Phosphate-buffered saline-based nucleofection of primary endothelial cells

Although various nonviral transfection methods are available, cell toxicity, low transfection efficiency, and high cost remain hurdles for in vitro gene delivery in cultured primary endothelial cells. Recently, unprecedented transfection efficiency for primary endothelial cells has been achieved due to the newly developed nucleofection technology that uses a combination of novel electroporation condition and specific buffer components that stabilize the cells in the electrical field. Despite superior transfection efficiency and cell viability, high cost of the technology has discouraged cardiovascular researchers from liberally adopting this new technology. Here we report that a phosphate-buffered saline (PBS)-based nucleofection method can be used for efficient gene delivery into primary endothelial cells and other types of cells. Comparative analyses of transfection efficiency and cell viability for primary arterial, venous, microvascular, and lymphatic endothelial cells were performed using PBS. Compared with the commercial buffers, PBS can support equally remarkable nucleofection efficiency to both primary and nonprimary cells. Moreover, PBS-mediated nucleofection of small interfering RNA (siRNA) showed more than 90% knockdown of the expression of target genes in primary endothelial cells. We demonstrate that PBS can be an unprecedented economical alternative to the high-cost buffers or nucleofection of various primary and nonprimary cells.     

Efficient transfection of primary zebrafish fibroblasts by nucleofection

Although various gene delivery techniques are available, their application in zebrafish cell cultures has not been extensively studied. Here, we report that nucleofection of zebrafish primary embryonic fibroblasts results in higher transfection efficiency in comparison to other non-viral gene delivery methods. The transfection was performed using green fluorescent protein (GFP) gene constructs of a different size. Greatest DNA uptake was obtained with 4.9-kb plasmid, resulting in 43% GFP positive cells. Nucleofection with 7.4-kb pH2B-GFP plasmid followed by geneticin (G418) selection was successfully used to establish a cell line expressing nuclear histone 2B-GFP fusion protein. Efficient transfection of zebrafish fibroblasts by nucleofection offers a non-viral technique of plasmid delivery and can be used to overexpress genes of interest in these cells.     

Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells

Human mesenchymal stromal cell (hMSC) is a potential target for cell and gene therapy-based approaches against a variety of different diseases. Whilst cationic lipofection has been widely experimented, the Nucleofector technology is a relatively new non-viral transfection method designed for primary cells and hard-to-transfect cell lines. Herein, we compared the efficiency and viability of nucleofection with cationic lipofection, and used the more efficient transfection method, nucleofection, to deliver a construct of minimalistic, immunologically defined gene expression encoding the erythropoietin (MIDGE-EPO) into hMSC. MIDGE construct is relatively safer than the viral and plasmid expression systems as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. Using a plasmid encoding the luciferase gene, we demonstrated a high transfection efficiency using the U-23 (21.79 ± 1.09%) and C-17 (5.62 ± 1.09%) pulsing program in nucleofection. The cell viabilities were (44.93 ± 10.10)% and (21.93 ± 5.72)%, respectively 24 h post-nucleofection. On the other hand, lipofection treatment only yielded less than 0.6% efficiencies despite showing higher viabilities. Nucleofection did not affect hMSC renewability, immunophenotype and differentiation potentials. Subsequently, we nucleofected MIDGE-EPO using the U-23 pulsing program into hMSC. The results showed that, despite a low nucleofection efficiency with this construct, the EPO protein was stably expressed in the nucleofected cells up to 55 days when determined by ELISA or immunocytochemical staining. In conclusion, nucleofection is an efficient non-viral transfection approach for hMSC, which when used in conjunction with a MIDGE construct, could result in extended and stable transgene expression in hMSC.     

