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At least 649 records · Page 36Linked to original sources
PubMed1995

Genetic approaches to the study of cytokine regulation of mucosal immunity.

Efforts to design effective mucosal vaccines have been hampered by an incomplete understanding of factors controlling the development of mucosal immunity. It is now clear, however, that T cell-derived cytokines play a major role. Recent developments in 'gene knockout' technology have allowed the generation of strains of mice in which particular genes have been inactivated. The availability of mice rendered deficient for production of Th2 cytokines has facilitated studies of the induction and development of mucosal immune responses in the absence of these factors. We have used several genetic approaches, including cytokine-deficient mice and recombinant vectors constructed to express genes for a range of different cytokines, to demonstrate the importance of these factors in the mucosa. Such genetic approaches appear to represent powerful tools for in vivo studies of the influence of cytokines in mucosal immunoregulation.

Animals↗
PubMed2003

Human adenovirus type 35: nucleotide sequence and vector development.

In this report, we describe the complete 34,794 base pair genomic sequence of the human adenovirus serotype 35 (Ad35) Holden strain. The viral genome exhibits a compact organization similar to other adenoviral serotypes, with overlapping genes on both strands. In all, 47 open reading frames (ORFs) were identified, including early (E1, 2, 3, 4) and late (L1, 2, 3, 4, 5) regions conserved among the adenoviridae family. In addition, 14 ORFs were identified that do not encode known adenoviral genes. Comparison of the predicted translational products of the conserved genes with those of other adenoviruses revealed that Ad35 has high homology to Ad7, Ad3, Ad21, Ad17, and simian Ads25. Based on the complete Ad35 DNA sequence, E3-, E1-, and E1/E3-deleted Ad35-based vector systems were developed. An HEK293-derived cell line was established for the propagation of the E1-deleted Ad35 vector, avoiding the emergence of replication-competent adenovirus. Moreover, production of the E1-deleted recombinant Ad35 vector was achieved by transient transduction of a plasmid encoding the Ad35 E1B gene in HEK293 cells. Testing showed that the Ad35-based vector efficiently infects both human and rhesus macaque dendritic cells. Our novel Ad35-based vectors and their corresponding packaging cell lines will provide a versatile and powerful system for DNA-based vaccine development and gene therapy applications.

Adenovirus E1 Proteins↗
PubMed2004

Induction of cellular immune responses to simian immunodeficiency virus gag by two recombinant negative-strand RNA virus vectors.

A recombinant Newcastle disease virus (rNDV) expressing simian immunodeficiency virus (SIV) Gag protein (rNDV/SIVgag) was generated. The rNDV/SIVgag virus induced Gag-specific cellular immune responses in mice, leading to a specific anti-Gag antiviral immunity. This was evidenced by the inhibition of growth of recombinant vaccinia virus expressing an identical Gag antigen (rVac/SIVgag) but not of wild-type vaccinia virus in rNDV/SIVgag-immunized mice. Among intravenous, intraperitoneal, or intranasal immunization routes, intranasal administration induced the strongest protective response against challenge with rVac/SIVgag. We further demonstrated that these immune responses were greatly enhanced after booster immunization with recombinant influenza viruses expressing immunogenic portions of SIV Gag. The magnitude of the protective immune response correlated with the levels of cellular immune responses to Gag, which were still evident 9 weeks after immunization. These results suggest that rNDV and influenza virus vectors are suitable candidate vaccines against AIDS as well as against other infectious diseases.

Amino Acid Sequence↗
PubMed1987

Construction of expression vectors for the production of interferons in yeast.

Expression vectors designed for the production of foreign proteins in yeast are constructed as a composite of functional DNA segments brought together in a single plasmid DNA molecule. Such a composite DNA molecule constitutes a complex entity in terms of its replication and selection functions (in both E. coli and S. cerevisiae) and its gene expression properties. The latter depend critically upon transcriptional control signals (the promoter and terminator regions from a highly expressed yeast gene) that are used to direct the expression of foreign genes in yeast cells. This paper describes the construction of such an expression vector based on the transcription control signals of the yeast phosphoglycerate kinase gene and its application to the production of human interferon-alpha's in yeast cells.

Cloning, Molecular↗
PubMed1994

Gene targeting by a vector with hairpin-shaped oligonucleotide caps.

