Plasmid Molecules Research Articles

Segregational Instability in E. Coli of Expression Plasmids Carrying Human Interferon Gamma Gene and its 3’-End Truncated Variants

ABSTRACTSegregation of the expression plasmids under non-selective conditions is due to the irregular distribution of the plasmid molecules between the daughter cells during cell division. Thus a heterogeneous population of cells appears in which the non-productive plasmid-free cells overgrow the plasmid-bearing once. The main factors that influence plasmid segregational instability are: plasmid structure, plasmid copy number, host cell genotype, level of protein expression, fermentation conditions, etc. The aim of this work is to investigate the effect of recombinant gene structure and level of expression on the segregation of expression plasmids based on pBR322. To this end the wild type human interferon gamma (hIFNγ) gene and a series of seven derivative genes obtained by systemic 3′-end deletion were inserted into an expression plasmid under a strong constitutive promoter. The segregational instability was studied under batch fermentation conditions in non-selective Luria Bertani medium, where the cell cultures were maintained at an exponential growth phase. To describe the population dynamics, a mathematical model proposed by Stewart & Levin was used and the parameters Δ (difference in specific growth rate between plasmid-free and plasmid-harbouring cells) and θ (relative plasmid loss rate) were determined for each plasmid. The obtained results showed that any variation in the 3′-end of the hIFNγ-gene strongly affected plasmid instability. We also found that the relative plasmid loss rate θ was proportional to the hIFNγ-mRNA content in the cell. Based on these results, we conclude that the structure of the constitutively expressed gene is of great importance for the segregational plasmid instability.

Organization of Plasmid DNA into Nucleosome-Like Structures after Transfection in Eukaryotic Cells

ABSTRACTOne of the most often used methods to monitor DNA repair in vivo is the host cell reactivation assay (HCR). It is based on the recovery of in vitro damaged plasmids after transfection in host cells. However, it is not clear to what extent plasmid molecules were degraded in the cells and whether they were packed with histones to form chromatin. Since these questions are important to evaluate the results obtained with HCR, in the present paper we studied the fate of the plasmid pEGFP-N1 after transfection in HEK 293 cells. To this end nuclei isolated from cells transfected with native and trioxsalen crosslinked pEGFP-N1 were digested with micrococcal nuclease (MNase) and DNA was subjected to electrophoresis. Southern blots were prepared and probed with digoxigenin-labeled plasmid DNA to reveal the plasmid DNA digestion pattern. Our results showed that nucleosome-like particles were formed on both native and damaged plasmid DNA after transfection. However, the nucleosome ladders were anomalous compared to the ladders generated by digestion of bulk cellular chromatin.

Analysis of plasmid genes by phylogenetic profiling and visualization of homology relationships using Blast2Network

BackgroundPhylogenetic methods are well-established bioinformatic tools for sequence analysis, allowing to describe the non-independencies of sequences because of their common ancestor. However, the evolutionary profiles of bacterial genes are often complicated by hidden paralogy and extensive and/or (multiple) horizontal gene transfer (HGT) events which make bifurcating trees often inappropriate. In this context, plasmid sequences are paradigms of network-like relationships characterizing the evolution of prokaryotes. Actually, they can be transferred among different organisms allowing the dissemination of novel functions, thus playing a pivotal role in prokaryotic evolution. However, the study of their evolutionary dynamics is complicated by the absence of universally shared genes, a prerequisite for phylogenetic analyses.ResultsTo overcome such limitations we developed a bioinformatic package, named Blast2Network (B2N), allowing the automatic phylogenetic profiling and the visualization of homology relationships in a large number of plasmid sequences. The software was applied to the study of 47 completely sequenced plasmids coming from Escherichia, Salmonella and Shigella spps.ConclusionThe tools implemented by B2N allow to describe and visualize in a new way some of the evolutionary features of plasmid molecules of Enterobacteriaceae; in particular it helped to shed some light on the complex history of Escherichia, Salmonella and Shigella plasmids and to focus on possible roles of unannotated proteins.The proposed methodology is general enough to be used for comparative genomic analyses of bacteria.

A radiation target method for size determination of supercoiled plasmid DNA

Supercoiled DNA plasmids were exposed in the frozen state to high-energy electrons. Surviving supercoiled molecules were separated from their degradation products (e.g., open circle and linear forms) by agarose gel electrophoresis and subsequently quantified by staining and image analysis. Complex survival curves were analyzed using radiation target theory, yielding the radiation-sensitive mass of each form. One of the irradiated plasmids was transfected into cells, permitting radiation analysis of gene expression. Loss of this function was associated with a mass much smaller than the entire plasmid molecule, indicating a lack of energy transfer in amounts sufficient to cause structural damage along the DNA polynucleotide. The method of radiation target analysis can be applied to study both structure and function of DNA.

