Established cell transfection via nucleofection relies on nucleofection buffers with unknown and proprietary makeup due to trade secrecy, inhibiting the possibility of using this otherwise effective method for developing cell therapy. We devised a three-step method for discovering an optimal formulation for the nucleofection of any cell-line. These steps include the selection of the best nucleofection program and known buffer type, selection of the best polymer for boosting the transfection efficiency of the best buffer, and the comparison with the optimal buffer from an established commercial vendor (Amaxa). Using this 3-step selection system, competitive nucleofection formulations were discovered for multiple cell lines, which are equal to or surpass the efficiency of the Amaxa nucleofector solution in a variety of cells and cell lines, including primary adipose stem cells, muscle cells, tumor cells, and immune cells. Through the use of scanning electron microscopy, we have revealed morphological changes, which predispose for the ability of these buffers to assist in transferring plasmid DNA into the nuclear space. Our formulation may greatly reduce the cost of electroporation study in laboratory and boosts the potential of application of electroporation-based cell therapies in clinical trials.
electroporation; cell transfection; cell therapy; adipose stem cells; formulation
The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected.
Studies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells) and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts). With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific.
In general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival.
Achieving efficient introduction of plasmid DNA into primary cultures of mammalian cells is a common problem in biomedical research. Human primary cranial suture cells are derived from the connective mesenchymal tissue between the bone forming regions at the edges of the calvarial plates of the skull. Typically they are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs in utero and postnatally. To better understand the molecular mechanisms involved in skull growth, and in abnormal growth conditions, such as craniosynostosis, caused by premature bony fusion, it is essential to be able to easily introduce genes into primary bone forming cells to study their function.
A comparison of several lipid-based techniques with two electroporation-based techniques demonstrated that the electroporation method known as nucleofection produced the best transfection efficiency. The parameters of nucleofection, including cell number, amount of DNA and nucleofection program, were optimized for transfection efficiency and cell survival. Two different genes and two promoter reporter vectors were used to validate the nucleofection method and the responses of human primary suture mesenchymal cells by fluorescence microscopy, RT-PCR and the dual luciferase assay. Quantification of bone morphogenetic protein (BMP) signalling using luciferase reporters demonstrated robust responses of the cells to both osteogenic BMP2 and to the anti-osteogenic BMP3.
A nucleofection protocol has been developed that provides a simple and efficient, non-viral alternative method for in vitro studies of gene and protein function in human skull growth. Human primary suture mesenchymal cells exhibit robust responses to BMP2 and BMP3, and thus nucleofection can be a valuable method for studying the potential competing action of these two bone growth factors in a model system of cranial bone growth.
Transfection; Nucleofection; Skull; Bone; Primary cell culture; Mesenchymal; BMP2; luciferase
Although various non-viral 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 utilizes a combination of novel electroporation conditions and specific buffer components that stabilize the cells in the electrical field. Despite its superior transfection efficiency and cell viability, high cost of the technology has discouraged the cardiovascular researchers to liberally adopt 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 by using PBS. Compared to the commercial buffers, PBS can support equally remarkable nucleofection efficiency to both primary and non-primary cells. Moreover, PBS-mediated nucleofection of siRNA showed more than 90% knockdown of the expression of target genes in primary endothelial cells. Together, we demonstrate that PBS can be an unprecedented economical alternative for the high-cost buffers for nucleofection of various primary and non-primary cells.
electroporation; nucleofection; primary endothelial cells; phosphate-buffered saline
Tumour homing capacity of engineered human adipose-derived mesenchymal stromal cells (ADMSCs) expressing anti-tumour agents might be the key for a much safer and yet efficient targeted tumour therapy. However, ADMSCs exhibit resistant to most gene transfection techniques and the use of highly efficient viral vectors has several disadvantages primarily concerning safety risk. Here, we optimized the use of highly efficient and safe nucleofection-based transfection using plasmid encoded for TNF-Related Apoptosis Inducing Ligand (TRAIL) into ADMSCs and investigated the potential anti-tumourigenic of TRAIL-expressing ADMSCs (ADMSCs-TRAIL) on selected cancer models in vitro.
Different concentration of TRAIL-encoded plasmid and ADMSCs were nucleofected and the percentage of fluorescence cells were analyzed to determine the optimal condition. TRAIL protein and mRNA were validated in nucloeofected ADMSCs using ELISA and RT-PCR respectively. Evaluation of TRAIL specific death receptors were performed on both tumours (A549/lung tumour, LN18/glioblastoma and HepG2/hepatocellular carcinoma) and haematological malignant lines (REH/acute lymphocytic leukaemia, K562/chronic myelogenous leukaemia and KMS-28BM/multiple myeloma) using flow cytometry. ADMSCs-TRAIL was subsequently assessed for anti-tumourigenic properties using both proliferation assay (MTS assay) and apoptosis assay (Annexin-V / Propidium Iodide staining).
