Intracellular Plasmid Research Articles

Furan-rich, biobased transfection agents as potential oligomeric candidates for intracellular plasmid DNA delivery.

Biobased, DNA delivery vectors have been synthesized with a core motif composed of 2,5-bishydroxymethylfuran (BHMF) readily available from an important biomass feedstock 5-hydroxymethyl furfural (HMF). To generate the product, BHMF was first converted to 2,5-furan bishydroxymethyl diacrylate (2,5-FDA), which was later conjugated with different types of secondary amines. Rich in tertiary nitrogen, these oligomeric FDA-amino esters demonstrated stable electrostatic interactions with negatively charged plasmid DNA in an aqueous environment. We evaluated synthetic routes toward these plasmid DNA-binding amino esters (pFASTs), identified their nanoscale features, and attempted to establish their structure-property relationship in the context of the DNA delivery. Our preliminary studies show that the pFASTs formed stable complexes with the plasmid DNA. Dynamic light scattering indicated that the DNA polyplexes of pFASTs have hydrodynamic diameters within the size range of 100-150 nm with a surface charge (ζ-potential) ranging from -10 to +33 mV, depending on pFAST type. These oligomeric amino esters rich in furan motif were also found to successfully transfect the GFP-expressing plasmid DNA intracellularly. Collectively, this study establishes a new route to produce DNA transfection agents from sustainable resources that can be used for transferring genetic materials for humans, veterinary, and agrochemical purposes.

Knockout of the lysosomal membrane protein, LAMP2C, improves transient gene expression in HEK293 cells via increased intracellular plasmid availability.

Plasmid-based transfection can be used in many applications such as transient gene expression (TGE)-based therapeutic protein production. These applications preferentially require maximization of intracellular plasmid availability. Here, we applied a lysosome engineering approach to alleviate lysosome-mediated nucleic acid degradation and enhance the TGE in mammalian cells. By knocking out the lysosomal membrane protein LAMP2C, which is known to be the main player in RNautophagy/DNautophagy (RDA), we significantly improved transient fluorescent protein expression in HEK293 cells by improving the retention rate of transfected plasmids; however, this effect was not observed in CHO cells. Additional knockout of a lysosomal membrane transporter and another RDA player, SIDT2, was ineffective, regardless of the presence of LAMP2C. LAMP2C knockout enhanced TGE-based mAb production in HEK293 cells by up to 2.82-fold increase in specific mAb productivity. Taken together, these results demonstrate that HEK293 cells can be engineered to improve the usage of the transfected plasmid via knockout of the lysosomal membrane protein LAMP2C and provide efficient host cells in TGE systems for therapeutic protein production.

Association between adeno‐associated virus genomic titers and intracellular plasmid levels

AbstractThe recombinant adeno‐associated viral (rAAV) vector is one of the most effective viral vectors in gene therapy because of its low immunogenicity, high transduction efficiency, broad tissue specificity, and long‐term transgene expression ability. HEK293T cells are widely used to produce rAAV vectors, and acquisition of high rAAV‐producing cells is one of the essential processes for higher rAAV vector production. To acquire such cells, a method to identify cells with high expression of transgene protein was developed; however, the relationship between high expression of transgene protein and increased production of rAAV has not been sufficiently studied to date. We used the fluorescent protein ZsGreen1 as a transgene, subdivided the adherent cells according to the intensity of transgene expression protein, and reconfirmed the relationship between rAAV vector production and the amount of intracellular plasmid. We found that the amount of rAAV produced was not correlated with the intensity of expression of the transgene protein but may be correlated with the amount of intracellular plasmid. We also found that cells with high expression of transgene protein might not necessarily produce large amounts of rAAV vector. Based on these results, it was suggested that intracellular plasmid copy number could be used as a new marker for cells producing high levels of rAAV vector.