Effects of the Incorporation of a Hydrophobic Middle Block into a PEG−Polycation Diblock Copolymer on the Physicochemical and Cell Interaction Properties of the Polymer−DNA Complexes

One-component homopolymers of cationic monomers (polycations) and diblock copolymers comprising poly(ethylene glycol) (PEG) and a polycation block have been the most widely used types of polymers for the formulation of polymer-based gene delivery systems. In this study, we incorporate a hydrophobic middle block into the conventional PEG-polycation architecture and investigate the effects of this hydrophobic modification on the physicochemical and cell-level biological properties of the polymer-DNA complexes that are relevant to gene delivery applications. The ABC-type triblock copolymer used in this study consists of (A) PEG, (B) hydrophobic poly( n-butyl acrylate) (PnBA), and (C) cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) component polymers. The properties of the triblock copolymer/DNA complexes are compared with those of two other more conventional DNA carriers derived, respectively, using a PDMAEMA homopolymer and a PEG-PDMAEMA diblock copolymer that had comparable molecular weights for individual blocks. In aqueous solution, the PEG-PnBA-PDMAEMA polymer forms positively charged spherical micelles. The electrostatic complexation of these micelles with plasmid DNA molecules results in the formation of stable small-sized DNA particles that are coated with a micelle monolayer, as confirmed by agarose gel electrophoresis, dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM). Proton nuclear magnetic resonance ( (1)H NMR) spectroscopy measurements indicate that the whole micelle-DNA assembly (named "micelleplex" for convenience) is shielded predominantly by the PEG chains. DLS and optical microscopy imaging measurements indicate that compared with PDMAEMA-DNA polyplexes, the micelleplexes have a significantly lower tendency to aggregate under physiological salt concentrations and show reduced interactions with negatively charged components in serum such as albumin and erythrocytes. While the micelleplexes are comparable to the PEG-PDMAEMA-based DNA polyplexes in terms of their stability against aggregation under high salt concentrations and in the presence of the albumin protein, they have a slightly higher tendency to interact with erythrocytes than the diblock copolymer polyplexes. Agarose gel electrophoresis measurements indicate that relative to the PEG-PDMAEMA polyplexes, the micelleplexes provide better protection of the encapsulated DNA from enzymatic degradation and also exhibit greater stability against disintegration induced by polyanionic additives; in these respects, the PDMAEMA homopolymer-based polyplexes show the best performance. In vitro studies in HeLa cells indicate that the PDMAEMA polyplexes show the highest gene transfection efficiency among the three different gene delivery systems. Between the micelleplexes and the PEG-PDMAEMA polyplexes, a higher gene transfection efficiency is observed with the latter system. All three formulations show comparable levels of cytotoxicity in HeLa cells.

DNA molecules resolved by electrical double layer force spectroscopy imaging

This paper deals with spatially resolved force spectroscopy applied to the imaging of the electrical double layer (EDL) force. It is focused on an important point that has not been elucidated yet, the resolution that can be achieved. For this purpose, single plasmid DNA molecules have been studied with an atomic force microscope operated in the force spectroscopy imaging mode. With this approach, important quantities deduced from the force spectra can be simultaneously mapped along with the topography image. Single DNA molecules have been probed obtaining a lateral resolution in the EDL force similar to that of the topography.

Christmas out of season: who is Kris Kringle and what has he wrought?