Efficient gene transfer into murine embryonic stem cells by nucleofection

Genetic manipulation of embryonic stem (ES) cells is performed by non-viral as well as viral transfection methods. We tested the recently developed nucleofection method delivering plasmid DNA directly into the nucleus for the introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into murine ES cells. Cell viability decreased from 77% before to 40% 24 h after nucleofection. Transfection effciencies in viable stem cells were between 85% and 96% with high levels of EGFP expression [mean fluorescence intensity (MFI): 630 +/- 90] 24 h after nucleofection. After a two week culture in geneticin (G418) selection medium, nearly 50% of the stem cells were EGFP positive and continued transgene expression (MFIs: 120-240) for a two further weeks. We conclude that nucleofection is an efficient nonviral gene transfer method for the introduction of genes into murine ES cells.     

Astrocytic Responses to DNA Delivery Using Nucleofection

Nucleofection is a powerful non-viral transfection technique that can deliver plasmid DNA with high efficiency to cells that are traditionally difficult to transfect. In this study, we demonstrate that nucleofection of astrocytes grown in primary cell culture resulted in 76 ± 9% transfected cells and low cytotoxicity. However, the nucleofected astrocytes showed a reduced re-attachment to the growth media when replated and subsequent impairment of proliferation. This led to substantially decreased cell densities during the initial 72 h following transfection. Furthermore, these cells were less efficient at producing wound closure in a scratch model of injury. Nucleofection also resulted in the generation of a small proportion of polynucleated cells. The findings demonstrate that nucleofection provides a valuable technique for delivering DNA to astrocytes in culture. However, considerable care is needed in designing and interpreting such studies because of long-lasting changes induced in key properties of these cells by the nucleofection process.     

Lipofection and nucleofection of substrate plasmid can generate widely different readings of DNA end-joining efficiency in different cell lines

In vivo plasmid end-joining assays are valuable tools for dissecting important qualitative and quantitative aspects of non-homologous end-joining (NHEJ) – a key mechanism for the repair of DNA double-strand breaks (DSBs) in higher eukaryotes. They enable the use of defined DNA ends as substrates for end-joining and the analysis by sequencing of the resulting junctions to identify the repair pathways engaged. Yet, plasmid assays have generated divergent results of end-joining capacity in the same DSB repair mutants when used under different conditions, which implies contributions from undefined and therefore uncontrolled parameters. To help standardize these assays, we searched for parameters underpinning these variations and identified transfection method as an important determinant. Here, we compare a lipid-based transfection method, lipofection, with an electroporation method, nucleofection, and find large, unanticipated and cell line-dependent differences in percent end-joining without recognizable trends. For example, in rodent cells, transfection using lipofection gives nearly WT end-joining in DNA-PKcs mutants and only mildly inhibited end-joining in Lig4 and Ku mutants. In contrast, transfection using nucleofection shows marked end-joining inhibition in all NHEJ mutants tested as compared to the WT. In human HCT116 cells, end-joining after nucleofection is strongly suppressed even in the WT and the differences to the mutants are small. After lipofection, in contrast, end-joining is high in WT cells and markedly suppressed in the mutants. We conclude that better understanding and control of the physicochemical/biological and analytical parameters underpinning these differences will be required to generate with plasmid assays results with quantitative power comparable to that of well-established methods of DSB analysis such as pulsed-field gel electrophoresis or γ-H2AX foci scoring. Until then, caution is needed in the interpretation of the results obtained – particularly with reference to pathway efficiency and residual damage – and confirmation of critical results with alternative transfection approaches is advisable.     

Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system

Porcine embryonic fibroblasts (PEF) are important as donor cells for nuclear transfer for generation of genetically modified pigs. In this study, we determined an optimal protocol for transfection of PEF with the Amaxa Nucleofection system, which directly transfers DNA into the nucleus of cells, and compared its efficiency with conventional lipofection and electroporation. Cell survival and transfection efficiency were assessed using dye-exclusion assay and a green fluorescent protein (GFP) reporter construct, respectively. Our optimized nucleofection parameters yielded survival rates above 60%. Under these conditions, FACS analysis demonstrated that 79% of surviving cells exhibited transgene expression 48 h after nucleofection when program U23 was used. This efficiency was higher than that of transfection of PEFs with electroporation (ca. 3-53%) or lipofection (ca. 3-8%). Transfected cells could be expanded as stably transgene-expressing clones over a month. When porcine nuclear transfer (NT) was performed using stable transformant expressing GFP as a donor cell, 5-6% of reconstituted embryos developed to blastocysts, from which 30-50% of embryos exhibited NT-embryo-derived green fluorescence. Under the conditions evaluated, nucleofection exhibited higher efficiency than conventional electroporation and lipofection, and may be a useful alternative for generation of genetically engineered pigs through nuclear transfer.     

Nucleofection-based gene targeting in human pre-B cells

Electroporation is a powerful and convenient means for transfection of nonviral vectors into mammalian cells, providing an essential tool for numerous applications including gene targeting via homologous recombination. Recent evidence clearly suggests that high-efficiency gene transfer can be achieved in most cell lines by nucleofection, an electroporation-based transfection method that allows transfected vectors to directly enter the nucleus. In this paper, we analyze the effectiveness of nucleofection for gene targeting using human pre-B cells. For this, we tested 93 different transfection conditions, and found several conditions that gave high (~ 80%) transfection efficiency with low cytotoxicity (~ 70% survival rate). Remarkably, under the optimal nucleofection conditions, the gene-targeting efficiency was ~ 2-5-fold higher than that achieved with conventional electroporation methods. We also found that nucleofection conditions with high transfection efficiency and low cytotoxicity tend to provide high gene-targeting efficiency. Our results provide significant implications for gene targeting, and suggest that nucleofection-based nonviral gene transfer is useful for systematic generation of human gene-knockout cell lines.     

Comparison of plasmid DNA versus PCR amplified gene of insert DNA for nucleofection in Kasumi-1 cells

Plasmid electroporation, or its optimized version nucleofection, is an important technique for gene transfection of cells in suspension. However, substantial cell death and/or low transfection efficiency are still common for some cell lines. By using enhanced green fluorescent protein (EGFP) as a reporter, we compared the use of PCR amplified EGFP (PaEGFP) and its parental plasmid (pEGFP-N2) for nucleofection in Kasumi-1 cells. We found that PaEGFP induced significantly lower cell death but had similar transfection efficiency compared to its parent plasmid (pEGFP-N2). Most importantly, contrary to the pEGFP-N2-nucleofected cells, the PaEGFP-nucleofected cells subsequently grew properly. Tests in other cell lines also implied that PaEGFP indeed induced consistently less cell death, but transfection efficiencies varied, being good in suspension cell lines but lower in adhesive cell lines. We suggest that direct transfection with PCR amplified genes can be a simple and useful approach for optimization of electropulse-based transfection not only of Kasumi-1 cells, but also may be useful for other cell lines that are difficult to transfect in suspension.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-013-9683-y) contains supplementary material, which is available to authorized users.     

Transient transfection of purified Babesia bovis merozoites

Transient transfection of intraerythrocytic Babesia bovis parasites has been previously reported. In this study, we describe the development and optimization of methods for transfection of purified B. bovis merozoites using either nucleofection (Amaxa) or conventional electroporation (Gene Pulser II, BioRad). Initially, the optimal buffer ("Plasmodium 88A6") and program (v-24) for nucleofection of free merozoites with a plasmid containing the luciferase gene as a reporter were determined. Using the same reporter plasmid, optimal voltage, capacitance and resistance for transfecting free merozoites by electroporation were defined to be 1.2 kV/25 microF/200 Omega. Using these optimal parameters, analysis of the time course of luciferase expression using either system to transfect free B. bovis merozoites showed high enzyme activity at 24h, with a rapid decline thereafter. Nucleofection was approximately five times more effective than electroporation when using a small quantity (2 microg) of DNA, while electroporation was twice as effective as nucleofection when a larger quantity of plasmid DNA (100 microg) was used. Parasite viability was significantly higher when using nucleofection when compared to electroporation regardless of the amount of DNA used. Comparison of luciferase expression after transfection of merozoites with circular, linearized, or double digested plasmid indicated that intact, circular plasmid was necessary for optimal luciferase expression. Overall, the results provide a basis for optimal transfection of purified B. bovis merozoites using either nucleofection or conventional electroporation. However, nucleofection is significantly more efficient when transfecting either circular or restriction digested DNA in the 2-10 microg range.     