Profile and actual transmissibility of Carbapenem resistance genes: Intracellular and extracellular DNA in hospital wastewater

The current worldwide spread of carbapenem resistance genes (CRGs) has posed a major public health threat, which continues to grow in severity. Hospital wastewaters (HWWs) are major reservoirs for antibiotic resistance genes, while resistomes in HWWs are still poorly characterized when it comes to CRGs. We comprehensively characterized the profile and actual transmissibility of extracellular CRGs (eCRGs) and intracellular CRGs (iCRGs) in HWWs for the first time. In this study, CRGs showed similar relative abundance in treated and untreated HWWs. Meanwhile, HWWs treatments led to the enrichment of blaIMP-8, probably attributed to the promotion of Novosphingobium and Prosthecobacter after treatment. To evaluate the transmission potential of CRGs, extracellular and intracellular carbapenem-resistant plasmids were captured from HWWs by transformation and conjugation, respectively. We found an interesting phenomenon regarding the transmission characteristics of CRGs: blaKPC-carrying plasmids could only be captured by transformation, while blaNDM-carrying plasmids were captured by conjugation. Further experiments showed that HWW treatments increased the conjugation ability of blaNDM. In conclusion, our study demonstrated that HWWs are significant reservoirs of CRGs and various CRGs exhibit different modes of transmission in HWWs. CRGs cannot be removed by membrane bioreactor and chlorine disinfection. An urgent need is to develop more efficient wastewater treatments to limit CRG dissemination.

A Novel Method of Inducible Directed Evolution to Evolve Complex Phenotypes.

Directed evolution is a powerful technique for identifying beneficial mutations in defined DNA sequences with the goal of improving desired phenotypes. Recent methodological advances have made the evolution of short DNA sequences quick and easy. However, the evolution of DNA sequences >5kb in length, notably gene clusters, is still a challenge for most existing methods. Since many important microbial phenotypes are encoded by multigene pathways, they are usually improved via adaptive laboratory evolution (ALE), which while straightforward to implement can suffer from off-target and hitchhiker mutations that can adversely affect the fitness of the evolved strain. We have therefore developed a new directed evolution method (Inducible Directed Evolution, IDE) that combines the specificity and throughput of recent continuous directed evolution methods with the ease of ALE. Here, we present detailed methods for operating Inducible Directed Evolution (IDE), which enables long (up to 85kb) DNA sequences to be mutated in a high throughput manner via a simple series of incubation steps. In IDE, an intracellular mutagenesis plasmid (MP) tunably mutagenizes the pathway of interest, located on the phagemid (PM). MP contains a mutagenic operon ( danQ926, dam, seqA, emrR, ugi , and cda1 ) that can be expressed via the addition of a chemical inducer. Expression of the mutagenic operon during a cell cycle represses DNA repair mechanisms such as proofreading, translesion synthesis, mismatch repair, and base excision and selection, which leads to a higher mutation rate. Induction of the P1 lytic cycle results in packaging of the mutagenized phagemid, and the pathway-bearing phage particles infect naïve cells, generating a mutant library that can be screened or selected for improved variants. Successive rounds of IDE enable optimization of complex phenotypes encoded by large pathways (as of this writing up to 36 kb), without requiring inefficient transformation steps. Additionally, IDE avoids off-target genomic mutations and enables decoupling of mutagenesis and screening steps, establishing it as a powerful tool for optimizing complex phenotypes in E. coli .

Notch Intracellular Domain Plasmid Delivery via Poly(Lactic-Co-Glycolic Acid) Nanoparticles to Upregulate Notch Pathway Molecules.

Notch signaling is a highly conserved signaling system that is required for embryonic development and regeneration of organs. When the signal is lost, maldevelopment occurs and leads to a lethal state. Delivering exogenous genetic materials encoding Notch into cells can reestablish downstream signaling and rescue cellular functions. In this study, we utilized the negatively charged and FDA approved polymer poly(lactic-co-glycolic acid) to encapsulate Notch Intracellular Domain-containing plasmid in nanoparticles. We show that primary human umbilical vein endothelial cells (HUVECs) readily uptake the nanoparticles with and without specific antibody targets. We demonstrated that our nanoparticles are non-toxic, stable over time, and compatible with blood. We further demonstrated that HUVECs could be successfully transfected with these nanoparticles in static and dynamic environments. Lastly, we elucidated that these nanoparticles could upregulate the downstream genes of Notch signaling, indicating that the payload was viable and successfully altered the genetic downstream effects.

