Single-cell electroporation for gene transfer in vivo

We report an electroporation technique for targeting gene transfer to individual cells in intact tissue. Electrical stimulation through a micropipette filled with DNA or other macromolecules electroporates a single cell at the tip of the micropipette. Electroporation of a plasmid encoding enhanced green fluorescent protein (GFP) into the brain of intact Xenopus tadpoles or rat hippocampal slices resulted in GFP expression in single neurons and glia. In vivo imaging showed morphologies, dendritic arbor dynamics, and growth rates characteristic of healthy cells. Coelectroporation of two plasmids resulted in expression of both proteins, while electroporation of fluorescent dextrans allowed direct visualization of transfer of molecules into cells. This technique will allow unprecedented spatial and temporal control over gene delivery and protein expression.     

Use of electroporation for high-molecular-weight DNA-mediated gene transfer

Electroporation was used to introduce high-molecular-weight DNA into murine hematopoietic cells and NIH3T3 cells. CCRF-CEM cells were stably transfected with SV2NEO plasmid and the genomic DNA from G-418-resistant clones (greater than 65 kb) was introduced into mouse bone marrow and NIH3T3 cells by electroporation. NEO sequences and expression were detected in the hematopoietic tissues of lethally irradiated mice, with 24% of individual spleen colonies expressing NEO. The frequency of genomic DNA transfer into NIH3T3 cells was 0.25 X 10(-3). Electroporation thus offers a powerful mode of gene transfer not only of cloned genes but also of high-molecular-weight DNA into cells.     

A comparison of calcium phosphate coprecipitation and electroporation

Plasmid-based transfection assays provide a rapid means to measure homologous and nonhomologous recombination in mammalian cells. Often it is of interest to examine the stimulation of recombination by DNA damage induced by radiation, genotoxic chemicals, or nucleases. Transfection is frequently performed by using calcium phosphate coprecipitation (CPP), because this method is well suited for handling large sample sets, and it does not require expensive reagents or equipment. Alternative transfection methods include lipofection, microinjection, and electroporation. Since DNA strand breaks are known to stimulate both homologous and nonhomologous recombination, the induction of nonspecific damage during transfection would increase background recombination levels and thereby reduce the sensitivity of assays designed to detect the stimulation of recombination by experimentally induced DNA damage. In this article, we compare the stimulatory effects of nuclease-induced double-strand breaks (DSBs) on homologous and nonhomologous recombination for molecules transfected by CPP and by electroporation. Although electroporation yielded fewer transfectants, both nonhomologous and homologous recombination were stimulated by nuclease-induced DSBs to a greater degree than with CPP. Ionizing radiation is an effective agent for inducing DNA strand breaks, but previous studies using CPP generally showed little or no stimulation of homologous recombination among plasmids damaged with ionizing radiation. By contrast, we found clear dose-dependent enhancement of recombination with irradiated plasmids transfected using electroporation. Thus, electroporation provides a higher signal-to-noise ratio for transfection-based studies of damage-induced recombination, possibly reflecting less nonspecific damage to plasmid DNA during transfection of mammalian cells.     

Instantaneous gene transfer from donor to recipient microorganisms via electroporation

Simplified electroporation methodologies have been developed that reliably yield transformants with only minutes of effort. Neither DNA purification, cells in specific phase of growth, cell washing nor chilled cuvettes are required to obtain transformants. Electroporation can be used to transfer plasmid or chromosomal DNA directly from donor to recipient cells. This simplified direct method of electroporation has been demonstrated to work for both intra- as well as interspecies transformations using a variety of microorganisms. The use of electroporation to purify plasmid DNA was also investigated and found to be inferior to conventional plasmid isolation procedures.     

Lentiviral vector-mediated gene transfer to endotherial cells compared with adenoviral and retroviral vectors

Human immunodeficiency virus (HIV, lentivirus) vector has attractive features for gene therapy, including the ability to transduce non-dividing cells and long-term transgene expression. We have already reported that lentivirus vector can transduce well-differentiated rat cardiac myocytes. Endothelial cells (EC) are an attractive target for gene therapy, both for the treatment of cardiovascular disease and for the systemic delivery of recombinant gene products directly into the circulation. There are several reports regarding application of adenovirus and retrovirus based vectors to EC. However, there have been few reports which show the effect to lentivirus-mediated gene transfer efficiency, compared with adenovirus and retrovirus. In this study, bovine aortic endothelial cells (BAECs) were infected, in vitro, with these virus vectors. Transduction efficiency (TE) of beta-Gal gene transfer in BAECs by adenovirus, lentivirus, or retrovirus at MOI10 (Multiplicity of infection) (determined on Hela cells) is 69+/-11, 33+/-8, or 22+/-6% respectively. In adenovirus and lentivirus, almost 100% of BAECs were transduced at MOI 50. However, in retrovirus, TE showed only 48+/-6% at MOI 50 and no increase at MOI 100. The percentage of beta-Gal positive cells was decreased rapidly at longer passage of cells after being transduced by adenovirus. However, lentivirus and retrovirus showed sustained higher percentage of positive cells. Furthermore, transduction by lentiviral vectors had no significant effect on viability of BAECs. Our results indicate that lentivirus showed high-level and long term gene expression in BAECs. Lentivirus can be an effective vector for the ex vivo, genetically modified EC implantation and in vivo gene therapy.     

