The successful establishment of an Agrobacterium-mediated transformation method and optimisation of six critical parameters known to influence the efficacy of Agrobacterium T-DNA transfer in the unicellular microalga Chlorella vulgaris (UMT-M1) are reported. Agrobacterium tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA1304 containing the gfp:gusA fusion reporter and a hygromycin phosphotransferase (hpt) selectable marker driven by the CaMV35S promoter were used for transformation. Transformation frequency was assessed by monitoring transient β-glucuronidase (GUS) expression 2 days post-infection. It was found that co-cultivation temperature at 24°C, co-cultivation medium at pH 5.5, 3 days of co-cultivation, 150 μM acetosyringone, Agrobacterium density of 1.0 units (OD(600)) and 2 days of pre-culture were optimum variables which produced the highest number of GUS-positive cells (8.8-20.1%) when each of these parameters was optimised individually. Transformation conducted with the combination of all optimal parameters above produced 25.0% of GUS-positive cells, which was almost a threefold increase from 8.9% obtained from un-optimised parameters. Evidence of transformation was further confirmed in 30% of 30 randomly-selected hygromycin B (20 mg L(-1)) resistant colonies by polymerase chain reaction (PCR) using gfp:gusA and hpt-specific primers. The developed transformation method is expected to facilitate the genetic improvement of this commercially-important microalga.
Gene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 μg/mL and 7 μg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.
Commonly used visible markers for transgenesis use fluorescent proteins expressed at the surface of the body, such as in eyes. One commonly used marker is the 3xP3-EGFP cassette containing synthetic binding sites for the eyeless/Pax6 conserved transcription factor. This marker cassette leads to fluorescent eyes in a variety of animals tested so far. Here we show that upon reaching adulthood, transgenic Bicyclus anynana butterflies containing this marker cassette exponentially loose fluorescence in their eyes. After 12 days, transgenic individuals are no longer distinguishable from wild type individuals. The decreased eye fluorescence is likely due to significantly decreased or halted eyeless/Pax6 expression observed in wild type animals upon adult emergence. Implications from these findings include care in screening transgenic animals before these reach adulthood, or shortly thereafter, and in using adult animals of the same age for quantitative screening of likely homozygote and heterozygote individuals.
Rice is one of the most important cereal crops with great potential for biotechnology progress. In transformation method, antibiotic resistance genes are routinely used as powerful markers for selecting transformed cells from surrounding non-transformed cells. In this study, the toxicity level of hygromycin was optimized for two selected mutant rice lines, MR219 line 4 and line 9. The mature embryos were isolated and cultured on an MS medium with different hygromycin concentrations (0, 20, 40, 60, 80 and 100 mg L(-1)). Evidently, above 60 mg L(-1) was effective for callus formation and observed completely dead. Further there were tested for specific concentration (0-60). Although, 21.28% calli survived on the medium containing 45 mg L(-1) hygromycin, it seemed suitable for the identification of putative transformants. These findings indicated that a system for rice transformation in a relatively high frequency and the transgenes are stably expressed in the transgenic plants. Green shoots were regenerated from the explant under hygromycin stress. RT-PCR using hptII and gus sequence specific primer and Southern blot analysis were used to confirm the presence of the transgene and to determine the transformation efficiency for their stable integration in regenerated plants. This study demonstrated that the hygromycin resistance can be used as an effective marker for rice transformation.
Morinda citrifolia, is a valuable medicinal plant with a wide range of therapeutic properties and extensive transformation study on this plant has yet been known. Present study was conducted to establish a simple and reliable transformation protocol for M. citrifolia utilising Agrobacterium tumefaciens via direct seed exposure. In this study, the seeds were processed by tips clipping and dried and subsequently incubated in inoculation medium. Four different parameters during the incubation such as incubation period, bacterial density, temperature and binary vectors harbouring beta-glucuronidase (GUS) gene (pBI121 and pGSA1131), were tested to examine its effect on transformation efficiency. The leaves from the treated and germinated seedlings were analysed via Polymerase Chain Reaction (PCR), histochemical assay of the GUS gene and reverse transcription-PCR (RT-PCR). Results of the study showed that Agrobacterium strain LBA4404 with optical density of 1.0 and 2 h incubation period were optimum for M. citrifolia transformation. It was found that various co-cultivation temperatures tested and type of vector used did not affect the transformation efficiency. The highest transformation efficiency for M. citrifolia direct seed transformation harbouring pBI121 and pGSA1131 was determined to be 96.8% with 2 h co-cultivation treatment and 80.4% when using bacterial density of 1.0, respectively. The transformation method can be applied for future characterization study of M. citrifolia.
