•Uniform electrotransfection obtained by sequential energizing of two electrode pairs.•Cells exposed to an overall equal intensity of electric field.•Seven different cell types successfully ...transfected.•Higher transfection efficiency and cell viability achieved.
Cell electrotransfection is an effective approach for transferring exogenous molecules into living cells by electric stimulation. The existing in-situ electrotransfection micro-devices for adherent cells exhibit the drawbacks of low transfection efficiency and low cell viability. An important reason for these drawbacks is the unequal exposure of cells to the electric field. It was found that cells growing directly below the energized electrodes experience a much lower electric field intensity when compared to the cells growing below the spacing area of the electrodes, resulting in low transfection with a strip-like pattern. Therefore, a new strategy for the in-situ electrotransfection of adherent cells growing in a standard 12-well plate is proposed in this study. By sequentially energizing electrodes arranged in a nested and non-contact manner, the cells were exposed to an overall equal intensity of the electric field, and thus a higher efficiency of transfection was achieved. The seven cell lines transfected using this method exhibited high transfection efficiency and high cell viability, demonstrating the potential for studying gene function.
Electrotransfection (ET) is a nonviral method for delivery of various types of molecules into cells both in vitro and in vivo. Close to 90 clinical trials that involve the use of ET have been ...performed, and approximately half of them are related to cancer treatment. Particularly, ET is an attractive technique for cancer immunogene therapy because treatment of cells with electric pulses alone can induce immune responses to solid tumors, and the responses can be further enhanced by ET of plasmid DNA (pDNA) encoding therapeutic genes. Compared to other gene delivery methods, ET has several unique advantages. It is relatively inexpensive, flexible, and safe in clinical applications, and introduces only naked pDNA into cells without the use of additional chemicals or viruses. However, the efficiency of ET is still low, partly because biological mechanisms of ET in cells remain elusive. In previous studies, it was believed that pDNA entered the cells through transient pores created by electric pulses. As a result, the technique is commonly referred to as electroporation. However, recent discoveries have suggested that endocytosis plays an important role in cellular uptake and intracellular transport of electrotransfected pDNA. This review will discuss current progresses in the study of biological mechanisms underlying ET and future directions of research in this area. Understanding the mechanisms of pDNA transport in cells is critical for the development of new strategies for improving the efficiency of gene delivery in tumors.
•A localized electroporation device based on microtrap technology was developed.•Cells are positioned in regions of high electric field by flow.•Successful delivery of small molecules and plasmid DNA ...achieved.•Maintains high cell viability and achieves high electroporation efficiency.
We developed a localized single-cell electroporation chip to deliver exogenous biomolecules with high efficiency while maintaining high cell viability. In our microfluidic device, the cells are trapped in a microtrap array by flow, after which target molecules are supplied to the device and electrotransferred to the cells under electric pulses. The system provides the ability to monitor the electrotransfer of exogenous biomolecules in real time. We reveal through numerical simulations that localized electroporation is the mechanism of permeabilization in the microtrap array electroporation device. We demonstrate the simplicity and accuracy of this microtrap technology for electroporation by delivery of both small molecules using propidium iodide and large molecules using plasmid DNA for gene expression, illustrating the potential of this minimally invasive method to be widely used for precise intracellular delivery purposes (from bioprocess engineering to therapeutic applications).
•A method was developed to remove extranuclear DNA in isolated cell nuclei.•The method enabled quantification of pDNA copy number per nucleus post electrotransfection.•The experimental data improved ...a theoretical framework of electrotransfection.•The study predicted spatiotemporal profile of pDNA in individual cells.