Santa Claus is sometimes referred to as Kris Kringle, a name most likely derived from Christ Kindel (Christ-child). The German name of the Christ Child is Christkind, commonly used in its diminutive form Christkindel. The Dutch– German protestant reform movement brought in the idea that the Christchild should be the standard bearer for Christmas. Traditionally, an image used for his messenger was a young child with a golden crown who holds a tiny “Tree of Light” and brings the gifts of the Christ Child. We currently enjoy July, not Christmas. However, could it be that Kringle has brought us a present out of season? Wound repair, regeneration of ischemic tissues, and the new discipline of “tissue engineering” all require improved vascularization of the regenerating tissues. Recent advances in understanding the process of blood vessel growth have offered significant tools for therapeutic neovascularization [1]. Several angiogenic growth factors including vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been used for the vascularization of regenerating and ischemic tissues in animal models. Three approaches have been used for vascularization of bioengineered tissue: incorporation of angiogenic factors in the bioengineered tissue, seeding endothelial cells with other cell types, and prevascularization of matrices prior to cell seeding [1]. One approach is to rationally design matrices that provide specific adhesion molecules such as a receptor for alpha v beta 3-integrin expressed on angiogenic endothelial cells that are able to store and release angiogenic growth factors such as bFGF that target cell type-specific responses [2]. Moreover, these matrices can be modified to release complexed plasmid DNA or other molecules that transfect surrounding cells and improve angiogenesis. During wound healing, cells infiltrate into the scaffold and degrade it, thereby releasing entrapped growth factors. The scaffold is completely removed when tissue healing is achieved. In this issue, Zhao et al. [3] demonstrate improved neovascularization and wound repair by targeting human bFGF to fibrin. bFGF is a potent mitogen for numerous cell types of mesodermal origin including fibroblasts, vascular endothelial cells, and vascular smooth muscle cells and therefore holds great promise as an agent to promote wound healing. bFGF can be produced recombinantly and therefore should be readily available therapeutically. The problem resides in bFGF delivery. Exogenous bolus injection seems to work as studies involving the heart and ischemic limb have shown. However, the bFGF diffuses rapidly from the injection site and bFGF delivery with a sustained site-targeting result has been elusive. Zhao et al.[3] fused bFGF to a kringle repeat in the plasminogen molecule termed kringle 1 (K1). They thereby constructed a fusion protein (K1-bFGF) that has a high tendency to bind to fibrin. Plasma clots of course develop at the sites of wound injury. However, the authors also developed a fibrin-scaffold/K1-bFGF construct. This system successfully induced neovascularization by delivering K1-bFGF in a sustained fashion. The result was a microenvironment that promoted cell growth, angiogenesis, and tissue regeneration. J Mol Med (2008) 86:1081–1083 DOI 10.1007/s00109-008-0389-0

Low‐copy episomal vector pFY20 and high‐saturation coverage genomic libraries for the fission yeast Schizosaccharomyces pombe

In fission yeast, as in many organisms, episomally replicating plasmid DNA molecules can be used for a wide variety of applications. However, replicating plasmids described previously are each propagated at a high copy number per cell. Plasmid fission yeast twenty (pFY20) contains the ura4(+) gene for positive and negative selection, an origin of replication (ars1) and a stability element (stb). Although this plasmid does not have a centromere, it is propagated with a copy number of about two plasmids per haploid genome equivalent and it is transmitted with relatively high fidelity in mitosis and meiosis. This low-copy vector is useful for screens and mutational studies where overexpression (e.g. from high copy plasmids) is undesirable. We therefore constructed multiple partial-digest, size-fractionated, fission yeast genomic DNA libraries in pFY20 and in the cloning vector pBluescript KS(+). These libraries have sufficient complexity (average of 2100 genome equivalents each) for saturation screening by complementation, plasmid shuffle or hybridization.

Conditional gene vectors regulated in cis

Non-integrating gene vectors, which are stably and extrachromosomally maintained in transduced cells would be perfect tools to support long-term expression of therapeutic genes but preserve the genomic integrity of the cellular host. Small extrachromosomal plasmids share some of these ideal characteristics but are primarily based on virus blueprints. These plasmids are dependent on viral trans-acting factors but they can replicate their DNA molecules in synchrony with the chromosome of the cellular host and segregate to daughter cells in an autonomous fashion. On the basis of the concept of the latent origin of DNA replication of Epstein-Barr virus, oriP, we devised novel derivatives, which exclusively rely on an artificial replication factor for both nuclear retention and replication of plasmid DNA. In addition, an allosteric switch regulates the fate of the plasmid molecules, which are rapidly lost upon addition of doxycycline. Conditional maintenance of these novel plasmid vectors allows the reversible transfer of genetic information into target cells for the first time.

The Making of a Scientist II (Nobel Lecture)

Perturbing genes: The incorporation of >100 HSV‐tk plasmid molecules into a single, ordered, concatemer when injected into somatic cells was the first demonstration of homologous recombination between cointroduced DNA molecules in cultured mammalian cells. From this came first the idea that manipulating this efficient machinery would allow any endogenous cellular gene in cultured cells to be mutated in any chosen way, and, eventually, the award of the 2007 Nobel Prize for Physiology or Medicine.