Segregational Drift Constrains the Evolutionary Rate of Prokaryotic Plasmids.

Plasmids are extrachromosomal genetic elements in prokaryotes that have been recognized as important drivers of microbial ecology and evolution. Plasmids are found in multiple copies inside their host cell where independent emergence of mutations may lead to intracellular genetic heterogeneity. The intracellular plasmid diversity is thus subject to changes upon cell division. However, the effect of plasmid segregation on plasmid evolution remains understudied. Here, we show that genetic drift during cell division—segregational drift—leads to the rapid extinction of novel plasmid alleles. We established a novel experimental approach to control plasmid allele frequency at the levels of a single cell and the whole population. Following the dynamics of plasmid alleles in an evolution experiment, we find that the mode of plasmid inheritance—random or clustered—is an important determinant of plasmid allele dynamics. Phylogenetic reconstruction of our model plasmid in clinical isolates furthermore reveals a slow evolutionary rate of plasmid-encoded genes in comparison to chromosomal genes. Our study provides empirical evidence that genetic drift in plasmid evolution occurs at multiple levels: the host cell and the population of hosts. Segregational drift has implications for the evolutionary rate heterogeneity of extrachromosomal genetic elements.

Degradation and deactivation of plasmid-encoded antibiotic resistance genes during exposure to ozone and chlorine

Degradation and deactivation kinetics of an antibiotic resistance gene (ARG) by ozone (O3) and free available chlorine (FAC) were investigated in phosphate-buffered solutions at pH 7 for O3 (in the presence of tert‑butanol), and pH 6.8 or 8.1 for FAC. We used a plasmid (pUC19)-encoded ampicillin resistance gene (ampR) in both extracellular (e-) and intracellular (i-) forms. The second-order rate constant (kO3) for degradation of 2686 base pair (bp) long e-pUC19 toward O3, which was determined by quantitative polymerase chain reaction assay, was calculated to be ~2 × 105 M−1s−1. The deactivation rate constants of e-pUC19 by O3 measured with various recipient E. coli strains were within a factor of 2 compared with the degradation rate constant for e-pUC19. The degradation/deactivation kinetics of i-pUC19 were similar to those of e-pUC19, indicating only a minor influence of cellular components on O3 reactivity toward i-pUC19. For FAC, the degradation and deactivation rates of e-pUC19 were decreased in the presence of tert‑butanol, implying involvement of direct FAC as well as some radical (e.g., •OH) reactions. The degradation rates of e-ampR segments by direct FAC reaction could be explained by a previously-reported two-step sequential reaction model, in which the rate constants increased linearly with e-ampR segment length. The deactivation rate constants of e-pUC19 during exposure to FAC were variable by a factor of up to 4.3 for the different recipient strains, revealing the role of DNA repair in the observed deactivation efficiencies. The degradation/deactivation of e-pUC19 were significantly faster at pH 6.8 than at pH 8.1 owing to pH-dependent FAC speciation variation, whereas i-pUC19 kinetics exhibited much smaller dependence on pH, demonstrating intracellular plasmid DNA reactions with FAC occurred at cytoplasmic pH (~7.5). Our results are useful for predicting and/or measuring the degradation/deactivation efficiency of plasmid-encoded ARGs by water treatment with ozonation and chlorination.