To improve the efficiency of gene targeting, a linearized vector containing part of the mouse transthyretin (ttr) gene, a bacterial neomycin-resistance gene and a herpes simplex virus thymidine kinase gene was capped with hairpin-shaped oligonucleotides. The capped vector not only improved the efficiency of positive-negative selection about 3.5-fold, but also increased about 2.5-fold the ratio of ttr gene targeted clones to the selectants.

Animals↗
PubMed1988

Expression of human calmodulin cDNA in Escherichia coli and characterization of the protein.

A cDNA clone of human calmodulin, isolated from liver, was subcloned into the expression vector pKK233-2. The resulting expression plasmid, designated pCWCaM1, produced human calmodulin in Escherichia coli SG5. The cDNA was sequenced using novel primers designed for use in plasmid-sequencing protocols with pKK233-2 and pKK223-3. The expressed calmodulin was purified and subjected to NMR analysis which revealed a structure essentially the same as natural calmodulin isolated from human tissue. The activation of myosin light chain kinase by the genetically engineered human calmodulin and bovine brain calmodulin was studied and found to be comparable to a high degree. The expressed calmodulin appears to be comparable to normal calmodulin and can be used for site-directed mutagenesis and structure/function investigations.

Base Sequence↗
PubMed2006

Circumventing recombination events encountered with production of a clinical-grade adenoviral vector with a double-expression cassette.

Delivery of multiple exogenous genes into target cells is important for a broad range of gene therapy applications, including combined therapeutic gene expression and noninvasive imaging. Previous studies ( Mol Ther 4: 223-231, 2001 ) have described the adenoviral vector RGDTKSSTR with a double-expression cassette that encodes herpes simplex virus thymidine kinase (HSVtk) for molecular chemotherapy and human somatostatin receptor subtype-2 (hSSTR2) for indirect imaging. In this vector, both genes are inserted in place of the E1 region of the adenoviral genome and expressed independently from two cytomegalovirus (CMV) promoters. During production of clinical-grade RGDTKSSTR, we found that the CMV promoters and simian virus 40 (SV40) poly(A) regions located in both expression cassettes provoked homologous recombination and deletion of one of the cassettes. To resolve this problem, we designed a strategy for substituting the duplicate promoters and poly(A) regions. We placed the hSSTR2 gene in the new Ad5.SSTR/TK.RGD vector under the control of a CMV promoter with a bovine growth hormone poly(A) region, whereas the SV40 promoter, enhancer, and poly(A) signal controlled HSVtk expression. This use of different regulatory sequences allowed independent expression of both transgenes from a single adenoviral vector and circumvented the recombination problem. Reconstruction of the vector with a double-expression cassette enables its use in human clinical trials.

Adenoviridae↗
PubMed1999

Escherichia coli skp chaperone coexpression improves solubility and phage display of single-chain antibody fragments.

Expression of single-chain antibody fragments (scAb)in the periplasm of Escherichia coli often results in low soluble product yield and cell lysis. We have increased scAb solubility and prevented cell culture lysis by coexpressing the E. coli Skp chaperone gene. A mutant Skp cistron was linked to a bacteriophage T7 gene 10 translational initiation region and placed either downstream of a scAb gene within an isopropyl beta-d-thiogalactopyranoside-inducible expression cassette or on a separate colE1-compatible arabinose-inducible vector. Increases in scAb solubility reflected the amount of coexpressed Skp. A bacteriophage display vector that was also engineered to coexpress Skp permitted display of a virtually undisplayable scAb and should prove useful in expanding library sizes.

Bacterial Proteins↗
PubMed2003

Generation of an adenoviral vector containing an addition of a heterologous ligand to the serotype 3 fiber knob.

As an initial assessment of the feasibility of employing the adenovirus serotype 3 (Ad3) fiber knob as a locale for introducing a tropism-modifying motif, we generated an adenoviral vector containing a six-histidine tag genetically fused to the carboxy-terminus of the Ad3 fiber knob. The heterologous tag proved to be accessible for binding in the context of the virion and, moreover, had rendered the modified vector capable of mediating gene transfer through an artificial, non-Ad3 receptor.

Adenoviridae↗
PubMed2005

In vitro pharmacodynamic evaluation of antiviral medicinal plants using a vector-based assay technique.