Nucleofection showed increased total plasmid concentration (2 μg to 8 μg) resulted in significantly higher reporter expression (11.33% to 39.7%) with slight reduction on cells viability (~10%). ADMSCs-TRAIL significantly inhibited ~50% of cell proliferation in LN18, signifying sensitivity of the cell to ADMSCs-TRAIL mediated inhibition. Inhibition of both tumour and malignant lines proliferation by ADMSCs-TRAIL conditioned medium noticed in HepG2, A549 and REH respectively, whereas K562 and KMS-28BM malignant lines exhibit resistant to ADMSCs-TRAIL mediated inhibition. Moreover, we found that native ADMSCs alone were capable of inducing apoptosis in both LN18 and HepG2 tumour lines, despite substantial increased on the percentage of apoptosis by ADMSCs-TRAIL.
ADMSCs-TRAIL selectively inhibit cancer model and markedly induces apoptosis. Through investigation of the specific TRAIL death receptors expression, we saw that the receptors expression did influence the sensitivity of some but not all cancer lines to TRAIL-mediated inhibition. This study provides further insight into the anti-tumourigenic potential of ADMSCs-TRAIL on different cancer models.
Human adipose derived mesenchymal stromal cells; Nucleofection; TNF-related apoptosis inducing ligand (TRAIL); Cancer cell lines; Proliferation; Apoptosis
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.
Bone marrow mesenchymal stromal cells; Nucleofection; Cationic lipofection; MIDGE; Erythropoietin
Genetic manipulation of human embryonic stem cells (hESC) has been limited by their general resistance to common methods used to introduce exogenous DNA or RNA. Efficient and high throughput transfection of nucleic acids into hESC would be a valuable experimental tool to manipulate these cells for research and clinical applications.
We investigated the ability of two commercially available electroporation systems, the Nucleofection® 96-well Shuttle® System from Lonza and the Neon™ Transfection System from Invitrogen to efficiently transfect hESC. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an ATP catalyzed luciferase reaction. The transfected cells were also analyzed by flow cytometry for common markers of pluripotency.
Both systems are capable of transfecting hESC at high efficiencies with little loss of cell viability. However, the reproducibility and the ease of scaling for high throughput applications led us to perform more comprehensive tests on the Nucleofection® 96-well Shuttle® System. We demonstrate that this method yields a large fraction of transiently transfected cells with minimal loss of cell viability and pluripotency, producing protein expression from plasmid vectors in several different hESC lines. The method scales to a 96-well plate with similar transfection efficiencies at the start and end of the plate. We also investigated the efficiency with which stable transfectants can be generated and recovered under antibiotic selection. Finally, we found that this method is effective in the delivery of short synthetic RNA oligonucleotides (siRNA) into hESC for knockdown of translation activity via RNA interference.
Our results indicate that these electroporation methods provide a reliable, efficient, and high-throughput approach to the genetic manipulation of hESC.
AIM: To enhance the differentiation of insulin producing cell (IPC) ability from embryonic stem (ES) cells in vitro.
METHODS: Four-day embryoid body (EB)-formatted ES cells were dissociated as single cells for the followed plasmid DNA delivery. The use of Nucleofector™electroporator (Amaxa biosystems, Germany) in combination with medium-contained G418 provided a high efficiency of gene delivery for advanced selection. Neucleofected cells were plated on the top of fibronectin-coated Petri dishes. Addition of Ly294002 and raised the glucose in medium at 24 h before examination. The differentiation status of these cells was monitored by semi-quantitative PCR (SQ-PCR) detection of the expression of relative genes, such as oct-4, sox-17, foxa2, mixl1, pdx-1, insulin 1, glucagons and somatostatin. The percentage of IPC population on d 18 of the experiment was investigated by immunohistochemistry (IHC), and the content/secretion of insulin was estimated by ELISA assay. The mice with severe combined immunodeficiency disease (SCID) pretreated with streptozotocin (STZ) were used to eliminate plasma glucose restoration after pax4+ ES implantation.
RESULTS: A high efficiency of gene delivery was demonstrated when neucleofection was used in the present study; approximately 70% cells showed DsRed expression 2 d after neucleofection. By selection of medium-contained G418, the percentage of DsRed expressing cells kept high till the end of study. The pancreatic differentiation seemed to be accelerated by pax4 nucleofection. When compared to the group of cells with mock control, foxa2, mixl1, pdx1, higher insulin and somatostatin levels were detected by SQ-PCR 4 d after nucleofection in the group of pax4 expressing plasmid delivery. Approximately 55% of neucleofected cells showed insulin expression 18 d after neucleofection, and only 18% of cells showed insulin expression in mock control. The disturbance was shown by nucleofected pax4 RNAi vector; only 8% of cells expressed insulin 18 d after nucleofection. A higher IPC population was also detected in the insulin content by ELISA assay, and the glucose dependency was demonstrated in insulin secretion level. In the animal model, improvement of average plasma glucose concentration was observed in the group of pax-4 expressed ES of SCID mice pretreated with STZ, but no significant difference was observed in the group of STZ-pretreated SCID mice who were transplanted ES with mock plasmid.