Targeting of gene expression by siRNA in CML primary cells

Development of array methods contributes to elucidation of many genes expressed during oncogenesis. Our array-based analyses of gene expression in patients with chronic myeloid leukemia (CML) revealed several genes (MMP8, MMP9, PCNA, JNK2, MAPK p38) with significant increased expression. We suppose that the genes may be implicated in the disease development and a siRNA-suppression can elucidate their functions in leukemogenesis. One of the crucial requirements for this purpose is a high efficiency of siRNA delivery into CML primary cells. Using fluorescein-labeled siRNAs we systematically tested a variety of physical and chemical non-vector based transfection methods in order to evaluate which of them gave the most suitable transfer. Chemically synthesized siRNAs against mentioned genes were transfected into the cells and level of knockdown was determined by real time RT-PCR. Chemical transfection reagents (Oligofectamine, Metafectene, siPORT Amine) commonly used to transfect siRNAs in CML cell lines showed very low siRNA delivery in CML primary cells—mRNA levels decreased at the most to 76%. Electroporation achieved better results (suppression to 63%) but it was associated with high degree of cell death (more than 60%). In the study we obtained the best transfection efficiency using nucleofector technology. Gene expressions ranged 22–37% that remained from original levels. According to our results, nucleofection appears to be the only suitable non-viral method for siRNA delivery into the hard-to-transfect CML primary cells.     

Optimization of Babesia bovis transfection methods

The tick borne Babesia parasites remain an important limitation for development of cattle industries worldwide. A stable transfection of Babesia bovis will be useful for functional analysis of the recently sequenced B. bovis genome and to design improved methods to control Babesia infections. In this study, we describe a novel system for nucleofection of B. bovis infected erythrocytes and we optimize methods to introduce plasmids encoding the luciferase reporter gene into Babesia infected erythrocytes or free merozoites using either a BioRad GenePulser II electroporation system or nucleofection technology (Amaxa) A comparative study among four different transfection methods: transfection of infected erythrocytes and purified merozoites with 2 or 100 microg of plasmid, using electroporation (BioRad GenePulser II) or nucleofection (Amaxa) indicates that electroporation of infected erythrocytes with 100 microg of plasmid or nucleofection with 2 microg of plasmid are the most efficient ways to transfect B. bovis parasites. The data also indicate that nucleofection is more efficient than electroporation for transfecting small quantities of plasmids (2 microg range), whereas the inverse is true for transfection of larger quantities (100 microg range). This information will facilitate further development of efficient stable transfection systems.     

mRNA-mediated gene delivery into human progenitor cells promotes highly efficient protein expression

Gene transfer into human CD34+ haematopoietic progenitor cells (HPC) and multi-potent mesenchymal stromal cells (MSC) is an essential tool for numerous in vitro and in vivo applications including therapeutic strategies, such as tissue engineering and gene therapy. Virus based methods may be efficient, but bear risks like tumorigenesis and activation of immune responses. A safer alternative is non-viral gene transfer, which is considered to be less efficient and accomplished with high cell toxicity. The truncated low affinity nerve growth factor receptor (ALNGFR) is a marker gene approved for human in vivo application. Human CD34+ HPC and human MSC were transfected with in vitro-transcribed mRNA for DeltaLNGFR using the method of nucleofection. Transfection efficiency and cell viability were compared to plasmid-based nucleofection. Protein expression was assessed using flow cytometry over a time period of 10 days. Nucleofection of CD34+ HPC and MSC with mRNA resulted in significantly higher transfection efficiencies compared to plasmid transfection. Cell differentiation assays were performed after selecting DeltaLNGFR positive cells using a fluorescent activating cell sorter. Neither cell differentiation of MSC into chondrocytes, adipocytes and osteoblasts, nor differentiation of HPC into burst forming unit erythroid (BFU-E) colony forming unit-granulocyte, erythrocyte, macrophage and megakaryocyte (CFU-GEMM), and CFU-granulocyte-macrophage (GM) was reduced. mRNA based nucleofection is a powerful, highly efficient and non-toxic approach for transient labelling of human progenitor cells or, via transfection of selective proteins, for transient manipulation of stem cell function. It may be useful to transiently manipulate stem cell characteristics and thus combine principles of gene therapy and tissue engineering.     