AIMS: Medicinal plants are increasingly being projected as suitable alternative sources of antiviral agents. The development of a suitable in vitro pharmacodynamic screening technique could contribute to rapid identification of potential bioactive plants and also to the standardization and/or pharmacokinetic-pharmacodynamic profiling of the bioactive components. METHODS AND RESULTS: Recombinant viral vectors (lentiviral, retroviral and adenoviral) transferring the firefly luciferase gene were constructed and the inhibition of viral vector infectivity by various concentrations of plant extracts was evaluated in HeLa or Hep2 cells by measuring the changes in luciferase activity. Cytotoxicity of the extracts was evaluated in parallel on HeLa or Hep2 cells stably expressing luciferase. Amongst the 15 extracts screened, only the methanol (ME) and the ethyl acetate (ET) fractions of the lichen, Ramalina farinacea specifically reduced lentiviral and adenoviral infectivity in a dose-dependent manner. Further, chromatographic fractionation of ET into four fractions (ET1-ET4) revealed only ET4 to be selectively antiviral with an IC50 in the 20 microg ml(-1) range. Preliminary mechanistic studies based on the addition of the extracts at different time points in the viral infection cycle (kinetic studies) revealed that the inhibitory activity was highest if extract and vectors were preincubated prior to infection, suggesting that early steps in the lentiviral or adenoviral replication cycle could be the major target of ET4. Inhibition of wild-type HIV-1 was also observed at a 10-fold lower concentration of the extract. CONCLUSIONS: The vector-based assay is a suitable in vitro pharmacodynamic evaluation technique for antiviral medicinal plants. The technique has successfully demonstrated the presence of antiviral principles in R. farinacea. SIGNIFICANCE AND IMPACT OF STUDY: Potential anti-HIV medicinal plants could rapidly be evaluated with the reported vector-based technique. The lichen, R. farinacea could represent a lead source of antiviral substances and is thus worthy of further studies.

Adenoviridae Infections↗
PubMed2006

A Gateway-based platform for multigene plant transformation.

The post-genomic era offers unrivalled opportunities for genetic manipulation of polygenic traits, multiple traits, and multiple gene products. However, remaining technical hurdles make the manipulation of multiple genes in plants difficult. Here we describe a Gateway-based vector system to enable multiple transgenes to be directly linked or fused. The vector system consists of a destination vector and two special attL-flanked entry vectors each containing an attR cassette incompatible with the attL. By multiple rounds of LR recombination reactions, which we call MultiRound Gateway, multiple transgenes can be delivered sequentially and indefinitely into the Gateway-compatible destination vector through alternate use of the two special entry vectors. In our proof-of-principle experiments we have used this vector system to construct a plant transformation vector containing seven functional DNA fragments, including a screening marker gene, two reporter genes and four matrix attachment region sequences. This system provides a platform for fully realizing the potential of plant genetic manipulation.

Arabidopsis↗
PubMed2000

Insulation from viral transcriptional regulatory elements improves inducible transgene expression from adenovirus vectors in vitro and in vivo.

Recombinant adenoviruses (Ad) are attractive vectors for gene transfer in vitro and in vivo. However, the widely used E1-deleted vectors as well as newer generation vectors contain viral sequences, including transcriptional elements for viral gene expression. These viral regulatory elements can interfere with heterologous promoters used to drive transgene expression and may impair tissue-specific or inducible transgene expression. This study demonstrates that the activity of a metal-inducible promoter is affected by Ad sequences both upstream and downstream of the transgene cassette in both orientations. Interference with expression from the heterologous promoter was particularly strong by viral regulatory elements located within Ad sequences nucleotides 1-341. This region is present in all recombinant Ad vectors, including helper-dependent vectors. An insulator element derived from the chicken gamma-globin locus (HS-4) was employed to shield the inducible promoter from viral enhancers as tested after gene transfer with first-generation Ad vectors in vitro and in vivo. Optimal shielding was obtained when the transgene expression cassette was flanked on both sides by HS-4 elements, except for when the HS-4 element was placed in 3'-->5' orientation in front of the promoter. The insulators reduced basal expression to barely detectable levels in the non-induced stage, and allowed for induction factors of approximately 40 and approximately 230 in vitro and in vivo, respectively. Induction ratios from Ad vectors without insulators were approximately 40-fold lower in vitro and approximately 15-fold lower in vivo. This study proves the potential of insulators to improve inducible or tissue-specific gene expression from adenovirus vectors, which is important for studying gene functions as well as for gene therapy approaches. Furthermore, our data show that insulators exert enhancer-blocking effects in episomal DNA.

