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Polyplex-induced cytosolic nuclease activation leads to differential transgene expression.

ABSTRACT: Cytosolic nucleases have been proposed to play an important role in limiting the effectiveness of polyplex-based gene delivery agents. In order to explore the effect of cell membrane disruption on nuclease activation, nuclease activity upon polyplex uptake and localization, and nuclease activity upon gene expression, we employed an oligonucleotide molecular beacon (MB). The MB was incorporated as an integral part of the polymer/DNA polyplex, and two-color flow cytometry experiments were performed to explore the relationship of MB cleavage with propidium iodide (PI) uptake, protein expression, and polyplex uptake. In addition, confocal fluorescence microcopy was performed to examine both polyplex and cleaved MB localization. The impact of cell membrane disruption was also probed using whole-cell patch clamp measurement of the plasma membrane's electrical conductance. Differential activation of cytosolic nuclease was observed with substantial activity for B-PEI and G5 PAMAM dendrimer (G5), less cleavage for jetPEI, and little activity for L-PEI. jetPEI and L-PEI exhibited substantially greater transgene expression, consistent with the lower amounts of MB oligonucleotide cleavage observed. Cytosolic nuclease activity, although dependent on the choice of polymer employed, was not related to the degree of cell plasma membrane disruption that occurred as measured by PI uptake or whole-cell patch clamp.

SUBMITTER: Rattan R

PROVIDER: S-EPMC3776314 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Polyplex-induced cytosolic nuclease activation leads to differential transgene expression.

Rattan Rahul R Vaidyanathan Sriram S Wu Gordon S-H GS Shakya Anisha A Orr Bradford G BG Banaszak Holl Mark M MM

Molecular pharmaceutics 20130724 8

Cytosolic nucleases have been proposed to play an important role in limiting the effectiveness of polyplex-based gene delivery agents. In order to explore the effect of cell membrane disruption on nuclease activation, nuclease activity upon polyplex uptake and localization, and nuclease activity upon gene expression, we employed an oligonucleotide molecular beacon (MB). The MB was incorporated as an integral part of the polymer/DNA polyplex, and two-color flow cytometry experiments were performe ...[more]

PMID: 23834286

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Project description:Background and aimsWater deficit is the most serious environmental factor limiting agricultural production. In this work, the tolerance to water stress (WS) of transgenic plum lines harbouring transgenes encoding cytosolic antioxidant enzymes was studied, with the aim of achieving the durable resistance of commercial plum trees.MethodsThe acclimatization process was successful for two transgenic lines: line C3-1, co-expressing superoxide dismutase (two copies) and ascorbate peroxidase (one copy) transgenes simultaneously; and line J8-1, harbouring four copies of the cytosolic ascorbate peroxidase gene (cytapx). Plant water relations, chlorophyll fluorescence and the levels of antioxidant enzymes were analysed in both lines submitted to moderate (7 d) and severe (15 d) WS conditions. Additionally, in line J8-1, showing the best response in terms of stress tolerance, a proteomic analysis and determination of the relative gene expression of two stress-responsive genes were carried out.Key resultsLine J8-1 exhibited an enhanced stress tolerance that correlated with better photosynthetic performance and a tighter control of water-use efficiency. Furthermore, this WS tolerance also correlated with a higher enzymatic antioxidant capacity than wild-type (WT) and line C3-1 plum plants. On the other hand, line C3-1 displayed an intermediate phenotype between WT plants and line J8-1 in terms of WS tolerance. Under severe WS, the tolerance displayed by J8-1 plants could be due to an enhanced capacity to cope with drought-induced oxidative stress. Moreover, proteomic analysis revealed differences between WT and J8-1 plants, mainly in terms of the abundance of proteins related to carbohydrate metabolism, photosynthesis, antioxidant defences and protein fate.ConclusionsThe transformation of plum plants with cytapx has a profound effect at the physiological, biochemical, proteomic and genetic levels, enhancing WS tolerance. Although further experiments under field conditions will be required, it is proposed that J8-1 plants would be an interesting Prunus rootstock for coping with climate change.

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Polyplex-induced cytosolic nuclease activation leads to differential transgene expression.

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Polyplex-induced cytosolic nuclease activation leads to differential transgene expression.

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