Search results for "Transgenes"
showing 10 items of 66 documents
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…
α-Synuclein expression levels do not significantly affect proteasome function and expression in mice and stably transfected PC12 cell lines
2004
α-Synuclein (α-syn) is a small protein of unknown function that is found aggregated in Lewy bodies, the histopathological hallmark of sporadic Parkinson disease and other synucleinopathies. Mutations in the α-syn gene and a triplication of its gene locus have been identified in early onset familial Parkinson disease. α-Syn turnover can be mediated by the proteasome pathway. A survey of published data may lead to the suggestion that overexpression of α-syn wild type, and/or their variants (A53T and A30P), may produce a decrease in proteasome activity and function, contributing to α-syn aggregation. To investigate the relationship between synuclein expression and proteasome function we have s…
Promoter activity of the sea urchin (Paracentrotus lividus) nucleosomal H3 and H2A and linker H1 a-histone genes is modulated by enhancer and chromat…
2009
Core promoters and chromatin insulators are key regulatory elements that may direct a transcriptional enhancer to prefer a specific promoter in complex genetic loci. Enhancer and insulator flank the sea urchin (Paracentrotus lividus) alpha-histone H2A transcription unit in a tandem repeated cluster containing the five histone genes. This article deals with the specificity of interaction between the H2A enhancer-bound MBF-1 activator and histone gene promoters, and with the mechanism that leads the H1 transcripts to peak at about one-third of the value for nucleosomal H3 and H2A mRNAs. To this end, in vivo competition assays of enhancer and insulator functions were performed. Our evidence su…
The relative role of the T-domain and flanking sequences for developmental control and transcriptional regulation in protein chimeras of Drosophila O…
2004
optomotor-blind (omb) and optomotor-blind related-1 (org-1) encode T-domain DNA binding proteins in Drosophila. Members of this family of transcription factors play widely varying roles during early development and organogenesis in both vertebrates and invertebrates. Functional specificity differs in spite of similar DNA binding preferences of all family members. Using a series of domain swap chimeras, in which different parts of OMB and ORG-1 were mutually exchanged, we investigated the relevance of individual domains in vitro and in vivo. In cell culture transfection assays, ORG-1 was a strong transcriptional activator, whereas OMB appeared neutral. The main transcriptional activation fun…
Regulation of the sea urchin early H2A histone gene expression depends on the modulator element and on sequences located near the 3' end
1999
Abstract Transcription of the sea urchin early histone genes occurs transiently during early cleavage, reaching the maximum at the morula stage and declining to an undetectable level at the gastrula stage. To identify the regulatory elements responsible for the timing and the levels of transcription of the H2A gene, we used promoter binding studies in nuclear extracts and microinjection of a CAT transgene driven by the early H2A promoter. We found that morula and gastrula nuclear proteins produced indistinguishable DNase I footprint patterns on the H2A promoter. Two sites of interactions, centred on the modulator/enhancer and on the CCAAT box respectively, were detected. Deletion of the mod…
The Arabidopsis Copper Transporter COPT1 Functions in Root Elongation and Pollen Development
2004
Copper plays a dual role in aerobic organisms, as both an essential and a potentially toxic element. To ensure copper availability while avoiding its toxic effects, organisms have developed complex homeostatic networks to control copper uptake, distribution, and utilization. In eukaryotes, including yeasts and mammals, high affinity copper uptake is mediated by the Ctr family of copper transporters. This work is the first report on the physiological function of copper transport in Arabidopsis thaliana. We have studied the expression pattern of COPT1 in transgenic plants expressing a reporter gene under the control of the COPT1 promoter. The reporter gene is highly expressed in embryos, tric…
Development of S/MAR minicircles for enhanced and persistent transgene expression in the mouse liver.
2010
We have previously described the development of a scaffold/matrix attachment region (S/MAR) episomal vector system for in vivo application and demonstrated its utility to sustain transgene expression in the mouse liver for at least 6 months following a single administration. Subsequently, we observed that transgene expression is sustained for the lifetime of the animal. The level of expression, however, does drop appreciably over time. We hypothesised that by eliminating the bacterial components in our vectors, we could improve their performance since bacterial sequences have been shown to be responsible for the immunotoxicity of the vector and the silencing of its expression when applied i…
Direct and long-term detection of gene doping in conventional blood samples
2010
The misuse of somatic gene therapy for the purpose of enhancing athletic performance is perceived as a coming threat to the world of sports and categorized as 'gene doping'. This article describes a direct detection approach for gene doping that gives a clear yes-or-no answer based on the presence or absence of transgenic DNA in peripheral blood samples. By exploiting a priming strategy to specifically amplify intronless DNA sequences, we developed PCR protocols allowing the detection of very small amounts of transgenic DNA in genomic DNA samples to screen for six prime candidate genes. Our detection strategy was verified in a mouse model, giving positive signals from minute amounts (20 μl)…
Functional characterization of the enhancer blocking element of the sea urchin early histone gene cluster reveals insulator properties and three esse…
2000
Insulator elements can be functionally identified by their ability to shield promoters from regulators in a position-dependent manner or their ability to protect adjacent transgenes from position effects. We have previously reported the identification of a 265 bp sns DNA fragment at the 3' end of the sea urchin H2A early histone gene that blocked expression of a reporter gene in transgenic embryos when placed between the enhancer and the promoter. Here we show that sns interferes with enhancer-promoter interaction in a directional manner. When sns is placed between the H2A modulator and the inducible tet operator, the modulator is barred from interaction with the basal promoter. However, th…
Paratransgenic manipulation of tsetsemiR275alters the physiological homeostasis of the fly’s midgut environment
2021
AbstractTsetse flies are vectors of parasitic African trypanosomes (Trypanosomaspp.). Current disease control methods include fly-repelling pesticides, trapping flies, and chemotherapeutic treatment of infected people. Inhibiting tsetse’s ability to transmit trypanosomes by strengthening the fly’s natural barriers can serve as an alternative approach to reduce disease. The peritrophic matrix (PM) is a chitinous and proteinaceous barrier that lines tsetse’s midgut. It protects the epithelial cells from the gut lumen content such as food and invading trypanosomes, which have to overcome this physical barrier to establish an infection. Bloodstream form trypanosomes shed variant surface glycopr…