different extents and characteristics of the metal coverage by manipulating the reaction conditions. TEM revealed either a continuous coverage with an electrodense thin film when using sodium citrate as reductant or a discrete coverage with well-dispersed metal nanoparticles of diameters between 2 and 9,nm when using sodium borohydride and short reaction times. At long reaction times and using sodium borohydride, the metal nanoparticles coalesced and resulted in a thick metal layer. HRTEM-EDS confirmed the identity of the metal nanoparticles. Compared to other non-recombinant viral scaffolds used until now, the recombinant VP6 nanotubes employed here have important advantages, including a longer axial dimension, a dynamic multifunctional hollow structure, and the possibility of producing them massively by a safe and efficient bioprocess. Such characteristics confer important potential applications in nanotechnology to the novel nanobiomaterials produced here. Biotechnol. Bioeng. 2009; 104: 871,881. © 2009 Wiley Periodicals, Inc. [source] Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium,BIOTECHNOLOGY & BIOENGINEERING, Issue 3 2007Rebekka Biedendieck Abstract A multiple vector system for the intracellular high-level production of affinity tagged recombinant proteins in Bacillus megaterium was developed. The N- and C-terminal fusion of a protein of interest to a Strep II and a His6 -tag is possible. Corresponding genes are expressed under the control of a xylose-inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N- or C-terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one-step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry. Biotechnol. Bioeng. 2007;96: 525,537. © 2006 Wiley Periodicals, Inc. [source] Gene therapy for diseases of the cornea , a reviewCLINICAL & EXPERIMENTAL OPHTHALMOLOGY, Issue 2 2010Keryn A Williams PhD Abstract The cornea is particularly suited to gene therapy. The cornea is readily accessible, normally transparent, and is somewhat sequestrated from the general circulation and the systemic immune system. The principle of genetic therapy for the cornea is to use an appropriate vector system to transfer a gene to the cornea itself, or to the ocular environs, or systemically, so that a transgenic protein will be expressed that will modulate congenital or acquired disease. The protein may be structural such as a collagen, or functionally active such as an enzyme, cytokine or growth factor that may modulate a pathological process. Alternatively, gene expression may be silenced by the use of modalities such as antisense oligonucleotides. Interestingly, despite a very considerable amount of work in animal models, clinical translation directed to gene therapy of the human cornea has been minimal. This is in contrast to gene therapy for monogenic inherited diseases of the retina, where promising early results of clinical trials for Leber's congenital amaurosis have already been published and a number of other trials are ongoing. [source] Performance evaluation of the SX-6 vector architecture for scientific computationsCONCURRENCY AND COMPUTATION: PRACTICE & EXPERIENCE, Issue 1 2005Leonid Oliker Abstract The growing gap between sustained and peak performance for scientific applications is a well-known problem in high-performance computing. The recent development of parallel vector systems offers the potential to reduce this gap for many computational science codes and deliver a substantial increase in computing capabilities. This paper examines the intranode performance of the NEC SX-6 vector processor, and compares it against the cache-based IBM Power3 and Power4 superscalar architectures, across a number of key scientific computing areas. First, we present the performance of a microbenchmark suite that examines many low-level machine characteristics. Next, we study the behavior of the NAS Parallel Benchmarks. Finally, we evaluate the performance of several scientific computing codes. Overall results demonstrate that the SX-6 achieves high performance on a large fraction of our application suite and often significantly outperforms the cache-based architectures. However, certain classes of applications are not easily amenable to vectorization and would require extensive algorithm and implementation reengineering to utilize the SX-6 effectively. Copyright © 2005 John Wiley & Sons, Ltd. [source] Indications for cell stress in response to adenoviral and baculoviral gene transfer observed by proteome profiling of human cancer cellsELECTROPHORESIS, Issue 11 2010Christopher Gerner Abstract Gene transfer to cultured cells is an important tool for functional studies in many areas of biomedical research and vector systems derived from adenoviruses and baculoviruses are frequently used for this purpose. In order to characterize how viral gene transfer vectors affect the functional state of transduced cells, we applied 2-D PAGE allowing quantitative determination of protein amounts and synthesis rates of metabolically labeled cells and shotgun proteomics. Using HepG2 human hepatoma cells we show that both vector types can achieve efficient expression of green fluorescent protein, which accounted for about 0.1% of total cellular protein synthesis 72,h after transduction. No evidence in contrast was found for expression of proteins from the viral backbones. With respect to the host cell response, both vectors induced a general increase in protein synthesis of about 50%, which was independent of green fluorescent protein expression. 