Electrotransfection is a non-viral method for delivery of nucleic acids into cells. In our previous study, we have determined the minimal copy number of plasmid DNA (pDNA) per cell required for transgene expression post electrotransfection, and developed a statistical framework to predict the pDNA copy number in the nucleus. To experimentally verify the prediction, the current study was designed to quantify the average copy number of pDNA per nucleus post electrotransfection. To achieve it, we developed a novel approach to effectively obtain isolated nuclei with minimal contamination by extranuclear pDNA. This sample preparation method enabled us to accurately measure intranuclear pDNA using quantitative real-time PCR. The data showed that the copy number of pDNA per nucleus was dependent on the period of cell culture post pulsing and the pDNA dose for electrotransfection. Additionally, the data were used to improve the statistical framework for understanding kinetics of pDNA transport in cells, and predicting how the kinetics depended on different factors. It is expected that the framework and the methodology developed in the current study will be useful for evaluating factors that may affect kinetics and mechanisms of pDNA transport in cells.
•A statistical framework was developed for quantitative analysis of non-viral gene delivery.•The framework was applied to analysis of electrotransfection data.•Average copy number of plasmid per cell ...was quantified experimentally under different conditions.•The study predicted the copy numbers of plasmid in different subcellular compartments.•The copy number in a cell had to be higher than a threshold to achieve transgene expression.
Plasmid DNA (pDNA) has been widely used for non-viral gene delivery. After pDNA molecules enter a mammalian cell, they may be trapped in subcellular structures or degraded by nucleases. Only a fraction of them can function as templates for transcription in the nucleus. Thus, an important question is, what is the minimal amount of pDNA molecules that need to be delivered into a cell for transgene expression? At present, it is technically a challenge to experimentally answer the question. To this end, we developed a statistical framework to establish the relationship between two experimentally quantifiable factors — average copy number of pDNA per cell among a group of cells after transfection and percent of the cells with transgene expression. The framework was applied to the analysis of electrotransfection under different experimental conditions in vitro. We experimentally varied the average copy number per cell and the electrotransfection efficiency through changes in extracellular pDNA dose, electric field strength, and pulse number. The experimental data could be explained or predicted quantitatively by the statistical framework. Based on the data and the framework, we could predict that the minimal number of pDNA molecules in the nucleus for transgene expression was on the order of 10. Although the prediction was dependent on the cell and experimental conditions used in the study, the framework may be generally applied to analysis of non-viral gene delivery.
Objectives
Guangdong Small-ear Spotted (GDSS) pigs are a pig breed native to China that possesses unfortunate disadvantages, such as slow growth rate, low lean-meat percentage, and reduced feed ...utilization. In contrast to traditional genetic breeding methods with long cycle time and high cost, CRISPR/Cas9-mediated gene editing for the modification of the pig genome can quickly improve production traits, and therefore this technique exhibits important potential in the genetic improvement and resource development of GDSS pigs. In the present study, we aimed to establish an efficient CRISPR/Cas9-mediated gene-editing system for GDSS pig cells by optimizing the electrotransfection parameters, and to realize efficient CRISPR/Cas9-mediated gene editing of GDSS pig cells.
Results
After optimization of electrotransfection parameters for the transfection of GDSS pig cells, we demonstrated that a voltage of 150 V and a single pulse with a pulse duration of 20 ms were the optimal electrotransfection parameters for gene editing in these cells. In addition, our study generated GDSS pig single-cell colonies with biallelic mutations in the myostatin (
MSTN
) gene and insulin-like growth factor 2 (
IGF2
) intron-3 locus, which play an important role in pig muscle growth and muscle development. The single-cell colonies showed no foreign gene integration or off-target effects, and maintained normal cell morphology and viability. These gene-edited, single-cell colonies can in the future be used as donor cells to generate
MSTN
- and
IGF2
-edited GDSS pigs using somatic cell nuclear transfer (SCNT).
Conclusions
This study establishes the foundation for genetic improvement and resource development of GDSS pigs using CRISPR/Cas9-mediated gene editing combined with SCNT.
Electrotransfection is based on application of high-voltage pulses that transiently increase membrane permeability, which enables delivery of DNA and RNA in vitro and in vivo. Its advantage in ...applications such as gene therapy and vaccination is that it does not use viral vectors. Skeletal muscles are among the most commonly used target tissues. While siRNA delivery into undifferentiated myoblasts is very efficient, electrotransfection of siRNA into differentiated myotubes presents a challenge. Our aim was to develop efficient protocol for electroporation-based siRNA delivery in cultured primary human myotubes and to identify crucial mechanisms and parameters that would enable faster optimization of electrotransfection in various cell lines.