Adenoviridae↗
PubMed1987

Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow.

Defective ecotropic and amphotropic retroviral vectors containing the cDNA for human hypoxanthine phosphoribosyltransferase (HPRT) were developed for efficient gene transfer and high-level cellular expression of HPRT. Helper cell clones which produced a high viral titer were generated by a simplified method which minimizes cell culture. We used the pZIP-NeoSV(X) vector containing a human hprt cDNA. Viral titers (1 X 10(3) to 5 X 10(4)/ml) of defective SVX HPRT B, a vector containing both the hprt and neo genes, were increased 3- to 10-fold by cocultivation of the ecotropic psi 2 and amphotropic PA-12 helper cells. Higher viral titers (8 X 10(5) to 7.5 X 10(6] were obtained when nonproducer NIH 3T3 cells or psi 2 cells carrying a single copy of SVX HPRT B were either transfected or infected by Moloney leukemia virus. The SVX HPRT B defective virus partially corrected the HPRT deficiency (4 to 56% of normal) of cultured rodent and human Lesch-Nyhan cells. However, instability of HPRT expression was detected in several infected clones. In these unstable variants, both retention and loss of the SVX HPRT B sequences were observed. In the former category, cells which became HPRT- (6-thioguanine resistant [6TGr]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418r could be regained by counterselection in hypoxanthine-aminopterin-thymidine. In vitro mouse bone marrow experiments indicated low-level expression of the neo gene in in vitro CFU assays. Individual CFU were isolated and pooled, and the human hprt gene was shown to be expressed. These studies demonstrated the applicability of vectors like SVX HPRT B for high-titer production of defective retroviruses required for hematopoietic gene transfer and expression.

Bone Marrow↗
PubMed2004

Cloning, sequencing and partial characterisation of sorbitol transporter (srlT) gene encoding phosphotransferase system, glucitol/sorbitol-specific IIBC components of Erwinia herbicola ATCC 21998.

A DNA fragment of approximately 1500 bp, harbouring the sorbitol transport gene (srlT), was amplified from the chromosomal DNA of Erwinia herbicola ATCC 21998 by PCR and cloned in Escherichia coli JM109. Degenerate oligonucleotide primers used were designed based on the conserved regions in the gene sequences within the gut operon of E. coli (Gene Bank accession no. J02708) and the srl operon of Erwinia amylovora (Gene Bank accession no. Y14603). The cloned DNA fragment was sequenced and found to contain an open reading frame of 1473 nucleotides coding for a protein of 491 amino acids, corresponding to a mass of 52410 Da. The nucleotide sequence of this ORF was highly homologous to that of the gutA gene of Escherichia coli gut operon, the srlE gene of Shigella flexrni and the sorbitol transporter gene sequence of Escherichia coli K12 (Gene Bank accession nos. J02708, AE016987 and D90892 respectively). The protein sequence showed significant homology to that of the phosphotransferase system i.e. the glucitol/sorbitol-specific IIBC components of Escherichia coli and Erwinia amylovora (P56580, O32522). The cloned DNA fragment was introduced into a pRA90 vector and the recombinant was used for developing srlT mutants of Erwinia herbicola, by homologous recombination. Mutants obtained were unable to grow on minimal medium with sorbitol. The insertion of the pRA90 vector inside the srlT gene sequence of the mutants was confirmed by DNA-DNA hybridisation.

Amino Acid Sequence↗
PubMed2005

Interleukin 7-engineered stromal cells: a new approach for hastening naive T cell recruitment.