A modified host-cell reactivation assay to quantify DNA repair capacity in cryopreserved peripheral lymphocytes

The host-cell reactivation assay (HCRA) is a functional assay that allows the identification of the genes responsible for DNA repair-deficient syndromes, such as Xeroderma pigmentosum, by cross-complementation experiments. It has also been used in molecular epidemiology studies to correlate the low nucleotide excision repair pathway function in peripheral blood lymphocytes with an increased risk of bladder, head and neck, skin and lung cancers. Herein, we present the technical validation of a newly modified HCRA, where nucleofection is used for the transfection of the pmaxGFP plasmid into cryopreserved peripheral blood lymphocytes (PBLs) or lymphoblastoid cell lines. In each sample, 20-24h after transfection, the relative DNA repair capacity (DRC) was quantified by flow cytometry, comparing the transfection efficiency of nucleoporated cells with undamaged plasmid to those transfected with UV-light damaged plasmid in the seven cell lines that were characterized by different DNA repair phenotypes. Dead cells were excluded from the analysis. We observed a high reproducibility of the relative DRC, transfection efficiency and cell viability. The inter-experimental normalization of the flow cytometry resulted in an increased data accuracy and reproducibility. The amount of cells required for each transfection reaction was reduced fourfold, without affecting the final relative DRC. Furthermore, our HCRA demonstrated strong discrimination power in the UV-light dose-response, both in lymphoblastoid cell lines and cryopreserved PBLs. We also observed a strong correlation of the relative DRC data, when samples were measured against two independent batches of both damaged and undamaged plasmid DNA. The relative DRC variable shows a normal distribution when analyzed in the cryopreserved PBLs from a cohort of 35 lung cancer patients and a 5.59-fold variation in the relative DRC is identified among our patients. The mitotic dynamic was discarded as a confounding factor for the relative DRC measurement in this cohort of patients. The results indicate that our method is highly sensitive, reliable and reproducible, and thus, it suitable for population-based studies to quantify in vitro DNA-repair deficiencies.     

Nucleofection as an efficient nonviral transfection method for human monocytic cells

Despite some progress in the field of gene transfer into hard-to-transfect cells, so far an efficient nonviral method for monocytes has not been available. A comparison of plasmid DNA with capped and polyadenylated mRNA for enhanced green fluorescent protein gene delivery into the commonly used monocytic cell lines U937 and THP-1 suggested that limited DNA trafficking may be the underlying cause of poor transfection results. As Nucleofector technology delivers DNA (or mRNA) straight into the nucleus, we obtained nucleofection efficiencies of up to 80% without significant cell toxicity. Moreover, as the DNA quickly reaches the nucleus, nucleofected cells were ready for analysis after only 2–6 h. The technique is suitable not only for monocytes but also for other hard-to-transfect cells.     