CONCLUSION: Enhancement of IPC differentiation from EB-dissociated ES cells can be revealed by simply using pax4 expressing plasmid delivery. Not only more IPCs but also pancreatic differentiation-related genes can be detected by SQ-PCR. Expression of relative genes, such as foxa 2, mixl 1, pdx-1, insulin 1 and somatostatin after nucleofection, suggests that pax4 accelerates the whole differentiation progress. The higher insulin production with glucose dependent modulation suggests that pax4 expression can drive more mature IPCs. Although further determination of the entire mechanism is required, the potential of pax-4-nucleofected cells in medical treatment is promising.
Diabetes mellitus; Nucleofection; pax4; Embryonic stem cells; Insulin producing cells
Technologies designed to allow manipulation and modification of human embryonic stem (hES) cells are numerous and vary in the complexity of their methods, efficiency, reliability, and safety. The most commonly studied and practiced of these methods include electroporation, lipofection, nucleofection, and lentiviral transduction. However, at present, it is unclear which protocol offers the most efficient and reliable method of gene transfer to hES cells. In this study, a bi-fusion construct with ubiquitin promoter driving enhanced green fluorescent protein reporter and the firefly luciferase (pUb-eGFP-Fluc) along with neomycin selection marker was used for in vitro and in vivo studies. In vitro studies examined the transfection efficiency and viability of each technique using two hES cell lines (male H1 and female H9 cells). Lentiviral transduction demonstrated the highest efficiency (H1: 25.3 ± 4.8%; H9: 22.4 ± 6.5%) with >95% cell viability. Nucleofection demonstrated transfection efficiency of 16.1 ± 3.6% (H1) and 5.8 ± 3.2% (H9). However, minimal transfection efficiency was observed with electroporation (2.1 ± 0.4% (H1) and 1.9 ± 0.3% (H9)) and lipofection (1.5 ± 0.5% (H1) and 1.3 ± 0.2% (H9); P < 0.05 vs. lentiviral transduction). Electroporation also demonstrated the highest cell death (62 ± 11% (H1) and 42 ± 10% (H9)) followed by nucleofection (25 ± 9% (H1) and 30 ± 15 (H9)). Importantly, lentiviral transduction generated a greater number of hES cell lines stably expressing the double-fusion reporter gene (hES-DF) compared to other transfection techniques. Finally, following subcutaneous transplantation into immunodeficient nude mice, the hES-eGFP-Fluc cells showed robust proliferation as determined by longitudinal bioluminescence imaging. In summary, this study demonstrates that lentiviral transduction and nucleofection are efficient, simple, and safe techniques for reliable gene transfer in hES cells. The double-fusion construct provides an attractive approach for generating stable hES cell lines and monitoring engraftment and proliferation in vitro and in vivo.
Human embryonic stem cell; Molecular imaging; Gene transfer; Plasmid transfection; Lentivirus transduction
Dendritic cells (DC) are potent antigen-presenting cells that hold promise as cell-based therapeutic vaccines for infectious diseases and cancer. Ideally, DC would be engineered to express autologous viral or tumor antigens to ensure the presentation of relevant antigens to host T cells in vivo; however, expression of wild-type viral genes in primary cell lines can be problematic. Nucleofection is an effective means of delivering transgenes to primary cell lines, but its use in transfecting DNA or mRNA into DC has not been widely investigated. We show that nucleofection is a superior means of transfecting human and monkey monocyte-derived DC with DNA and mRNA compared to lipofection and conventional electroporation. However, the delivery of DNA and mRNA had significantly different outcomes in transfected DC. DC nucleofected with DNA encoding green fluorescent protein (GFP) had poor antigen expression and viability and were refractory to maturation with CD40 ligand. In contrast, >90% of DC expressed uniform and high levels of GFP from 3 h to 96 h postnucleofection with mRNA while maintaining a normal maturation response to CD40 ligation. Monkey DC nucleofected with wild-type, non-codon-optimized mRNA encoding simian immunodeficiency virus Gag stimulated robust antigen-specific effector T-cell responses at 24 h and 48 h postnucleofection, reflecting sustained antigen presentation in transfected DC, whereas no detectable T-cell response was noted when DC were nucleofected with DNA encoding the same Gag sequence. These data indicate that mRNA nucleofection may be an optimal means of transfecting DC with autologous tumor or viral antigen for DC-based immunotherapy.
