Intranuclear trafficking of plasmid DNA is mediated by nuclear polymeric proteins lamins and actin.

Ondřej, Vladan; Lukášová, Emı́lie; Krejčí, Jana; Kozubek, Stanislav

doi:10.18388/abp.2008_3077

Cited by 7 publications

(3 citation statements)

References 27 publications

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“…To promote the intracellular unpackaging of DNA, current approaches include sulfide-conjugated polymers that promote complex disassembly within the cytoplasm and “charge-shifting” polymers that release DNA upon polymer hydrolysis . Unpackaging of the DNA in the endosome or cytoplasm, however, is not ideal given that plasmid DNA cannot effectively diffuse in the cytoplasm , and has a cytoplasmic half-life of approximately 60 min . Recent studies have shown that total cellular DNA release correlates poorly with transfection efficiency .…”

Section: Introductionmentioning

confidence: 99%

Using the Epigenetic Code To Promote the Unpackaging and Transcriptional Activation of DNA Polyplexes for Gene Delivery

2012

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Nonviral gene delivery has seen limited clinical application due in part to the inefficiency with which most nonviral vehicles navigate the intracellular gene delivery pathway. One key problem is the inability of most DNA-packaging materials to release DNA and enable its efficient transcription. Thus, our aim was to develop gene delivery polyplexes capable of initiating their own transcription upon arrival in the nucleus. We created nuclease-resistant polyplexes with plasmid DNA (pDNA) and post-translationally modified histone 3 (H3K4Me3) tail peptides known to signal transcriptional activation on chromosomal DNA. When the H3K4Me3–pDNA polyplexes were directly microinjected into the nuclei of NIH/3T3 mouse fibroblasts, protein expression occurred earlier and in a greater fraction of cells than when polyethylenimine–pDNA polyplexes were microinjected. The rate of protein expression initiated by the H3K4Me3–pDNA polyplexes was also significantly accelerated in comparison with the rate initiated by non-trimethylated H3–pDNA polyplexes. These differences in protein expression rates were quantified by the development of a noncompartmentalized cellular kinetics model. These results highlight the importance of polyplex unpackaging as a gene delivery barrier, and demonstrate for the first time that the epigenetic code can be utilized in nonviral gene delivery.

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Section: Introductionmentioning

confidence: 99%

Using the Epigenetic Code To Promote the Unpackaging and Transcriptional Activation of DNA Polyplexes for Gene Delivery

2012

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“…Despite the existence of plastids in various forms, all plastids in a particular organism have common DNA, inherited from the maternal parent, showing that the nuclear genome is vital regulatory role over the plastid morphology and function [69]. Furthermore, the plastids are estimated to contain at least 1000, and possibly as many as 5000 different proteins, the vast majority of these proteins are encoded within the nucleus and their corresponding DNA sequences are believed to have migrated from the plastid genome during the course of evolution [70]. Thus, the high number of the cotton NLP proteins predicted to be sublocalized within the nucleus could possibly be explained by either duplication or transfer events from the plastids to the nucleus.…”

Section: Discussionmentioning

confidence: 99%

Map-Based Functional Analysis of the GhNLP Genes Reveals Their Roles in Enhancing Tolerance to N-Deficiency in Cotton

