Plasmid DNA entry into postmitotic nuclei of primary rat myotubes.

Dowty, Martin E.; Williams, Phillip; Zhang, Guofeng; Hagstrom, James E.; Wolff, Jon A.

doi:10.1073/pnas.92.10.4572

Cited by 248 publications

(157 citation statements)

References 30 publications

(31 reference statements)

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“…However, both of these promoters generate very high levels of gene expression when injected into mouse quadricep muscles that are made up of post-mitotic, terminally differentiated myotubes (10,11). Thus, these plasmids gain access to the nuclei of these cells (33). One possible explanation for this paradox is that plasmids lacking the SV40 enhancer sequences are preferentially degraded in the cytoplasm, decreasing their numbers so that any entrance into the nucleus would be undetectable.…”

Section: Discussionmentioning

confidence: 99%

Sequence Requirements for Plasmid Nuclear Import

Dean

Müller³

et al. 1999

Experimental Cell Research

211

244

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SUMMARYThe nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of non-viral gene delivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366 bp fragment containing the SV40 origin of replication and early promoter. In this report, we show that, although fragments throughout this region can support varying degrees of nuclear import, the 72 bp repeats of the SV40 enhancer facilitate maximal transport. The functions of the promoter and the origin of replication are not needed for nuclear localization of plasmid DNA. In contrast to the import activity of the SV40 enhancer, two other strong promoter and enhancer sequences, the human cytomegalovirus (CMV) immediate early promoter and the Rous sarcoma virus LTR, were unable to direct nuclear localization of plasmids. The inability of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent protein (GFP) in microinjected cells. At times before cell division, as few as 3 to 10 copies per cell of cytoplasmically injected plasmids containing the SV40 enhancer gave significant GFP expression, while no expression was obtained with more than 1000 copies per cell of plasmids lacking the SV40 sequence. However, the levels of expression were the same for both plasmids after cell division in cytoplasmically injected cells and at all times in nuclear injected cells. Thus, the inclusion this SV40 sequence in non-viral vectors may greatly increase their ability to be transported into the nucleus, especially in non-dividing cells.

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Section: Discussionmentioning

confidence: 99%

Sequence Requirements for Plasmid Nuclear Import

Dean

Müller³

et al. 1999

Experimental Cell Research

211

244

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“…Alternatively, it is possible that inefficient nuclear delivery processes independent of the cell cycle exist which facilitate transfection in cell cycle arrested cells. Indeed, Dowty et al 27 have shown that plasmid DNA microinjected into the cytosol of post-mitotic rat myotubules was capable of yielding transgene expression. From their studies, cell-cycle independent nuclear delivery of plasmid DNA clearly exists in certain cell types.…”

Section: Figure 7 Semliki Forest Virus-mediated-gene Expression Sk-omentioning

confidence: 99%

Cationic lipid-mediated transfection of cells in culture requires mitotic activity

et al. 1999

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Cationic lipid-based delivery systems such as lipoplexes or aphidicolin exhibited 20-fold lower reporter gene activity stabilized plasmid-lipid particles (SPLP) represent a safer than asynchronous control cells upon incubation with lipoalternative to viral systems for gene therapy applications.plexes. When cells arrested in the G1 phase were allowed We studied the impact of cell cycle status on the efficiency to proceed though the cell cycle in the presence of the lipoof transfection of human ovarian carcinoma tumor cells plex or SPLP, transgene expression was found to coincide using two cationic-lipid based delivery systems. Cells with the transition of cells from the G2/M phase into the arrested in the G1 phase of the cell cycle by treatment with G1 phase of the subsequent cell cycle. In addition, higher aphidicolin were compared with an asynchronous dividing levels of reporter gene expression were observed when the population of cells. Treatment of the cells with aphidicolin cells were incubated with lipoplexes or SPLP during, or just had no effect on the rate of internalization of the lipid forbefore, mitosis. These results suggest that it may be possmulated DNA or on the level of gene expression observible to augment cationic lipid-mediated transfection by able in stably transfected cells. However, cells treated with manipulating the cell cycle status of the target cells.

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“…We, and others, have subsequently shown that nuclear import of plasmid DNA is sequence dependent, requires karyopherins (importins) and the small GTPase RAN, and occurs through the nuclear pore complex (NPC). [14][15][16][17][18] In our model of plasmid nuclear import, we propose that transcription factors present in the cytoplasm bind to the DTS and coat the plasmid with nuclear localization sequences (NLSs) that utilize importins to cross the NPC, which regulates the nucleocytoplasmic shuttling of macromolecules during interphase. [19][20][21] We have also developed a tissue-specific DTS, utilizing the smooth muscle g-actin (SMGA) promoter, that mediates nuclear import of pDNA specifically in smooth muscle cells (SMCs).…”

Section: Introductionmentioning

confidence: 99%

Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences

Miller

Dean

2008

Gene Ther

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Two shortcomings of nonviral gene therapy are a lack of tissue-specific targeting of vectors and low levels of gene transfer. Our laboratory has begun to address these limitations by designing plasmids that enter the nucleus of specific cell types in the absence of cell division, thereby enhancing expression in a controlled manner. We have shown that a 176 bp portion of the smooth muscle g-actin (SMGA) promoter can mediate plasmid nuclear import specifically in smooth muscle cells (SMCs). Here, we demonstrate that the binding sites for serum response factor (SRF) and NKX3-1/3-2 within this DNA nuclear targeting sequence (DTS) are required for plasmid nuclear import. Knockdown of these factors with siRNA abrogates plasmid nuclear import, indicating that they are necessary cofactors. In addition, coinjection of recombinant SRF and Nkx3.2 with the vector in TC7 epithelial cells rescues import. Finally, we show that the SRF nuclear localization sequence (NLS) is required for vector nuclear import. We propose that SRF and NKX3-1/3-2 bind the SMGA DTS in the cytoplasm, thus coating the plasmid with NLSs that mediate translocation across the nuclear pore complex. This discovery could aid in the development of more efficient nonviral vectors for gene transfer to SMCs.

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Plasmid DNA entry into postmitotic nuclei of primary rat myotubes.

Cited by 248 publications

References 30 publications

Sequence Requirements for Plasmid Nuclear Import

Sequence Requirements for Plasmid Nuclear Import

Cationic lipid-mediated transfection of cells in culture requires mitotic activity

Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences

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