Darwinian individuality of extrachromosomal genetic elements calls for population genetics tinkering

Plasmids are extrachromosomal genetic elements commonly found in prokaryotic (and sometime eukaryotic) cells. Small plasmids are often considered cryptic and their effect on the host is elusive, while large plasmids may encode functions that are essential for the host lifestyle and attain a secondary chromosome status. Plasmids are thus an important source of raw material for microbial genome evolution outside the mainstream of bacterial chromosomes. The discovery of plasmid-mediated antibiotic resistance led to extensive research on the contribution of plasmids to the environmental dimensions of antibiotic resistance and the evolution of plasmid-host interactions following the acquisition of plasmids encoding for antibiotic resistance. Recent experimental studies revealed the importance of intracellular plasmid diversity for plasmid-host interactions. Here we describe the evolutionary forces at play during plasmid evolution in a top-down approach: this includes the effect of processes at the level of the host population and the consideration of plasmids as Darwinian individuals within the host cell.

Intracellular Competitions Reveal Determinants of Plasmid Evolutionary Success.

Plasmids are autonomously replicating genetic elements that are ubiquitous in all taxa and habitats where they constitute an integral part of microbial genomes. The stable inheritance of plasmids depends on their segregation during cell division and their long-term persistence in a host population is thought to largely depend on their impact on the host fitness. Nonetheless, many plasmids found in nature are lacking a clear trait that is advantageous to their host; the determinants of plasmid evolutionary success in the absence of plasmid benefit to the host remain understudied. Here we show that stable plasmid inheritance is an important determinant of plasmid evolutionary success. Borrowing terminology from evolutionary biology of cellular living forms, we hypothesize that Darwinian fitness is key for the plasmid evolutionary success. Performing intracellular plasmid competitions between non-mobile plasmids enables us to compare the evolutionary success of plasmid genotypes within the host, i.e., the plasmid fitness. Intracellular head-to-head competitions between stable and unstable variants of the same model plasmid revealed that the stable plasmid variant has a higher fitness in comparison to the unstable plasmid. Preemptive plasmid competitions reveal that plasmid fitness may depend on the order of plasmid arrival in the host. Competitions between plasmids characterized by similar stability of inheritance reveal plasmid fitness differences depending on the plasmid-encoded trait. Our results further reveal that competing plasmids can be maintained in coexistence following plasmid fusions that maintain unstable plasmid variants over time. Plasmids are not only useful accessory genetic elements to their host but they are also evolving and replicating entities, similarly to cellular living forms. There is a clear link between plasmid genetics and plasmid evolutionary success – hence plasmids are evolving entities whose fitness is quantifiable.

Near-infrared optogenetic engineering of photothermal nanoCRISPR for programmable genome editing

We herein report an optogenetically activatable CRISPR-Cas9 nanosystem for programmable genome editing in the second near-infrared (NIR-II) optical window. The nanosystem, termed nanoCRISPR, is composed of a cationic polymer-coated Au nanorod (APC) and Cas9 plasmid driven by a heat-inducible promoter. The APC not only serves as a carrier for intracellular plasmid delivery but also can harvest external NIR-II photonic energy and convert it into local heat to induce the gene expression of the Cas9 endonuclease. Due to high transfection activity, the APC shows strong ability to induce a significant level of disruption in different genomic loci upon optogenetic activation. Moreover, the precise control of genome-editing activity can be simply programmed by finely tuning exposure time and irradiation time in vitro and in vivo and also enables editing at multiple time points, thus proving the sensitivity and inducibility of such an editing modality. The NIR-II optical feature of nanoCRISPR enables therapeutic genome editing at deep tissue, by which treatment of deep tumor and rescue of fulminant hepatic failure are demonstrated as proof-of-concept therapeutic examples. Importantly, this modality of optogenetic genome editing can significantly minimize the off-target effect of CRISPR-Cas9 in most potential off-target sites. The optogenetically activatable CRISPR-Cas9 nanosystem we have developed offers a useful tool to expand the current applications of CRISPR-Cas9, and also defines a programmable genome-editing strategy toward high precision and spatial specificity.

Plasmid-loadable magnetic/ultrasound-responsive nanodroplets with a SPIO-NP dispersed perfluoropentane core and lipid shell for tumor-targeted intracellular plasmid delivery.