Development of One Novel Multiple-Target Plasmid for Duplex Quantitative PCR Analysis of Roundup Ready Soybean

To enforce the labeling regulations of genetically modified organisms (GMOs), the application of reference molecules as calibrators is becoming essential for practical quantification of GMOs. However, the reported reference molecules with tandem marker multiple targets have been proved not suitable for duplex PCR analysis. In this study, we developed one unique plasmid molecule based on one pMD-18T vector with three exogenous target DNA fragments of Roundup Ready soybean GTS 40-3-2 (RRS), that is, CaMV35S, NOS, and RRS event fragments, plus one fragment of soybean endogenous Lectin gene. This Lectin gene fragment was separated from the three exogenous target DNA fragments of RRS by inserting one 2.6 kb DNA fragment with no relatedness to RRS detection targets in this resultant plasmid. Then, we proved that this design allows the quantification of RRS using the three duplex real-time PCR assays targeting CaMV35S, NOS, and RRS events employing this reference molecule as the calibrator. In these duplex PCR assays, the limits of detection (LOD) and quantification (LOQ) were 10 and 50 copies, respectively. For the quantitative analysis of practical RRS samples, the results of accuracy and precision were similar to those of simplex PCR assays, for instance, the quantitative results were at the 1% level, the mean bias of the simplex and duplex PCR were 4.0% and 4.6%, respectively, and the statistic analysis ( t-test) showed that the quantitative data from duplex and simplex PCR had no significant discrepancy for each soybean sample. Obviously, duplex PCR analysis has the advantages of saving the costs of PCR reaction and reducing the experimental errors in simplex PCR testing. The strategy reported in the present study will be helpful for the development of new reference molecules suitable for duplex PCR quantitative assays of GMOs.

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls. The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 µm and extends over ~15 µm around the mean penetration depth, which is adjustable between 0 and 50 µm. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches. For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery. When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls. The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of approximately 50-150 microm and extends over approximately 15 microm around the mean penetration depth, which is adjustable between 0 and 50 microm. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches. For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery. When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

Development of a pilot-scale bacterial fermentation for plasmid-based biopharmaceutical production using a stoichiometric medium

The recognition of the potential efficacy of plasmid DNA (pDNA) molecules as vectors in the treatment and prevention of emerging diseases has birthed the confidence to combat global pandemics. This is due to the close-to-zero safety concern associated with pDNA vectors compared to viral vectors in cell transfection and targeting. Considerable attention has been paid to the potential of pDNA vectors but comparatively less thought has been given to the practical challenges in producing large quantities to meet current rising demands. A pilot-scale fermentation scheme was developed by employing a stoichiometrically-designed growth medium whose exceptional plasmid yield performance was attested in a shake flask environment for pUC19 and pEGFP-N1 transformed into E. coliDH5α and E. coliJM109, respectively. Batch fermentation of E. coliDH5α-pUC19 employing the stoichiometric medium displayed a maximum plasmid volumetric and specific yield of 62.6 mg/L and 17.1 mg/g (mg plasmid/g dry cell weight), respectively. Fed-batch fermentation of E. coliDH5α-pUC19 on a glycerol substrate demonstrated one of the highest ever reported pilot-scale plasmid specific yield of 48.98 mg/g and a volumetric yield of 0.53 g/L. The attainment of high plasmid specific yields constitutes a decrease in plasmid manufacturing cost and enhances the effectiveness of downstream processes by reducing the proportion of intracellular impurities. The effect of step-rise temperature induction was also considered to maximize ColE1-origin plasmid replication.

DNA Interstrand Crosslinks Repair in Mammalian Cells

We studied the formation of double strand breaks (DSBs) as intermediates in the repair of DNA interstrand crosslinks (ICLs) by homologous recombination (HR). The plasmid EGFP-N1 was crosslinked with trioxsalen to give one ICL per plasmid on average. HeLa cells were transfected with the crosslinked plasmids and the ICL repair was monitored by following the restoration of the GFP expression. It was accompanied by gamma-H2AX foci formation suggesting that DSBs were formed during the process. However, the same amount of gamma-H2AX foci was observed when cells were transfected with native plasmid, which indicated that gamma-H2AX foci appearance could not be used to determine the amount of DSBs connected with the ICL repair in this system. For this reason we further monitored the DSB formation by determining the amount of linearized plasmids, since having one crosslink per plasmid on average, any ICL-driven DSB formation would lead to plasmid linearization. Native and crosslinked plasmids were incubated in repair-competent cell-free extracts from G1 and S phase HeLa cells. Although a considerable part of the ICLs was repaired, no linearization of the plasmids was observed in the extracts, which was interpreted that DSBs were not formed as intermediates in the process of ICL repair. In another set of experiments HR-proficient HeLa and HR-deficient irs3 cells were transfected with native and crosslinked plasmids, and 6 h and 12 h later the plasmid DNA was isolated and analyzed by electrophoresis. The same amount of linear plasmid molecules was observed in both cell lines, regardless of whether they were transfected with native or crosslinked pEGFP-N1, which further confirmed that DSB formation was not an obligatory step in the process of ICL repair by HR.