2-D PAGE autoradiographs identified a 3.6-fold increase of ,-actin synthesis in adenovirus transduced cells. In addition shotgun proteomics of cytoplasmic and nuclear extract fractions identified a slight induction of several proteins related to inflammatory activation, cell survival and chromatin function by both virus types. These data demonstrate that commonly used gene transfer vectors induce a response reminiscent of stress activation in host cells, which needs to be taken into account when performing functional assays with transduced cells. [source] Haematopoietic progenitor cells from the common marmoset as targets of gene transduction by retroviral and adenoviral vectorsEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2001Hitoshi Hibino Abstract: To establish a new non-human primate model for human cytokine and gene therapy, we characterized lymphocytes and haematopoietic progenitor cells of the small New World monkey, the common marmoset. We first assessed the reactions of marmoset bone marrow (BM) and peripheral blood (PB) cells to mouse anti-human monoclonal antibodies (mAbs) for the purpose of isolating marmoset lymphocytes and haematopoietic progenitor cells. Both cell fractions stained with CD4 and CD8 mAbs were identified as lymphocytes by cell proliferation assay and morphological examination. Myeloid-specific mAbs such as CD14 and CD33 did not react with marmoset BM and PB cells. No available CD34 and c-kit mAbs could be used to purify the marmoset haematopoietic progenitor cells. Furthermore, we studied the in vitro transduction of the bacterial ,-galactosidase (LacZ) gene into CFU-GM derived from marmoset BM using retroviral and adenoviral vectors. The transduction efficiency was increased by using a mixed culture system consisting of marmoset BM stromal cells and retroviral producer cells. It was also possible to transduce LacZ gene into marmoset haematopoietic progenitor cells with adenoviral vectors as well as retroviral vectors. The percentage of adenovirally transduced LacZ-positive clusters was 15% at day 4 (multiplicity of infection=200), but only 1,2% at day 14. The differential use of viral vector systems is to be recommended in targeting different diseases. Our results suggested that marmoset BM progenitor cells were available to examine the transduction efficiency of various viral vectors in vitro. [source] Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivoEXPERIMENTAL PHYSIOLOGY, Issue 1 2005Z. Shevtsova The brain parenchyma consists of several different cell types, such as neurones, astrocytes, microglia, oligodendroglia and epithelial cells, which are morphologically and functionally intermingled in highly complex three-dimensional structures. These different cell types are also present in cultures of brain cells prepared to serve as model systems of CNS physiology. Gene transfer, either in a therapeutic attempt or in basic research, is a fascinating and promising tool to manipulate both the complex physiology of the brain and that of isolated neuronal cells. Viral vectors based on the parvovirus, adeno-associated virus (AAV), have emerged as powerful transgene delivery vehicles. Here we describe highly efficient targeting of AAV vectors to either neurones or astrocytes in cultured primary brain cell cultures. We also show that transcriptional targeting can be achieved by the use of small promoters, significantly boosting the transgene capacity of the recombinant viral genome. However, we also demonstrate that successful targeting of a vector in vitro does not necessarily imply that the same targeting works in the adult brain. Cross-packaging the AAV-2 genome in capsids of other serotypes adds additional benefits to this vector system. In the brain, the serotype-5 capsid allows for drastically increased spread of the recombinant vector as compared to the serotype-2 capsid. Finally, we emphasize the optimal targeting approach, in which the natural tropism of a vector for a specific cell type is employed. Taken together, these data demonstrate the flexibility which AAV-based vector systems offer in physiological research. [source] Hairy Root and Its Application in Plant Genetic EngineeringJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 2 2006Zhi-Bi Hu Abstract Agrobacterium rhizogenes Conn. causes hairy root disease in plants. Hairy root-infected A. rhizogenes is characterized by a high growth rate and genetic stability. Hairy root cultures have been proven to be an efficient means of producing secondary metabolites that are normally biosynthesized in roots of differentiated plants. Furthermore, a transgenic root system offers tremendous potential for introducing additional genes along with the Ri plasmid, especially with modified genes, into medicinal plant cells with A. rhizogenes vector systems. The cultures have turned out to be a valuable tool with which to study the