Genomic integration of adenoviral gene transfer vectors following transduction of fertilized mouse oocytes

Adenoviral vectors (AdV) are popular tools to deliver foreign genes into a wide range of cells. They have also been used in clinical gene therapy trials. Studies on AdV-mediated gene transfer to mammalian oocytes and transmission through the germ line have been reported controversially. In the present study we investigated whether AdV sequences integrate into the mouse genome by microinjecting AdV into the perivitelline space of fertilized oocytes. We applied a newly developed PCR technique (HiLo-PCR) for identification of chromosomal junctions next to the integrated AdV. We demonstrate that mouse oocytes can be transduced by different recombinant adenoviral vectors (first generation and gutless). In one transgenic mouse line using the first generation adenoviral vector, the genome has integrated into a highly repetitive cluster located on the Y chromosome. While the transgene (GFP) was expressed in early embryos, no expression was detected in adult transgenic mice. The use of gutless AdV resulted in expression of the transgene, albeit the vector was not transmitted to progeny. These results indicate that under optimized conditions fertilized mouse oocytes are transduced by AdV and give rise to transgenic founder animals. Therefore, adequate precautions should be taken in gene therapy protocols of reproductive patients since transduction of oocytes or early embryos and subsequent chromosomal integration cannot be ruled out entirely.     

Optimization of ectopic gene expression in skeletal muscle through DNA transfer by electroporation

Background  

Electroporation (EP) is a widely used non-viral gene transfer method. We have attempted to develop an exact protocol to maximize DNA expression while minimizing tissue damage following EP of skeletal muscle in vivo. Specifically, we investigated the effects of varying injection techniques, electrode surface geometry, and plasmid mediums.     

Comparison of lipid-mediated and adenoviral gene transfer in human monocyte-derived macrophages and COS-7 cells

Lipid-mediated transfection was compared to adenoviral-mediated gene transfer in COS-7 cells as well as human monocyte-derived macrophages (HMDM). For this purpose, we monitored enhanced green fluorescent protein (EGFP) expression by fluorescence microscopy and quantified gene transfer by competitive PCR. Transfection of COS-7 cells with a novel lipid formulation for DNA transfer was highly effective in COS-7 cells. On average, 30% of the cells were fluorescent 48 h after transfection. In HMDM, the same formulation resulted in the expression of EGFP in less than 0.5% of cells. We measured plasmid DNA by quantitative PCR in lipid-transfected macrophages and found that each macrophage contained on average 2 fg of plasmid DNA 24 h after transfection, that is, more than 400 molecules of plasmid DNA entered each cell. Despite the high level of reporter DNA in lipid transfected cells, expression of the fluorescent protein was suppressed in more than 99.5% of the macrophages. We also used adenoviral gene transfer to introduce the foreign DNA into both COS-7 cells and HMDM. Even though the multiplicity of infection was less than 30, expression of EGFP was observed in nearly all COS-7 cells and in more than 80% of HMDM 48 h after transfection. Despite major advances in the field of lipid-mediated transfection of HMDM, the lipid formulations that are available commercially cannot compete with the efficiency of adenoviral gene transfer.     

