Proteomic and Functional Analyses of Protein–DNA Complexes During Gene Transfer
Department of Pediatrics, Division of Neonatology, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA.Molecular Therapy (Impact Factor: 6.23). 11/2012; 21(4). DOI: 10.1038/mt.2012.231
One of the barriers to successful nonviral gene delivery is the crowded cytoplasm, which plasmids need to actively traverse for gene expression. Relatively little is known about how this process occurs, but our lab and others have shown that the microtubule network and motors are required for plasmid movement to the nucleus. To further investigate how plasmids exploit normal physiological processes to transfect cells, we have taken a proteomics approach to identify the proteins that comprise the plasmid-trafficking complex. We have developed a live cell DNA-protein pull-down assay to isolate complexes at certain time points post-transfection (15 minutes to 4 hours) for analysis by mass spectrometry (MS). Plasmids containing promoter sequences bound hundreds of unique proteins as early as 15 minutes post-electroporation, while a plasmid lacking any eukaryotic sequences failed to bind many of the proteins. Specific proteins included microtubule-based motor proteins (e.g., kinesin and dynein), proteins involved in protein nuclear import (e.g., importin 1, 2, 4, and 7, Crm1, RAN, and several RAN-binding proteins), a number of heterogeneous nuclear ribonucleoprotein (hnRNP)- and mRNA-binding proteins, and transcription factors. The significance of several of the proteins involved in protein nuclear localization and plasmid trafficking was determined by monitoring movement of microinjected fluorescently labeled plasmids via live cell particle tracking in cells following protein knockdown by small-interfering RNA (siRNA) or through the use of specific inhibitors. While importin β1 was required for plasmid trafficking and subsequent nuclear import, importin α1 played no role in microtubule trafficking but was required for optimal plasmid nuclear import. Surprisingly, the nuclear export protein Crm1 also was found to complex with the transfected plasmids and was necessary for plasmid trafficking along microtubules and nuclear import. Our results show that various proteins involved in nuclear import and export influence intracellular trafficking of plasmids and subsequent nuclear accumulation.Molecular Therapy (2012); doi:10.1038/mt.2012.231.
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ABSTRACT: Electroporation is a physical method of transferring molecules into cells and tissues. It takes advantage of the transient permeabilization of the cell membrane induced by electric field pulses, which gives hydrophilic molecules access to the cytoplasm. This method offers high transfer efficiency for small molecules that freely diffuse though electrically permeabilized membrane. Larger molecules, such as plasmid DNA, face several barriers (plasma membrane, cytoplasmic crowding, nuclear envelope) which reduce transfection efficiency and engender a complex mechanism of transfer. Our work provides insight into the way electrotransferred DNA crosses the cytoplasm to reach the nucleus. For this purpose, single particle tracking experiments of fluorescently labeled DNA were performed. Investigations were focused on the involvement of the cytoskeleton using drugs disrupting or stabilizing actin and tubulin filaments as the two relevant cellular networks for particle transport. The analysis of 315 movies (~ 4000 trajectories) reveals that DNA is actively transported via the cytoskeleton. The large number of events allows a statistical quantification of the DNA motion kinetics inside the cell. Disruption of both filament types reduces active transport occurrence, velocities, and displacements of DNA particles. Interestingly, stabilization of both networks does not enhance DNA transport.Molecular Therapy (2013); doi:10.1038/mt.2013.182.
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ABSTRACT: Electroporation is a safe, efficient, and inexpensive method to transfer naked plasmid DNA into various tissues. For electroporation-mediated gene transfer to the mouse lung, a plasmid solution is delivered to the lungs via the trachea. Immediately after plasmid delivery, eight square wave pulses are delivered by two pre-gelled electrodes placed on each side of the chest. An optimal field strength in mice is 200 V/cm, with a pulse duration of 10 ms each and a 1 s interval between pulses. High level gene expression can be achieved within 24 h in all cell types in the lung with very little inflammation and no apparent trauma.
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ABSTRACT: Membrane electropermeabilization is the observation that the permeability of a cell membrane can be transiently increased when a micro-millisecond external electric field pulse is applied on a cell suspension or on a tissue. Applicative aspects for the transfer of foreign molecules (macromolecules) into the cytoplasm are routinely used. But only a limited knowledge about what is really occurring in the cell and its membranes at the molecular levels is available. This chapter is a critical attempt to report the present state of the art and to point out some of the still open problems. The experimental facts associated to membrane electropermeabilization are firstly reported. They are valid on biological and model systems. Secondly, soft matter approaches give access to the bioelectrochemical description of the thermodynamical constraints supporting the destabilization of simplified models of the biological membrane. It is indeed described as a thin dielectric leaflet, where a molecular transport takes place by electrophoresis and then diffusion. This naïve approach is due to the lack of details on the structural aspects affecting the living systems as shown in a third part. Membranes are part of the cell machinery. The critical property of cells as being an open system from the thermodynamical point of view is almost never present. Computer simulations are now contributing to our knowledge on electropermeabilization. The last part of this chapter is a (very) critical report of all the efforts that have been performed. The final conclusion remains that we still do not know all the details on the reversible structural and dynamical alterations of the cell membrane (and cytoplasm) supporting its electropermeabilization. We have a long way in basic and translational researches to reach a pertinent description.
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