Magwanga

Kirungu

et al. 2019

IJMS

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Nitrogen is a key macronutrient needed by plants to boost their production, but the development of cotton genotypes through conventional approaches has hit a bottleneck due to the narrow genetic base of the elite cotton cultivars, due to intensive selection and inbreeding. Based on our previous research, in which the BC2F2 generations developed from two upland cotton genotypes, an abiotic stress-tolerant genotype, G. tomentosum (donor parent) and a highly-susceptible, and a highly-susceptible, but very productive, G. hirsutum (recurrent parent), were profiled under drought stress conditions. The phenotypic and the genotypic data generated through genotyping by sequencing (GBS) were integrated to map drought-tolerant quantitative trait loci (QTLs). Within the stable QTLs region for the various drought tolerance traits, a nodule-inception-like protein (NLP) gene was identified. We performed a phylogenetic analysis of the NLP proteins, mapped their chromosomal positions, intron-exon structures and conducted ds/dn analysis, which showed that most NLP genes underwent negative or purifying selection. Moreover, the functions of one of the highly upregulated genes, Gh_A05G3286 (Gh NLP5), were evaluated using the virus gene silencing (VIGS) mechanism. A total of 226 proteins encoded by the NLP genes were identified, with 105, 61, and 60 in Gossypium hirsutum, G. raimondii, and G. arboreum, respectively. Comprehensive Insilico analysis revealed that the proteins encoded by the NLP genes had varying molecular weights, protein lengths, isoelectric points (pI), and grand hydropathy values (GRAVY). The GRAVY values ranged from a negative one to zero, showing that proteins were hydrophilic. Moreover, various cis-regulatory elements that are the binding sites for stress-associated transcription factors were found in the promoters of various NLP genes. In addition, many miRNAs were predicted to target NLP genes, notably miR167a, miR167b, miR160, and miR167 that were previously shown to target five NAC genes, including NAC1 and CUC1, under N-limited conditions. The real-time quantitative polymerase chain reaction (RT-qPCR) analysis, revealed that five genes, Gh_D02G2018, Gh_A12G0439, Gh_A03G0493, Gh_A03G1178, and Gh_A05G3286 were significantly upregulated and perhaps could be the key NLP genes regulating plant response under N-limited conditions. Furthermore, the knockdown of the Gh_A05G3286 (GhNLP5) gene by virus-induced silencing (VIGS) significantly reduced the ability of these plants to the knockdown of the Gh_A05G3286 (GhNLP5) gene by virus-induced gene silencing (VIGS) significantly reduced the ability of the VIGS-plants to tolerate N-limited conditions compared to the wild types (WT). The VIGS-plants registered lower chlorophyll content, fresh shoot biomass, and fresh root biomass, addition to higher levels of malondialdehyde (MDA) and significantly reduced levels of proline, and superoxide dismutase (SOD) compared to the WT under N-limited conditions. Subsequently, the expression levels of the Nitrogen-stress responsive gen...

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“…It was found that all Cy3-pDNA clusters in the nuclear region were associated with the NE staining, suggesting that these clusters were, at least partially, entrapped in nuclear invaginations. Interestingly, Ondrej et al [116] observed that fluorescently labeled DNA signals, which seemed to be situated inside the nucleus were colocalizing with actin fibers and lamins. The authors concluded that the nucleus must therefore contain an actin cytoskeleton, which, to our knowledge, has never been reported.…”

Section: Nuclear Uptakementioning

confidence: 99%

The intracellular visualization of exogenous DNA in fluorescence microscopy

Greitens,

Leroux,

Burger

2023

Preprint

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In the development of non-viral gene delivery vectors, it is essential to reliably localize and quantify transfected DNA inside the cell. To track DNA, fluorescence microscopy methods are commonly applied. These mostly rely on fluorescently labeled DNA, DNA binding proteins fused to a fluorescent protein, or fluorescence in situ hybridization (FISH). In addition, co-stainings are often used to determine the colocalization of the DNA in specific cellular compartments, such as the endolysosomes or the nucleus. We provide an overview of these DNA tracking methods, advice on how they should be combined, and indicate which co-stainings or additional methods are required to draw precise conclusions from a DNA tracking experiment. Some emphasis is given to the localization of exogenous DNA inside the nucleus, which is the last step of DNA delivery. We argue that suitable tools which allow for the nuclear detection of faint signals are still missing, hampering the rational development of more efficient non-viral transfection systems.

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Intranuclear trafficking of plasmid DNA is mediated by nuclear polymeric proteins lamins and actin.

Cited by 7 publications

References 27 publications

Using the Epigenetic Code To Promote the Unpackaging and Transcriptional Activation of DNA Polyplexes for Gene Delivery

Using the Epigenetic Code To Promote the Unpackaging and Transcriptional Activation of DNA Polyplexes for Gene Delivery

Map-Based Functional Analysis of the GhNLP Genes Reveals Their Roles in Enhancing Tolerance to N-Deficiency in Cotton

The intracellular visualization of exogenous DNA in fluorescence microscopy

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