Physiological and histological changes in skeletal muscle following in vivo gene transfer by electroporation

Electroporation (EP) is used to transfect skeletal muscle fibers in vivo, but its effects on the structure and function of skeletal muscle tissue have not yet been documented in detail. We studied the changes in contractile function and histology after EP and the influence of the individual steps involved to determine the mechanism of recovery, the extent of myofiber damage, and the efficiency of expression of a green fluorescent protein (GFP) transgene in the tibialis anterior (TA) muscle of adult male C57Bl/6J mice. Immediately after EP, contractile torque decreased by ～80% from pre-EP levels. Within 3 h, torque recovered to ～50% but stayed low until day 3. Functional recovery progressed slowly and was complete at day 28. In muscles that were depleted of satellite cells by X-irradiation, torque remained low after day 3, suggesting that myogenesis is necessary for complete recovery. In unirradiated muscle, myogenic activity after EP was confirmed by an increase in fibers with central nuclei or developmental myosin. Damage after EP was confirmed by the presence of necrotic myofibers infiltrated by CD68+ macrophages, which persisted in electroporated muscle for 42 days. Expression of GFP was detected at day 3 after EP and peaked on day 7, with ～25% of fibers transfected. The number of fibers expressing green fluorescent protein (GFP), the distribution of GFP+ fibers, and the intensity of fluorescence in GFP+ fibers were highly variable. After intramuscular injection alone, or application of the electroporating current without injection, torque decreased by ～20% and ～70%, respectively, but secondary damage at D3 and later was minimal. We conclude that EP of murine TA muscles produces variable and modest levels of transgene expression, causes myofiber damage due to the interaction of intramuscular injection with the permeabilizing current, and that full recovery requires myogenesis.     

Retrovirus vector-mediated gene transfer into the chick optic vesicle by in ovo electroporation

Owing to its external position in the embryo, the chick eye has been used as a readily accessible model for studying the molecular mechanisms behind the patterning of the central nervous system. Although methods of genetic analysis have not been established as in the mouse, the chick is convenient for analyzing the functions of genes by in ov o electroporation of retroviral vectors. In this review, we describe the retroviral vector-mediated transfer of genes into the chick optic vesicle by in ovo electroporation. A rapid, efficient, and sustained expression of transgenes is achieved by this approach.     

脂质体法和电穿孔法在转染Cos-7,Vero和Namalwa细胞中的比较

用脂质体法和电穿孔法分别转染Cos-7,Vero和Namalwa细胞。发现脂质体法在转染效率和操作方便方面比电穿孔法优越,而电穿孔法对细胞种类的适应性方面比脂质体法广。电穿孔法能转染Cos-7、Namalwa和Vero细胞,而用脂质体法只能转染Cos-7和Vero细胞。     

Testis mediated gene transfer: In vitro transfection in goat testis by electroporation

Testis mediated gene transfer (TMGT) is a potential tool for making transgenic mice having more than 90% success rate. However, this method needs further standardization before it can be adapted in other species including livestock. In order to standardize the TMGT in goat, buck testes (n = 20) collected from the slaughter house were injected with a vector driving green fluorescent protein (GFP) expression under a cytomegalovirus (CMV) promoter. Then, the testes were subjected to electroporation with predetermined voltage, pulse length, pulse interval and number of pulses. Seminiferous tubules were isolated from the electroporated testis and cultured in-vitro. The expression was checked at regular intervals. Green fluorescence was observed on different days in different samples. It suggests transient integration of the plasmid into the seminiferous tubules. This in-vitro transfection of seminiferous tubule using electroporation will provide valuable baseline information.     

The effect of cell synchronization on the efficiency of stable gene transfer by electroporation

We synchronized thymidine kinase deficient mouse Ltk-cells by two different methods, hydroxyurea double-block treatment or aphidicolin single-block treatment and transformed them with the cloned herpes simplex virus thymidine kinase gene at various time intervals by the electroporation technique. Marked enhancement of stable transformation efficiency was observed at the time corresponding to the peak of G2/M phase. These results suggest that the G2/M phase is the most efficient period for stable gene transfer by electroporation.     

Spatially and temporally controlled gene transfer by electroporation into adherent cells on plasmid DNA-loaded electrodes

Functional characterization of human genes is one of the most challenging tasks in current genomics. Owing to a large number of newly discovered genes, high-throughput methodologies are greatly needed to express in parallel each gene in living cells. To develop a method that allows efficient transfection of plasmids into adherent cells in spatial- and temporal-specific manners, we studied electric pulse-triggered gene transfer using a plasmid-loaded electrode. A plasmid was loaded on a gold electrode surface having an adsorbed layer of poly(ethyleneimine), and cells were then plated directly onto this modified surface. The plasmid was detached from the electrode by applying a short electric pulse and introduced into the cells cultured on the electrode, resulting in efficient gene expression, even in primary cultured cells. The location of transfected cells could be restricted within a small area on a micropatterned electrode, showing the versatility of the method for spatially controlled transfection. Plasmid transfection could also be performed in a temporally controlled manner without a marked loss of the efficiency when an electric pulse was applied within 3 days after cell plating. The method described here will provide an efficient means to transfer multiple genes, in parallel, into cultured mammalian cells for high-throughput reverse genetics research.