biochemical properties and the gene expression profile of metabolic pathways. Moreover, the cultures can be used to elucidate the intermediates and key enzymes involved in the biosynthesis of secondary metabolites. The present article discusses various applications of hairy root cultures in plant genetic engineering and potential problems associated with them. (Managing editor: Wei Wang) [source] Expression screening of integral membrane proteins from Helicobacter pylori 26695PROTEIN SCIENCE, Issue 12 2007Georgios Psakis Abstract The efficiency of Helicobacter pylori as a mucosal pathogen is caused by unique soluble and integral membrane proteins, which allow its survival at acidic pH and successful colonization of the gastric environment. With about one-fourth of the H. pylori's proteome comprising integral membrane proteins, the need for solution of their three-dimensional (3D) structures becomes persistent as it can potentially drive the generation of more effective drugs. This study presents a medium-throughput approach for cloning and expression screening of integral membrane proteins from H. pylori (26695) using Escherichia coli as the expression host. One-hundred sixteen H. pylori targets were cloned into two different vector systems and heterologously expressed in E. coli. Eighty-four percent of these proteins displayed medium to high expression. No clear-cut correlation was found between expression levels and number of putative transmembrane spans, predicted functionality, and molecular mass. Nonetheless, expression of transporters and hypothetical proteins ,40 kDa with two to four transmembrane spans displayed generally high expression levels. To statistically strengthen the quality of the data from the medium-throughput approach, a comparison with data derived from robotic-based methodologies was conducted. Optimization of expression and solubilization conditions for selected targets was also performed. Seventeen targets have been purified and subjected to crystallization so far. Eighteen percent of these targets (2/17) produced crystals under specific sets of crystallization conditions. [source] Growth factors improve gene expression after lentiviral transduction in human adult and fetal hepatocytesTHE JOURNAL OF GENE MEDICINE, Issue 2 2007Clare Selden Abstract Background Lentiviral vectors may be vectors of choice for transducing liver cells; they mediate integration in quiescent cells and offer potential for long-term expression. In adult liver, hepatocytes are generally mitotically quiescent. There has been controversy as to the necessity for lentiviral vector target cells to be in the cell cycle; currently, there is consensus that effective transduction can be achieved in quiescent hepatocytes, by using virus at high titre. However, transduction approaches which reduce the multiplicities of infection (MOIs) required provide potential benefit of cost and safety for therapeutic use. Methods We used two late-generation HIV-based lentiviral vector systems (pHR-SIN-cppT SGW and pRRLSIN.cPPT.PGK.WPRE) encoding LacZ/GFP reporter genes to transduce adult and fetal human hepatocytes in vitro + /, growth factors, hepatocyte growth factor (HGF) and epidermal growth factor (EGF). Green fluorescent protein (GFP) expression was observed microscopically, and quantified by fluorescence spectrometry for protein expression, fluorescence-activated cell sorting (FACS) analysis to identify the proportion of cells expressing GFP, and real-time quantitative polymerase chain reaction (PCR) for number of integrations. Results Gene expression following lentiviral transduction of human liver cells in vitro was markedly enhanced by the growth factors HGF and EGF. In adult cells growth factors led to a greater proportion of cells expressing more GFP per cell, from more integration events. In human fetal cells, the proportion of transduced hepatocytes remained identical, but cells expressed more GFP protein. Conclusions This has implications for the design of regimes for liver cell gene therapy, allowing marked reduction of MOIs, and reducing both cost and risk of viral-mediated toxicity. Copyright © 2007 John Wiley & Sons, Ltd. [source] Novel two-stage screening procedure leads to the identification of a new class of transfection enhancersTHE JOURNAL OF GENE MEDICINE, Issue 6 2006Birgit Neukamm Abstract Background Non-viral gene transfer efficiency is low as compared to viral vector systems. Here we describe the discovery of new drugs that are capable of enhancing non-viral gene transfer into mammalian cells using a novel two-stage screening procedure. Methods First, potential candidates are preselected from a molecular library at various concentrations by a semi-automated yeast transfection screen (YTS). The maximal transfection efficiency of every positive drug is subsequently determined in independent experiments at the optimal concentration and compared to the inhibitory effect of the drug on cell growth (IC50). In a subsequent mammalian cell transfection screen (MTS), the maximal transfection efficiency and the IC50 are determined for all preselected drugs using a human cell line and a luciferase reporter gene construct. Results Employing our novel