Using ultrasound activating contrast agents to induce sonoporation is a potential strategy for effective lesion-targeted gene delivery. Previous reports have proven that submicron nanodroplets have a better advantage than microbubbles in that they can pass through tumor vasculature endothelial gaps by passive targeting; however, they cannot achieve an adequate dose in tumors to facilitate ultrasound-enhanced gene delivery. Additionally, a few studies focused on delivering macromolecular genetic materials (i.e. overexpression plasmid and CRISPR plasmid) have presented more unique advantages than small-molecular genetic materials (i.e. miRNA mimics, siRNA and shRNA etc.), such as enhancing the expression of target genes with long-term effectiveness. Thereby, we constructed novel plasmid-loadable magnetic/ultrasound-responsive nanodroplets, where superparamagnetic iron oxide nanoparticle dispersed perfluoropentane was encapsulated with lipids to which plasmids could be adhered, and branched polyethylenimine was used to protect the plasmids from enzymolysis. Furthermore, in vitro and in vivo studies were performed to verify the magnetic tumor-targeting ability of the plasmid-loadable magnetic/ultrasound-responsive nanodroplets and focused ultrasound enhanced intracellular plasmid delivery. The plasmid-loadable magnetic/ultrasound-responsive nanodroplets, carrying 16-19 plasmids per droplet, had desirable diameters less than 300 nm, and integrated the merits of excellent magnetic targeting capabilities and phase transition sensitivity to focused ultrasound. Under programmable focused ultrasound exposure, the plasmid-loadable magnetic/ultrasound-responsive nanodroplets underwent a phase-transition into echogenic microbubbles and the subsequent inertial cavitation of the microbubbles achieved an ∼40% in vitro plasmid delivery efficiency. Following intravenous administration, T2-weighted magnet resonance imaging, scanning electron microscopy and inductively coupled plasma optical emission spectrometry of the tumors showed significantly enhanced intratumoral accumulation of the plasmid-loadable magnetic/ultrasound-responsive nanodroplets under an external magnetic field. And a GFP ELISA assay and immunofluorescence staining indicated that focused ultrasound-induced inertial cavitation of the plasmid-loadable magnetic/ultrasound-responsive nanodroplets significantly enhanced the intracellular delivery of plasmids within the tumor after magnet-assisted accumulation, while only lower GFP levels were observed in the tumors on applying focused ultrasound or an external magnet alone. Taken together, utilizing the excellent plasmid-loadable magnetic/ultrasound-responsive nanodroplets combined with magnetism and ultrasound could efficiently deliver plasmids to cancer cells, which could be a potential strategy for macromolecular genetic material delivery in the clinic to treat cancer.

A High-Resolution View of Adaptive Event Dynamics in a Plasmid.

Coadaptation between bacterial hosts and plasmids frequently results in adaptive changes restricted exclusively to host genome leaving plasmids unchanged. To better understand this remarkable stability, we transformed naïve Escherichia coli cells with a plasmid carrying an antibiotic-resistance gene and forced them to adapt in a turbidostat environment. We then drew population samples at regular intervals and subjected them to duplex sequencing—a technique specifically designed for identification of low-frequency mutations. Variants at ten sites implicated in plasmid copy number control emerged almost immediately, tracked consistently across the experiment’s time points, and faded below detectable frequencies toward the end. This variation crash coincided with the emergence of mutations on the host chromosome. Mathematical modeling of trajectories for adaptive changes affecting plasmid copy number showed that such mutations cannot readily fix or even reach appreciable frequencies. We conclude that there is a strong selection against alterations of copy number even if it can provide a degree of growth advantage. This incentive is likely rooted in the complex interplay between mutated and wild-type plasmids constrained within a single cell and underscores the importance of understanding of intracellular plasmid variability.