The delivery of a full plasmid, encoding the green fluorescent protein gene into African monkey kidney (Vero3) cells, was successfully achieved using nanobiocomposites based on layered double hydroxides. This demonstrated the potential of using the system as an alternative DNA delivery vector. Intercalation of the circular plasmid DNA, pEGFP-N2, into Mg/Al-NO(3)(-) layered double hydroxides (LDH) was accomplished through anion exchange routes to form the nanobiocomposite material. The host was previously synthesized at the Mg(2+) to Al(3+) molar ratio R(i) = 2 and subsequently intercalated with plasmid DNA. Size expansion of the interlamellae host from 8.8 A in LDH to 42 A was observed in the resulting nanobiocomposite, indicating stable hybridization of the plasmid DNA. The powder x-ray diffraction (PXRD) results, supplemented with Fourier-transform infrared (FTIR) spectroscopy, compositional and electrophoresis studies confirmed the encapsulation episode of the biomaterial. In order to elucidate the use of this resulting nanobiocomposite as a delivery vector, an MTT assay was performed to determine any cytotoxic effects of the host towards cells. The intercalated pEGFP-N2 anion was later successfully recovered through acidification with HNO(3) after treatment with DNA-degrading enzymes, thus also showing the ability of the LDH host to protect the intercalated biomaterial from degradation. Cell transfection studies on Vero3 cells were then performed, where cells transfected with the nanobiocomposite exhibited fluorescence as early as 12 h post-treatment compared to naked delivery of the plasmid itself.
In the past decade, interest in the production of recombinant pharmaceutical proteins in plants has tremendously progressed because plants do not harbor mammalian viruses, are economically competitive, easily scalable, and capable of carrying out complex post-translational modifications required for recombinant pharmaceutical proteins. Mucuna bracteata is an essential perennial cover crop species widely planted as an underground cover in oil palm and rubber plantations. As a legume, they have high biomass, thrive in its habitat, and can fix nitrogen. Thus, M. bracteata is a cost-efficient crop that shows ideal characteristics as a platform for mass production of recombinant protein. In this study, we established a new platform for the transient production of a recombinant protein in M. bracteata via vacuum-assisted agro-infiltration. Five-week-old M. bracteata plants were vacuum infiltrated with Agrobacterium tumefaciens harboring a plasmid that encodes for an anti-toxoplasma immunoglobulin (IgG) under different parameters, including trifoliate leaf positional effects, days to harvest post-infiltration, and the Agrobacterium strain used. Our results showed that vacuum infiltration of M. bracteata plant with A. tumefaciens strain GV3101 produced the highest concentration of heterologous protein in its bottom trifoliate leaf at 2 days post-infiltration. The purified anti-toxoplasma IgG was then analyzed using Western blot and ELISA. It was demonstrated that, while structural heterogeneity existed in the purified anti-toxoplasma IgG from M. bracteata, its transient expression level was two-fold higher than the model platform, Nicotiana benthamiana. This study has laid the foundation towards establishing M. bracteata as a potential platform for the production of recombinant pharmaceutical protein.
Matched MeSH terms: Gene Transfer Techniques/instrumentation
Transgenic oil palm (Elaeis guineensis Jacq.) plantlets are regenerated after Agrobacterium tumefaciens-mediated transformation of embryogenic calli derived from young leaves of oil palm. The calli are transformed with an Agrobacterium strain, LBA4404, harboring the plasmid pUBA, which carries a selectable marker gene (bar) for resistance to the herbicide Basta and is driven by a maize ubiquitin promoter. Modifications of the transformation method, treatment of the target tissues using acetosyringone, exposure to a plasmolysis medium, and physical injury via biolistics are applied. The main reasons for such modifications are to activate the bacterial virulence system and, subsequently, to increase the transformation efficiency. Transgenic oil palm cells are selected and regenerated on a medium containing herbicide Basta. Molecular analyses revealed the presence and integration of the introduced bar gene into the genome of the transformants.