Enhanced Nanomagnetic Gene Transfection of Human Prenatal Cardiac Progenitor Cells and Adult Cardiomyocytes

Magnetic nanoparticle-based gene transfection has been shown to be an effective, non-viral technique for delivery of both plasmid DNA and siRNA into cells in culture. It has several advantages over other non-viral delivery techniques, such as short transfection times and high cell viability. These advantages have been demonstrated in a number of primary cells and cell lines. Here we report that oscillating magnet array-based nanomagnetic transfection significantly improves transfection efficiency in both human prenatal cardiac progenitor cells and adult cardiomyocytes when compared to static magnetofection, cationic lipid reagents and electroporation, while maintaining high cell viability. In addition, transfection of adult cardiomyocytes was improved further by seeding the cells onto Collagen I-coated plates, with transfection efficiencies of up to 49% compared to 24% with lipid reagents and 19% with electroporation. These results demonstrate that oscillating nanomagnetic transfection far outperforms other non-viral transfection techniques in these important cells.     

Optimizing Electroporation Conditions in Primary and Other Difficult-to-Transfect Cells

Electroporation is a valuable tool for nucleic acid delivery because it can be used for a wide variety of cell types. Many scientists are shifting toward the use of cell types that are more relevant to in vivo applications, including primary cells, which are considered difficult to transfect. The ability to electroporate these cell types with nucleic acid molecules of interest at a relatively high efficiency while maintaining cell viability is essential for elucidating the pathway(s) in which a gene product is involved. We present data demonstrating that by optimizing electroporation parameters, nucleic acid molecules can be delivered in a highly efficient manner. We display transfection results for primary and difficult-to-transfect cell types including human primary fibroblasts, human umbilical vein endothelial cells, Jurkat cells, and two neuroblastoma cell lines [SK-N-SH (human) and Neuro-2A (mouse)] with plasmid DNAs and siRNAs. Our data demonstrate that by determining proper electroporation conditions, glyceraldehyde phosphate dehydrogenase mRNA was silenced in Jurkat cells when compared with negative control siRNA electroporations as early as 4 h post-transfection. Other experiments demonstrated that optimized electroporation conditions using a fluorescently labeled transfection control siRNA resulted in 75% transfection efficiency for Neuro-2A, 93% for human primary fibroblasts, and 94% for HUVEC cells, as analyzed by flow cytometry.     

Exogenous nucleic acids aggregate in non-P-body cytoplasmic granules when transfected into cultured cells

To modulate gene expression in research studies or in potential clinical therapies, transfection of exogenous nucleic acids including plasmid DNA and small interference RNA (siRNA) are generally performed. However, the cellular processing and the fate of these nucleic acids remain elusive. By investigating the cellular behavior of transfected nucleic acids using confocal imaging, here we show that when siRNA was co-transfected into cultured cells with other nucleic acids, including single-stranded RNA oligonucleotides, single and double-stranded DNA oligonucleotides, as well as long double-stranded plasmid DNA, they all aggregate in the same cytoplasmic granules. Interestingly, the amount of siRNA aggregating in granules was found not to correlate with the gene silencing activity, suggesting that assembly of cytoplasmic granules triggered by siRNA transfection may be separable from the siRNA silencing event. Our results argue against the claim that the siRNA-aggregating granules are the functional site of RNA interference (RNAi). Taken together, our studies suggest that, independent of their types or forms, extraneously transfected nucleic acids are processed through a common cytoplasmic pathway and trigger the formation of a new type of cytoplasmic granules “transfection granules”.     

Competitive DNA transfection formulation via electroporation for human adipose stem cells and mesenchymal stem cells

Background

Adipose stem cells have a strong potential for use in cell-based therapy, but the current nucleofection technique, which relies on unknown buffers, prevents their use.

Results

We developed an optimal nucleofection formulation for human adipose stem cells by using a three-step method that we had developed previously. This method was designed to determine the optimal formulation for nucleofection that was capable of meeting or surpassing the established commercial buffer (Amaxa), in particular for murine adipose stem cells. By using this same buffer, we determined that the same formulation yields optimal transfection efficiency in human mesenchymal stem cells.

Conclusions

Our findings suggest that transfection efficiency in human stem cells can be boosted with proper formulation.