Ultrasound and microbubble mediated plasmid DNA uptake: A fast, global and multi-mechanisms involved process

Ultrasound application combined with microbubbles has shown great potential for intracellular gene delivery. However, the fundamental mechanistic question of how plasmid DNA enters the intracellular space mediated by ultrasound and microbubble has not been fully explored and understood. The goal of this study is to unveil the detailed intracellular uptake process of plasmid DNA stimulated by ultrasound and microbubbles, uniquely highlighting the role of microbubbles play in this process. The usage of targeted microbubbles pinpointed the subcellular membrane site, where ultrasound exerted acoustic force onto the cell membrane. With the combination of high-speed video microscopy and 3D confocal fluorescence microscopy, we show the spatiotemporal correlation between the microbubble dynamics and intracellular plasmid DNA distribution. Two ultrasound modes (high pressure short pulse and low pressure long pulse) were chosen to trigger different plasmid DNA uptake routes. We found that reversible cell membrane disruption, induced by high pressure short pulse ultrasound, permitted plasmid DNA passage across cell membrane, but not in an exclusive way. Under both ultrasound modes, with or without cell membrane disruption, global plasmid DNA internalization, even nuclear-localization, was observed immediately post ultrasound application. Our results show that plasmid DNA uptake evoked by localized acoustically excited microbubbles is a fast (<2min), global (not limited to the site where microbubbles were attached), and multi-mechanisms involved process.

Intracellular plasmid DNA delivery by self-assembled nanoparticles of amphiphilic PHML-b-PLLA-b-PHML copolymers and the endocytosis pathway analysis.

This work presents a new series of polycationic nanoparticles of (l-)-lysine conjugated amphiphilic triblock copolymer poly(hydroxyletheyl methacrylate-L-lysine)-b-poly(L-lactide)-b-poly(hydroxyletheyl methacrylate-L-lysine)s (PHML-b-PLLA-b-PHML) as potent low cytotoxic vectors for intracellular plasmid DNA delivery. First, the triblock PHML-b-PLLA-b-PHML copolymers were prepared via a combination of metal-free controlled ring opening polymerization and successive atom transfer radical polymerization. Then the cationic PHML-b-PLLA-b-PHML nanoparticles were further prepared by solution self-assembly. The particle size, zeta potential and morphology of as-prepared PHML-b-PLLA-b-PHML nanoparticles were characterized by dynamic light scattering and atomic force microscopy, respectively. The plasmid DNA binding affinities and polyplex stabilities were separately explored by agarose gel retardation and DNase I degradation assays. Then invitro cytotoxicity and gene transfection efficiency of the PHML-b-PLLA-b-PHML nanoparticles vectors as well as relevant polyplex endocytosis pathway were investigated with H1299 cells. It was revealed that the PHML-b-PLLA-b-PHML nanoparticles exhibited low cytotoxicity, strong plasmid DNA binding affinity, high polyplex stability and efficient plasmid DNA transfection even under serum conditions (10% FBS). Moreover, the endocytosis analysis results disclosed that the PHML30-b-PLLA-b-PHML30 nanoparticle/plasmid DNA polyplexes were predominantly involved in lipid-raft-mediated endocytosis pathway, similar to that of SV40 virus-based vectors.

Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells

Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

Modulating APOBEC expression enhances DNA vaccine immunogenicity.

DNA vaccines have failed to induce satisfactory immune responses in humans. Several mechanisms of double-stranded DNA (dsDNA) sensing have been described, and modulate DNA vaccine immunogenicity at many levels. We hypothesized that the immunogenicity of DNA vaccines in humans is suppressed by APOBEC (apolipoprotein B (APOB) mRNA-editing, catalytic polypeptide)-mediated plasmid degradation. We showed that plasmid sensing via STING (stimulator of interferon (IFN) genes) and TBK-1 (TANK-binding kinase 1) leads to IFN-β induction, which results in APOBEC3A mRNA upregulation through a mechanism involving protein kinase C signaling. We also showed that murine APOBEC2 expression in HEK293T cells led to a 10-fold reduction in intracellular plasmid levels and plasmid-encoded mRNA, and a 2.6-fold reduction in GFP-expressing cells. A bicistronic DNA vaccine expressing an immunogen and an APOBEC2-specific shRNA efficiently silenced APOBEC2 both in vitro and in vivo, increasing the frequency of induced IFN-γ-secreting T cells. Our study brings new insights into the intracellular machinery involved in dsDNA sensing and how to modulate it to improve DNA vaccine immunogenicity in humans.