Negative regulation of pPS10 plasmid replication: origin pairing by zipping‐up DNA‐bound RepA monomers

In many plasmid replicons of gram-negative bacteria, Rep protein dimers are transcriptional self-repressors of their genes, whereas monomers are initiators of DNA replication. Switching between both functions implies conformational remodelling of Rep, and is promoted by Rep binding to the origin DNA repeats (iterons) or chaperones. Rep proteins play another key role: they bridge together two iteron DNA stretches, found either on the same or on different plasmid molecules. These so-called, respectively, 'looped' and 'handcuffed' complexes are thought to be negative regulators of plasmid replication. Although evidence for Rep-dependent plasmid handcuffing has been found in a number of replicons, the structure of these Rep-DNA assemblies is still unknown. Here, by a combination of proteomics, electron microscopy, genetic analysis and modelling, we provide insight on a possible three-dimensional structure for two handcuffed arrays of the iterons found at the origin of pPS10 replicon. These are brought together in parallel register by zipping-up DNA-bound RepA monomers. We also present evidence for a distinct role of RepA dimers in DNA looping. This work defines a new regulatory interface in Rep proteins.

Critical condition for the occurrence of a noise-reduction effect

Based on a copy number control model of bacterial plasmid, the internal noise near a given steady state is investigated by using the linear noise approximation. All the parameters are restricted to a certain region so that the time spent near the steady state is long enough and the absorbing state can be neglected. For the noise in the plasmid molecules, a transition occurs with increasing the noise in the signal molecules under certain conditions. A noise-reduction mechanism, noise suppression by noise, is found. More importantly, the critical condition for the occurrence of the noise-reduction effect is given in our theoretical treatment.

Effects of low energy carbon ions on plasmid DNA

The damage induced in supercoiled plasmid DNA molecules by low energy (< 1 keV u-1) singly and doubly charged carbon ions has been investigated as a function of ion exposure. The production of short linear fragments through multiple double strand breakage is indicated and exponential exposure responses for each of the topoisomers are presented. The damage produced by C2+ is apparent at much lower ion exposures that with C+.

Prediction of diffusion coefficients of plasmids

The ability to predict diffusion coefficients is important in the design, analysis, and operation of plasmid downstream processing operations such as membrane and fixed-bed chromatography. A correlation is proposed to predict the diffusion coefficient, D, of supercoiled plasmid DNA molecules in dilute solutions on the basis of the molecular weight, M, or size. Experimental data (18 points) collected from the literature confirmed the proposed variation of D with plasmid molecular weight as D proportional, variant M(-2/3), for molecules within the 1,800-287,100 base-pair range. The correlation was able to estimate the available experimental results with an average error of 6.3%.

Nuclear‐targeted chimeric vector enhancing nonviral gene transfer into skeletal muscle of Fabry mice in vivo

Poor nuclear entry, especially into nondividing cells, is a limiting factor in nonviral gene delivery. We have engineered a novel chimeric vector relying on the controlled assembly of a TAT-tagged multisubunit DNA binding protein (EcoR124I) with expression plasmids containing the EcoR124I recognition site. Molecular interactions of this molecular assembly were studied by electrophoretic mobility shift assay and atomic force microscopy. Maintenance of nanocomplexes in an appropriate stoichiometric ratio was both necessary and sufficient to produce a significant (>8-fold) increase in the activity of the therapeutic alpha-galactosidase A enzyme after intramuscular administration in the mouse model of Fabry disease. To our knowledge, this is the first molecular targeting system significantly enhancing plasmid-based expression in skeletal muscle. Coinjection with pluronic SP1017 produced further enhancement of gene expression, demonstrating cumulative effects of the increased nuclear delivery by TAT chimeras and transcription activation by the pluronic. Cell penetration peptides (CPP), such as TAT, have been shown to improve delivery of macromolecules, when linked directly. However, in our system TAT-enhanced targeting took place even though it was linked to the plasmid DNA molecule indirectly via two noncovalent bonds. Therefore, this proof-of principle result indicates that TAT (and potentially other CPP) can be used for targeting modular chimeric vectors and therapeutic nanodevices.