system we have been able to identify a new class of transfection enhancers, the tricyclic antidepressants (i.e. doxepin, maprotiline, desipramine and amoxapine). All positive drugs enhanced gene transfer in both yeast and human cell lines, but lower concentrations were sufficient for mammalian cells. With a triple combination of doxepin, amoxapine and chloroquine we obtained a transfection efficiency that exceeded that of chloroquine, one of the best-known transfection enhancers of mammalian cells, by nearly one order of magnitude. Conclusions Non-viral gene transfer efficiency can be increased significantly using new transfection enhancers that are identified by a novel, semi-automated two-stage screening system employing yeast cells in the first and specific human target cells in the second round. Copyright © 2006 John Wiley & Sons, Ltd. [source] Development and characterization of a minimal inducible packaging cell line for simian immunodeficiency virus-based lentiviral vectorsTHE JOURNAL OF GENE MEDICINE, Issue 4 2002Seraphin Kuate Abstract Background Lentiviral vectors allow gene transfer into non-dividing cells. Further development of these vector systems requires stable packaging cell lines that enable adequate safety testing. Methods To generate a packaging cell line for vectors based on simian immunodeficiency virus (SIV), expression plasmids were constructed that contain the codon-optimized gag-pol gene of SIV and the gene for the G protein of vesicular stomatitis virus (VSV-G) under the control of an ponasterone-inducible promoter. Stable cell lines expressing these packaging constructs were established and characterized. Results The RT activity and vector titers of cell clones stably transfected with the inducible gag-pol expession plasmid could be induced by ponasterone by more than a factor of 1000. One of these clones was subsequently transfected with the ponasterone-inducible VSV-G expression plasmid to generate packaging cells. Clones of the packaging cells were screened for vector production by infection with an SIV vector and subsequent induction by ponasterone. In the supernatant of selected ponasterone-induced producer clones vector titers of more than 1×105 transducing units/ml were obtained. Producer cell clones were stable for at least five months, as tested by vector production. Conclusions The packaging cells described should be suitable for most preclinical applications of SIV-based vectors. By avoiding regions of high homology between the vector and the packaging constructs, the design of the SIV packaging cell line should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the SIV vector constructs that can be packaged. Copyright © 2002 John Wiley & Sons, Ltd. [source] Least path criterion (LPC) for unique indexing in a two-dimensional decagonal quasilatticeACTA CRYSTALLOGRAPHICA SECTION A, Issue 5 2002N. K. Mukhopadhyay The least path criterion or least path length in the context of redundant basis vector systems is discussed and a mathematical proof is presented of the uniqueness of indices obtained by applying the least path criterion. Though the method has greater generality, this paper concentrates on the two-dimensional decagonal lattice. The order of redundancy is also discussed; this will help eventually to correlate with other redundant but desirable indexing sets. [source] New strategies for cancer gene therapy: Progress and opportunitiesCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 1 20102nd Australia, China Biomedical Research Conference (ACBRC2009) Summary 1.,To date, cancer persists as one of the most devastating diseases worldwide. Problems such as metastasis and tumour resistance to chemotherapy and radiotherapy have seriously limited the therapeutic effects of existing clinical treatments. 2.,To address these problems, cancer gene therapy has been developing over the past two decades, specifically designed to deliver therapeutic genes to treat cancers using vector systems. So far, a number of genes and delivery vehicles have been evaluated and significant progress has been made with several gene therapy modalities in clinical trials. However, the lack of an ideal gene delivery system remains a major obstacle for the successful translation of regimen to the clinic. 3.,Recent understanding of hypoxic and necrotic regions within solid tumours and rapid development of recombinant DNA technology have reignited the idea of using anaerobic bacteria as novel gene delivery systems. These bacterial vectors have unique advantages over other delivery systems and are likely to become the vector of choice for cancer gene therapy in the near future. 4.,Meanwhile, complicated tumour pathophysiology and associated metastasis make it hard to rely on a single therapeutic modality for complete tumour eradication. Therefore, the combination of cancer gene therapy with other conventional treatments has become paramount. 5.,The present review introduces important cancer gene therapy strategies and major vector systems that have been studied so far with an emphasis on bacteria-mediated cancer gene therapy. In addition, exemplary combined therapies are briefly reviewed. [source] | ||||||||||||||||||||||||||||