The effectiveness of mannose (using phosphomannose isomerase [pmi] gene) as a positive selection agent to preferably allow the growth of transformed oil palm embryogenic calli was successfully evaluated. Using the above selection agent in combination with the previously optimized physical and biological parameters and the best constitutive promoter, oil palm embryogenic calli were transformed with pmi gene for producing transgenic plants. Bombarded embryogenic calli were exposed to embryogenic calli medium containing 30:0 g/L mannose to sucrose 3 weeks postbombardment. Selectively, proliferating embryogenic calli started to emerge around 6 months on the above selection medium. The proliferated embryogenic calli were individually isolated once they reached a specific size and regenerated to produce complete plantlets. The complete regenerated plantlets were evaluated for the presence of transgenes by PCR and Southern analyses.
Gene delivery using invasive bacteria as vectors is a robust method that is feasible for plasmid and artificial chromosome DNA construct delivery to human cells presenting β1 integrin receptors. This technique is relatively underutilized owing to the inefficiency of gene transfer to targeted cell populations. Bacterial vectors must successfully adhere to the cell membrane, internalize into the cytoplasm, undergo lysis, and deliver DNA to the nucleus. There are limited studies on the use of exogenous reagents to improve the efficiency of bacteria-mediated gene delivery to mammalian cells. In this chapter, we describe how cationic lipids, conventionally used for DNA and protein transfection, as well as antimicrobial compounds, can be used to synergistically enhance the adherence of invasive bacterial vectors to the cell membrane and improve their predisposition to internalize into the cytoplasm to deliver DNA. Using simple combinatorial methods, functional DNA transfer can be improved by up to four-fold of invaded cell populations. These methods are easy to perform and are likely to be applicable for other bacterial vectors including Listeria and Salmonella.
Visual analysis of the gene delivery process when using invasive bacteria as a vector has been conventionally performed using standard light and fluorescence microscopy. These microscopes can provide basic information on the invasiveness of the bacterial vector including the ability of the vector to successfully adhere to the cell membrane. Standard microscopy techniques however fall short when finer details including membrane attachment as well as internalization into the cytoplasm are desired. High-resolution visual analysis of bacteria-mediated gene delivery can allow accurate measurement of the adherence and internalization capabilities of engineered vectors. Here, we describe the use of scanning electron microscopy (SEM) to directly quantify vectors when they are external to the cell wall, and confocal microscopy to evaluate the vectors when they have internalized into the cytoplasm. By performing the invasion procedure on microscope coverslips, cells can be easily prepared for analysis using electron or confocal microscopes. Imaging the invasion complexes in high resolution can provide important insights into the behavior of bacterial vectors including E. coli, Listeria, and Salmonella when invading their target cells to deliver DNA and other molecules.
The application of nanoparticles (NPs) has attracted considerable attention as targeted delivery systems. CaCO3 has become the focus due to its advantages including affordability, low toxicity, biocompatibility, cytocompatibility, pH sensitivity and sedate biodegradability and environment friendly materials. In this article, we will discuss the po- tential roles of CaCO3-NPs in three major therapeutic applications; as antimicrobial, for drug delivery, and as gene delivery nanocarrier.
Cancer treatment has been always considered one of the most critical and vital themes of clinical issues. Many approaches have been developed, depending on the type and the stage of tumor. Gene therapy has the potential to revolutionize different cancer therapy. With the advent of recent bioinformatics technologies and genetic science, it become possible to identify, diagnose and determine the potential treatment using the technology of gene delivery. Several approaches have been developed and experimented in vitro and vivo for cancer therapy including: naked nucleic acids based therapy, targeting micro RNAs, oncolytic virotherapy, suicide gene based therapy, targeting telomerase, cell mediated gene therapy, and CRISPR/Cas9 based therapy. In this review, we present a straightforward introduction to cancer biology and occurrence, highlighting different viral and non-viral gene delivery systems for gene therapy and critically discussed the current and various strategies for cancer gene therapy.