We established optimal electroporation parameters for efficient siRNA delivery in cultured myotubes and achieved efficient knock-down of HIF-1α while preserving cells viability. The results show that electropermeabilization is a crucial step for siRNA electrotransfection in myotubes. Decrease in viability was observed for higher electric energy of the pulses, conversely lower pulse energy enabled higher electrotransfection silencing yield. Experimental data together with the theoretical analysis demonstrate that siRNA electrotransfer is a complex process where electropermeabilization, electrophoresis, siRNA translocation, and viability are all functions of pulsing parameters. However, despite this complexity, we demonstrated that pulse parameters for efficient delivery of small molecule such as PI, can be used as a starting point for optimization of electroporation parameters for siRNA delivery into cells in vitro if viability is preserved.
The optimized experimental protocol provides the basis for application of electrotransfer for silencing of various target genes in cultured human myotubes and more broadly for electrotransfection of various primary cell and cell lines. Together with the theoretical analysis our data offer new insights into mechanisms that underlie electroporation-based delivery of short RNA molecules, which can aid to faster optimisation of the pulse parameters in vitro and in vivo.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Dysregulation of iron metabolism is observed in animal models of retinitis pigmentosa (RP) and in patients with age-related macular degeneration (AMD), possibly contributing to oxidative damage of ...the retina. Transferrin (TF), an endogenous iron chelator, was proposed as a therapeutic candidate. Here, the efficacy of TF non-viral gene therapy based on the electrotransfection of pEYS611, a plasmid encoding human TF, into the ciliary muscle was evaluated in several rat models of retinal degeneration. pEYS611 administration allowed for the sustained intraocular production of TF for at least 3 and 6 months in rats and rabbits, respectively. In the photo-oxidative damage model, pEYS611 protected both retinal structure and function more efficiently than carnosic acid, a natural antioxidant, reduced microglial infiltration in the outer retina and preserved the integrity of the outer retinal barrier. pEYS611 also protected photoreceptors from N-methyl-N-nitrosourea-induced apoptosis. Finally, pEYS611 delayed structural and functional degeneration in the RCS rat model of RP while malondialdehyde (MDA) ocular content, a biomarker of oxidative stress, was decreased. The neuroprotective benefits of TF non-viral gene delivery in retinal degenerative disease models further validates iron overload as a therapeutic target and supports the continued development of pEY611 for treatment of RP and dry AMD.
Electrotransfection (ET) is a non-viral approach widely used for delivery of naked nucleic acids. Its efficiency can be increased
by treatment of cells with various small molecule enhancers. However, ...these enhancers often fail to improve ET
, presumably due to rapid clearance in tissues after local injection, reducing their cellular uptake. To this end, we propose to develop a new type of ET enhancers, which we term nanoenhancer, that diffuse slowly in tissues and are poorly absorbed by blood and lymph microvessels.
Two nanoenhancers were synthesized with alginate (Alg) and chitosan (Chi) with or without poly (ethylene imine) (PEI). They were used to treat cells
or mouse muscle in the hind leg
prior to ET of plasmid DNA coding reporter genes. At 24 hours post ET, the efficiency of ET was quantified, and compared with that in the untreated controls. Changes in lysosomal size and acidity post nanoenhancer treatment were measured with fluorescence microscopy techniques.
We observed that the pretreatment of cells with the nanoenhancers could enhance the ET efficiency and cell viability in both C2C12 and HCT116 cells
, and the nanoenhancer pretreatment had similar effects on the ET efficiency
. Mechanisms of the enhancement were related to transient inactivation of lysosomal functions triggered by the nanoenhancer treatment. The concept of nanoenhancer will lead to development of new enhancers that can be used to improve ET efficiency
, highlighting its potential in clinical applications.
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