Bone Morphogenetic Protein 2 Gene Delivery via Polyethylene Imine Grafted Chondroitin Sulfate Non-viral Gene Carrier

Background: Bone morphogenetic protein 2 (BMP-2) has been frequently used for bone regeneration. The purpose of this study was to design a gene vector to deliver the BMP-2 plasmid into cells. Polyethylene imine (PEI) is commonly used for gene transfer, however the high cytotoxicity often leads to decreased transfection efficiency. Solutions to this problem have been widely discussed, one of which being the grafting of a highly biocompatibility polymer such as chondroitin sulfate (CS). Methods: Different molecular weights (1,800 and 25,000 g/mol) of PEI were grafted onto CS (PEI1.8K-g-CS and PEI25K-g-CS, respectively) through the ethyldimethylaminopropyl carbodiimide/ N-hydroxysuccinimide reaction. This modification resulted in a non-viral gene carrier with improved biocompatibility as well as enhanced transfection efficiency compared to PEI alone. Cytotoxicity toward human embryonic kidney cell line (293T) was assayed. Results: Cell viability in the PEI-grafted-CS group was higher than that of the PEI group, and this was particularly evident at high concentrations. The efficiency of transfection and the amount of intracellular plasmid DNA were monitored using green fluorescent protein. The expression of BMP-2 was measured by enzyme-linked immunosorbent assay. A polymer:DNA weight ratio of 4:1 in the PEI25K-g-CS/DNA polyplexes exhibited the highest transfection efficiency. Conclusions: The low toxicity and high transfection efficiency of PEI25K-g-CS make it ideal for application in gene delivery and therapy.

Loss of the Inducible Hsp70 Delays the Inflammatory Response to Skeletal Muscle Injury and Severely Impairs Muscle Regeneration

Skeletal muscle regeneration following injury is a highly coordinated process that involves transient muscle inflammation, removal of necrotic cellular debris and subsequent replacement of damaged myofibers through secondary myogenesis. However, the molecular mechanisms which coordinate these events are only beginning to be defined. In the current study we demonstrate that Heat shock protein 70 (Hsp70) is increased following muscle injury, and is necessary for the normal sequence of events following severe injury induced by cardiotoxin, and physiological injury induced by modified muscle use. Indeed, Hsp70 ablated mice showed a significantly delayed inflammatory response to muscle injury induced by cardiotoxin, with nearly undetected levels of both neutrophil and macrophage markers 24 hours post-injury. At later time points, Hsp70 ablated mice showed sustained muscle inflammation and necrosis, calcium deposition and impaired fiber regeneration that persisted several weeks post-injury. Through rescue experiments reintroducing Hsp70 intracellular expression plasmids into muscles of Hsp70 ablated mice either prior to injury or post-injury, we confirm that Hsp70 optimally promotes muscle regeneration when expressed during both the inflammatory phase that predominates in the first four days following severe injury and the regenerative phase that predominates thereafter. Additional rescue experiments reintroducing Hsp70 protein into the extracellular microenvironment of injured muscles at the onset of injury provides further evidence that Hsp70 released from damaged muscle may drive the early inflammatory response to injury. Importantly, following induction of physiological injury through muscle reloading following a period of muscle disuse, reduced inflammation in 3-day reloaded muscles of Hsp70 ablated mice was associated with preservation of myofibers, and increased muscle force production at later time points compared to WT. Collectively our findings indicate that depending on the nature and severity of muscle injury, therapeutics which differentially target both intracellular and extracellular localized Hsp70 may optimally preserve muscle tissue and promote muscle functional recovery.

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (n=96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (n=751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68=56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (n=48) or GFP fluorescence (n=94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.