PURPOSE: In order to enhance cellular uptake and to facilitate transdermal delivery of DNA vaccine, polyamidoamine (PAMAM) dendrimers conjugated with HIV transactivator of transcription (TAT) was developed.
METHODS: First, the plasmid DNA (pIRES-H5/GFP) nanoparticle was formulated using PAMAM dendrimer and TAT peptide and then characterized for surface charge, particle size, DNA encapsulation and protection of the pIRES-H5/GFP DNA plasmid to enzymatic digestion. Subsequently, the potency of the TAT-conjugated dendrimer for gene delivery was evaluated through in vitro transfection into Vero cells followed by gene expression analysis including western blotting, fluorescent microscopy and PCR. The effect of the TAT peptide on cellular uptake of DNA vaccine was studied by qRT-PCR and flow cytometry. Finally, the ability of TAT-conjugated PAMAM dendrimer for transdermal delivery of the DNA plasmid was assessed through artificial membranes followed by qRT-PCR and flow cytometry.
RESULTS: TAT-conjugated PAMAM dendrimer showed the ability to form a compact and nanometre-sized polyplexes with the plasmid DNA, having the size range of 105 to 115 nm and a positive charge of +42 to +45 mV over the N/P ratio of 6:1(+/-). In vitro transfection analysis into Vero cells confirms the high potency of TAT-conjugated PAMAM dendrimer to enhance the cellular uptake of DNA vaccine. The permeability value assay through artificial membranes reveals that TAT-conjugated PAMAM has more capacity for transdermal delivery of the DNA compared to unmodified PAMAM dendrimer (P<0.05).
CONCLUSIONS: The findings of this study suggest that TAT-conjugated PAMAM dendrimer is a promising non-viral vector for transdermal use.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
Cellulose acetate phthalate (CAP) microcapsules were formulated to deliver plasmid DNA (pDNA) to the intestines. The microcapsules were characterized and were found to have an average diameter of 44.33 ± 30.22 μm, and were observed to be spherical with smooth surface. The method to extract pDNA from CAP was modified to study the release profile of the pDNA. The encapsulated pDNA was found to be stable. Exposure to the acidic and basic pH conditions, which simulates the pH environment in the stomach and the intestines, showed that the release occurred in a stable manner in the former, whereas it was robust in the latter. The loading capacity and encapsulation efficiency of the microcapsules were low but the CAP recovery yield was high which indicates that the microcapsules were efficiently formed but the loading of pDNA can be improved. In vitro transfection study in 293FT cells showed that there was a significant percentage of green-fluorescent-protein-positive cells as a result of efficient transfection from CAP-encapsulated pDNA. Biodistribution studies in BALB/c mice indicate that DNA was released at the stomach and intestinal regions. CAP microcapsules loaded with pDNA, as described in this study, may be useful for potential gene delivery to the intestines for prophylactic or therapeutic measures for gastrointestinal diseases.
E. coli mediated gene delivery faces a major drawback of low efficiency despite of being a safer alternative to viral vectors. This study showed a novel, simple and effective strategy to enhance invasive E. coli DH10B vector's efficiency in human epithelial cells. The bactofection efficiency of invasive E .coli vector was analyzed in nine cell lines. It demonstrated highest (16%) reporter gene (GFP) expression in cervical cells. Methods were employed to further enhance its efficiency by adding transfection reagents (trans-bactofection method) to promote entry into host cells, lysosomotropic reagents for escape from lysosomal degradation or antibiotics to lyse internalized bacteria. Increased bacterial entry, as elucidated from nil to 3% expression in liver cells, was obtained upon complexing bacteria with PULSin. Chloroquine mediated endosomal escape resulted in 7.2 folds increase whereas tetracycline addition to lyse internalized bacteria caused ≈90% of GFP in HeLa. Eventually, the combined effect of these three methods exhibited close to 100% GFP in cervical and remarkable increase of 138 folds in breast cells. This is the first study showing comparative study of vector's gene delivery ability in various epithelial cells of the human body with improving its delivery efficiency. These data demonstrated the potential of developed bactofection method to boost up the efficiency of other bacterial vectors also, which could further be used for effectual therapeutic gene delivery in human cells.
