RESUMO
The transience of transgene expression is a major obstacle in the development of nonviral vectors. The CpG-free and pLIVE plasmids reportedly achieve long-term transgene expression in mouse liver. In this work, the anti-silencing elements within these plasmids were studied. The effects of plasmid that was being silenced on transgene expression from the CpG-free plasmid and those of transgene expression at early time points on silencing were also examined. The results suggested that the backbone sequence of the CpG-free plasmid and the 3' untranslated region of the albumin gene of the pLIVE plasmid contribute to durable expression. In addition, no influence of the silencing of another plasmid on the duration of CpG-free plasmid expression or of transgene expression at early time points on silencing was detected.
Assuntos
Ilhas de CpG/genética , DNA/genética , Inativação Gênica/fisiologia , Plasmídeos/genética , Elementos Silenciadores Transcricionais/genética , Animais , Feminino , Fígado/fisiologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Plasmid DNA was chromatinized with core histones (H2A, H2B, H3, and H4) in vitro and was delivered into mouse liver by hydrodynamics-based administration. Transgene expression from the chromatinized plasmid DNA was more efficient than that from plasmid DNA delivered in the naked form. The use of acetylation-enriched histones isolated from cells treated with a histone deacetylase inhibitor (trichostatin A) seemed to be more effective. These results indicated that chromatinized plasmid DNA is useful for efficient transgene expression in vivo.
Assuntos
DNA/administração & dosagem , Histonas/metabolismo , Fígado/metabolismo , Transgenes , Animais , Cromatina/química , Feminino , Regulação da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , PlasmídeosRESUMO
Although Cd(2+) is a more effective inducer of phytochelatin (PC) synthesis than Zn(2+) in higher plants, we have observed greater induction of PC synthesis by Zn(2+) than Cd(2+) in the marine green alga, Dunaliella tertiolecta. To elucidate this unique regulation of PC synthesis by Zn(2+), we investigated the effects of Zn(2+) and Cd(2+) on the activities of both phytochelatin synthase (PC synthase) and enzymes in the GSH biosynthetic pathway. PC synthase was more strongly activated by Cd(2+) than by Zn(2+), but the difference was not very big. On the other hand, gamma-glutamylcysteine synthetase (gamma-ECS) and glutathione synthetase (GS) were activated by both heavy metals, but their activities were higher in Zn-treated cells than in Cd-treated cells. Dose-dependent stimulation of intracellular reactive oxygen species (ROS) production was observed with Zn(2+), but not Cd(2+) treatment. These results suggest that Zn(2+) strongly promotes the synthesis of GSH through indirect activation of gamma-ECS and GS by stimulating ROS generation. This acceleration of the flux rate for GSH synthesis might mainly contribute to high level PC synthesis.
