Search results for "Genetic Vector"
showing 10 items of 158 documents
Functional roles of the membrane-associated AAV protein MAAP
2021
AbstractWith a limited coding capacity of 4.7 kb, adeno-associated virus (AAV) genome has evolved over-lapping genes to maximise the usage of its genome. An example is the recently found ORF in the cap gene, encoding membrane-associated accessory protein (MAAP), located in the same genomic region as the VP1/2 unique domain, but in a different reading frame. This 13 KDa protein, unique to the dependovirus genus, is not homologous to any known protein. Our studies confirm that MAAP translation initiates from the first CTG codon found in the VP1 ORF2. We have further observed MAAP localised in the plasma membrane, in the membranous structures in close proximity to the nucleus and to the nuclea…
Yeast vectors for the integration/expression of any sequence at theTYR1 locus
2007
We have constructed new yeast vectors for targeted integration and conditional expression of any sequence at the Saccharomyces cerevisiae TYR1 locus which becomes disrupted. We show that vector integration is not neutral, causing prototrophy for tyrosine and auxotrophy for the vector's selectable marker (uracil or leucine, depending on the vector used). This feature allows a double screening of transformed yeast cells, improving the identification of colonies with the desired chromosomal structure. The GAL10 gene promoter has been added to drive conditional expression of cloned sequences. Using these vectors, chromosomal structure verification of recombinant clones is no longer necessary, s…
A novel cell wall protein specific to the mycelial form of Yarrowia lipolytica.
1996
A cDNA clone specifying a cell wall protein was isolated from a Yarrowia lipolytica cDNA library. The cDNA library was constructed in the expression vector lambda gt 11, with the RNA isolated from actively growing mycelial cells. The deduced amino acid sequence shows that the encoded protein contains an N-terminal hydrophobic signal peptide. We have designated this protein YWP1 for Yarrowia lipolytica cell Wall Protein. Northern hybridization identified YWP1 transcript only when Y. lipolytica was growing in the mycelial form. The encoded protein seems to be covalently bound to the glucan cell wall since it is not released from the cell walls by sodium dodecyl sulphate extraction, but it is …
Altered intracellular sorting signals do not influence the efficacy of genetic melanoma vaccines incorporating helper determinants in mice.
2004
Background A genetic melanoma vaccine consisting of cDNA encoding the model self-antigen tyrosinase-related protein 2 (TRP2) fused in-frame to the immunogenic enhanced green fluorescent protein (EGFP) was able to break immune tolerance and stimulate CD8+ T cells in vivo. In the present study we investigated whether alteration of the intracellular antigen localization as a result of the linkage with immune-enhancing helper proteins affects the resulting immune response. Methods Expression plasmids and recombinant adenoviruses were constructed encoding various fusion proteins with different intracellular sorting signals which direct the antigen to the cytosol, the endoplasmic reticulum or the…
Gene transfer of the Co-stimulatory molecules B7-1 and B7-2 enhances the immunogenicity of human renal cell carcinoma to a different extent.
1999
Stimulation of a specific antitumour immune response with recruitment and induction of T-cell effector functions represents an attractive concept in human cancer therapy. Different cytokines and the B7 co-stimulatory molecules are both able to provide proliferation and activation signals for T cells. In the present study, we first demonstrated the absence of both B7-1 and B7-2 expression in human renal cell carcinoma (RCC) cell lines. The lack of B7 expression was associated with a low or absent proliferative response of allogeneic and autologous T cells upon stimulation with tumour cells. In order to investigate the role of B7-1 and B7-2, the human RCC cell line, MZ1257RC, which expresses …
Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector
2008
An ideal gene therapy vector should enable persistent transgene expression without limitations of safety and reproducibility. Here we report the development of a non-viral episomal plasmid DNA (pDNA) vector that appears to fulfil these criteria. This pDNA vector combines a scaffold/matrix attachment region (S/MAR) with a human liver-specific promoter (alpha1-antitrypsin (AAT)) in such a way that long-term expression is enabled in murine liver following hydrodynamic injection. Long-term expression is demonstrated by monitoring the longitudinal luciferase expression profile for up to 6 months by means of in situ bioluminescent imaging. All relevant control pDNA constructs expressing luciferas…
Transcriptional targeting of dendritic cells in gene gun-mediated DNA immunization favors the induction of type 1 immune responses
2003
Cutaneous dendritic cells (DC) are pivotal for the elicitation of antigen-specific immune responses following gene gun-mediated biolistic transfection of the skin. We transcriptionally targeted transgene expression to DC using vectors containing the murine fascin promoter (pFascin) to control antigen production and compared the immune response elicited with conventional DNA immunization using plasmid constructs with the ubiquitously active CMV promoter (pCMV). Biolistic transfection with pFascin initiated a marked type 1 immune response characterized by the occurrence of a large population of IFN-gamma-producing T helper (Th) cells in spleen and draining lymph nodes. Consistently, immunoglo…
Role of hepatocyte nuclear factor 3γ in the expression of human CYP2C genes
2004
Hepatocyte nuclear factor 3 gamma (HNF-3 gamma) is an important transcription factor for the maintenance of specific liver functions. However, its relevance in the expression of human cytochrome P450 (CYP) genes has not yet been explored. Several HNF3 putative binding sites can be identified in human CYP2C 5'-flanking regions. Gene reporter experiments with proximal promoters revealed that HNF-3 gamma transactivated CYP2C8, CYP2C9, and CYP2C19 (25-, 4-, and 4-fold, respectively), but it did not transactivate CYP2C18. However, overexpression of HNF-3 gamma in hepatoma cells by means of a recombinant adenovirus induced CYP2C9, CYP2C18, and CYP2C19 mRNA (4.5-, 20-, and 50-fold, respectively) b…
Transcriptional Regulation of Human CYP3A4 Basal Expression by CCAAT Enhancer-Binding Protein α and Hepatocyte Nuclear Factor-3γ
2003
Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of more than 50% of currently used therapeutic drugs, yet the mechanisms that control CYP3A4 basal expression in liver are poorly understood. Several putative binding sites for CCAAT/enhancer-binding protein (C/EBP) and hepatic nuclear factor 3 (HNF-3) were found by computer analysis in CYP3A4 promoter. The use of reporter gene assays, electrophoretic mobility shift assays, and site-directed mutagenesis revealed that one proximal and two distal C/EBP alpha binding sites are essential sites for the trans-activation of CYP3A4 promoter. No trans-activation was found in similar reporter gene experiments with a HNF-3 gamma expression vec…
Plasmid conjugation from Proteobacteria as evidence for the origin of xenologous genes in Cyanobacteria
2014
Comparative genomics have shown that 5% of Synechococcus elongatus PCC 7942 genes are of probable proteobacterial origin. To investigate the role of interphylum conjugation in cyanobacterial gene acquisition, we tested the ability of a set of prototype proteobacterial conjugative plasmids (RP4, pKM101, R388, R64, and F) to transfer DNA from Escherichia coli to S. elongatus. A series of BioBrick-compatible, mobilizable shuttle vectors was developed. These vectors were based on the putative origin of replication of the Synechococcus resident plasmid pANL. Not only broad-host-range plasmids, such as RP4 and R388, but also narrower-host-range plasmids, such as pKM101, all encoding MPFT-type IV …