Alginate, a natural polysaccharide, was explored in this study as an oral delivery vehicle of a mammalian expression vector into the murine intestinal mucosa. Alginate microspheres were produced through water-in-oil (W/O) emulsification method. Average diameter sizes of microspheres were 46.88 μm±3.07 μm with significant size reduction upon utilization of 1.0% Span80. Plasmid DNA (pDNA) carrying green fluorescent protein reporter gene (GFP), pVAX-GFP, was encapsulated within microspheres at efficiencies of 72.9 to 74.4%, carrying maximum load of 6 μg pDNA. Alginate microspheres demonstrated shrinkage in pH 1.2 and swelling in pH 9.0 with pDNA release about twice the amount released in acidic environment. Oral delivery of pVAX-GFP loaded-microspheres, at 50 μg, 100 μg and 150 μg dose, was performed on BALB/c mice. Tissue biodistribution, investigated through flow cytometric analysis, demonstrated GFP positive intestinal cells (<1.0%) with 1.3-fold higher levels for the 100 μg dose; therefore suggesting feasibility of the approach for oral gene delivery and vaccination.
A novel cationic polymer, dextran-spermine (D-SPM), has been found to mediate gene expression in a wide variety of cell lines and in vivo through systemic delivery. Here, we extended the observations by determining the optimal conditions for gene expression of D-SPM/plasmid DNA (D-SPM/pDNA) in cell lines and in the lungs of BALB/c mice via instillation delivery. In vitro studies showed that D-SPM could partially protect pDNA from degradation by nuclease and exhibited optimal gene transfer efficiency at D-SPM to pDNA weight-mixing ratio of 12. In the lungs of mice, the levels of gene expression generated by D-SPM/pDNA are highly dependent on the weight-mixing ratio of D-SPM to pDNA, amount of pDNA in the complex, and the assay time postdelivery. Readministration of the complex at day 1 following the first dosing showed no significant effect on the retention and duration of gene expression. The study also showed that there was a clear trend of increasing size of the complexes as the amount of pDNA was increased, where the sizes of the D-SPM/pDNA complexes were within the nanometer range.
Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells has emerged as an invaluable method for generating patient-specific stem cells of any lineage without the use of embryonic materials. Following the first reported generation of iPS cells from murine fibroblasts using retroviral transduction of a defined set of transcription factors, various new strategies have been developed to improve and refine the reprogramming technology. Recent developments provide optimism that the generation of safe iPS cells without any genomic modification could be derived in the near future for the use in clinical settings. This review summarizes current and evolving strategies in the generation of iPS cells, including types of somatic cells for reprogramming, variations of reprogramming genes, reprogramming methods, and how the advancement iPS cells technology can lead to the future success of reproductive medicine.
Aptamers, nucleic acid ligands that are specific against their corresponding targets are increasingly employed in a variety of applications including diagnostics and therapeutics. The specificity of the aptamers against their targets is also used as the basis for the formulation of the aptamer-based drug delivery system. In this review, we aim to provide an overview on the chaperoning roles of aptamers in acting as the cargo or load carriers, delivering contents to the targeted sites via cell surface receptors. Internalization of the aptamer-biomolecule conjugates via receptor-mediated endocytosis and the strategies to augment the rate of endocytosis are underscored. The cargos chaperoned by aptamers, ranging from siRNAs to DNA origami are illuminated. Possible impediments to the aptamer-based drug deliveries such as susceptibility to nuclease resistance, potentiality for immunogenicity activation, tumor heterogeneity are speculated and the corresponding amendment strategies to address these shortcomings are discussed. We prophesy that the future of the aptamer-based drug delivery will take a trajectory towards DNA nanorobot-based assay.