In this study we determined whether human stromal cells could be engineered with a retroviral vector carrying the interleukin 7 (IL-7) gene and investigated the effects on T cells in vitro and in vivo in a murine model. Transduced mesenchymal cells strongly express CD90 (98.15%), CD105 (87.6%), and STRO-1 (86.7%). IL-7 production was 16.37 (+/-2 SD) pg/ml, which remained stable for 60 days. In vitro-immunoselected naive T cells maintained the CD45RA+ CD45RO- naive phenotype (4.2 times more than controls) after 7 days of culture with IL-7-engineered stromal cells. The apoptosis rate (4.7%) of the naive T cells cultured with transduced stromal cells overlapped with that of freshly isolated cells. Immunohistological analysis detected stromal cells in bone marrow, spleen, and thymus. Cotransplantation of IL-7-engineered stromal cells with CD34+ cells improved engraftment in terms of CD45+ cells and significantly increased the CD3+ cell count in peripheral blood, bone marrow, and spleen. These data demonstrate the following: (1) human stromal cells can be transduced, generating a normal layer; (2) transduced stromal cells in vitro maintain the naive T cell phenotype; and (3) IL-7-transduced stromal cells in vivo home to lymphoid organs and produce sufficient IL-7 in loco, supporting T cell development in a cotransplantation model. Because of their efficient cytokine production and homing, IL-7-engineered stromal cells might be an ideal vehicle to hasten immunological reconstitution in T cell-depleted hosts.

Animals↗
PubMed1999

Cyclosporin A reduces the inflammatory response to a multi-mutant herpes simplex virus type-1 leading to improved transgene expression in sympathetic preganglionic neurons in hamsters.

Herpes simplex virus type 1 (HSV-1) based vectors hold great promise for gene transfer to CNS neurons. Problems such as loss of transgene expression, vector-associated cytotoxicity and the immune response to the vector or encoded transgene still remain obstacles to success. We used a replication-defective, HSV-1 vector (14Hdelta3vhsZ) that was engineered to have reduced cytotoxicity and express recombinant beta-galactosidase. A previous study in our laboratory showed no evidence for cytotoxicity in infected neurons although an inflammatory infiltrate occurred around infected cells and transgene expression was lost between 5 and 8 days. The immune response consisted of a primary response at the site of inoculation (adrenal gland), and a secondary immune response in the spinal cord around infected adrenal sympathetic preganglionic neurons due to retrograde transport of the vector. We tested whether conventional immunosuppressants could reduce the secondary immune response, leading to improved transgene expression at the secondary CNS site. 14Hdelta3vhsZ was injected into the adrenal gland in hamsters 1 day after immunosuppressant treatment began. Non-drug treated, 14Hdelta3vhzZ-infected hamsters were used as controls. Cyclosporin A administration led to the most persistent beta-galactosidase activity in neurons at 5 and 8 days. Methylprednisolone treatment resulted in the greatest reduction in the inflammatory cell infiltrate but the numbers of infected neurons did not increase concomitantly. This suggested no direct relationship between extent of the inflammatory cell infiltrate and level of transgene expression. These data demonstrate the potential of cyclosporin A as an immunosuppressant adjunct treatment for HSV-1 vector-mediated gene transfer from a peripheral site to neurons in the spinal cord.

Adrenal Glands↗
PubMed1987

Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template.

The important features of the protocol described here are as follows: First, the procedure consists of a few simple steps and results in a reasonably high frequency of mutagenesis. Second, using two primers, there is no need to isolate covalently closed double-stranded molecules as in our previous method. Third, the use of vectors derived from single-stranded phage facilitates template preparation, mutagenesis efficiency, screening, and DNA sequencing. Fourth, the same basic steps can be directly applied when using the single-stranded pUC derivatives.

Base Sequence↗
PubMed2006

Intra-ventricular infusion of rAAV1-EGFP resulted in transduction in multiple regions of adult rat brain: a comparative study with rAAV2 and rAAV5 vectors.

Most gene transfer studies conducted in the central nervous system (CNS) with recombinant adeno-associated virus (rAAV) vectors have been carried out by direct intra-parenchymal injection. However, this delivery method usually results in transduction of cells in only a limited region and is quite invasive, which may hamper its potential clinical application. Injection of viral vectors into the cerebrospinal fluid (CSF) may provide an alternative strategy for widespread gene delivery to the CNS via the subarachnoid space. In this study we compared the transduction abilities of rAAV types 1, 2, and 5 when infused directly into the right lateral cerebral ventricle of adult rats. Multiple structures in the vicinity of the lateral ventricle were transduced by rAAV1, but not by rAAV2 or rAAV5 vectors. Double immunolabeling showed that the transduced cells included not only neurons, but also glia. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) experiments demonstrated that rAAV1-mediated EGFP mRNA expression was significantly higher than that induced by either rAAV2 or 5. Our data suggest that intra-ventricular infusion of rAAV1 vectors provides a useful method for broad gene delivery to cells in the adult rat CNS.

Animals↗