Differentiating stem cells using gene delivery is a key strategy in tissue engineering and regenerative medicine applications. Nonviral gene delivery bypasses several safety concerns associated with viral gene delivery; however, leading nonviral techniques, such as electroporation, subject cells to high stress and can result in poor cell viabilities. Inhibition of Rho-associated coiled-coil kinase (ROCK) has been shown to mitigate apoptotic mechanisms associated with detachment and freezing of induced pluripotent stem cells and embryonic stem cells; however, inhibiting ROCK in mesenchymal stromal cells (MSCs) for improving gene delivery applications has not been reported previously. In this study, we hypothesized that ROCK Inhibitor (RI) would improve cell viability and gene expression in primary human umbilical cord mesenchymal stromal cells (hUCMSCs) when transfected via Nucleofection™. As hypothesized, the pre-treatment and post-treatment of hUCMSCs transfected via nucleofection with Y-27632-RI significantly improved survival rates of hUCMSCs and gene expression as measured by green fluorescent protein intensity. This study provides the first comparative look at the effect of Y-27632-RI on hUCMSCs that underwent transfection via nucleofection and shows that using Y-27632-RI in concert with nucleofection could greatly enhance the utility of differentiating and reprogramming hUCMSCs for tissue engineering applications.
Background. Transfection efficacy after nonviral gene transfer in primary epithelial cells is limited. The aim of this study was to compare transfection efficacy of the recently available method of nucleofection with the established transfection reagent FuGENE6. Methods. Primary human keratinocytes (HKC), primary human fibroblasts (HFB), and a human keratinocyte cell line (HaCaT) were transfected with reporter gene construct by FuGENE6 or Amaxa Nucleofector device. At corresponding time points, β-galactosidase expression, cell proliferation (MTT-Test), transduction efficiency (X-gal staining), cell morphology, and cytotoxicity (CASY) were determined.
Results. Transgene expression after nucleofection was significantly higher in HKC and HFB and detected earlier (3 h vs. 24 h) than in FuGENE6. After lipofection 80%–90% of the cells remained proliferative without any influence on cell morphology. In contrast, nucleofection led to a decrease in keratinocyte cell size, with only 20%–42% proliferative cells.
Conclusion. Related to the method-dependent increase of cytotoxicity, transgene expression after nucleofection was earlier and higher than after lipofection.
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 (ÄLNGFR) is a marker gene approved for human in vivo application. Human CD34+ HPC and human MSC were transfected with in vitro-transcribed mRNA for ΔLNGFR 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 ΔLNGFR 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.
adult stem cells; mRNA transfection; in vivo application; ΔLNGFR
Gene transfer into primary human CD4 T lymphocytes is a critical tool in studying the mechanism of T cell-dependent immune responses and human immunodeficiency virus-1 (HIV-1) infection. Nucleofection® is an electroporation technique that allows efficient gene transfer into primary human CD4 T cells that are notoriously resistant to traditional electroporation. Despite its popularity in immunological research, careful characterization of its impact on the physiology of CD4 T cells has not been documented. Herein, using freshly-isolated primary human CD4 T cells, we examine the effects of Nucleofection® on CD4 T cell morphology, intracellular calcium levels, cell surface activation markers, and transcriptional activity. We find that immediately after Nucleofection®, CD4 T cells undergo dramatic morphological changes characterized by wrinkled and dilated plasma membranes before recovering 1 hour later. The intracellular calcium level also increases after Nucleofection®, peaking after 1 hour before recovering 8 hours post transfection. Moreover, Nucleofection® leads to increased expression of T cell activation markers, CD154 and CD69, for more than 24 hours, and enhances the activation effects of phytohemagglutinin (PHA) stimulation. In addition, transcriptional activity is increased in the first 24 hours after Nucleofection®, even in the absence of exogenous stimuli. Therefore, Nucleofection® significantly alters the activation state of primary human CD4 T cells. The effect of transferred gene products on CD4 T cell function by Nucleofection® should be assessed after sufficient resting time post transfection or analyzed in light of the activation caveats mentioned above.
Nucleofection®; gene transfer; T cell activation; calcium; HIV-1; plasmid
Primary autologous B-lymphocytes, following ex vivo gene transfer and re-implantation, have been successfully utilized to prevent autoimmune disease and adaptive responses to therapeutic proteins in several animal models. However, efficient gene transfer to primary B cells requires use of retroviral vectors, which increase the risk of insertional mutagenesis. Here, we evaluated several alternative gene transfer approaches. Resting splenic B cells were purified and activated with LPS, and ex vivo GFP gene transfer was performed by means of nucleofection, lipofectamine, adenoviral infection, or murine retroviral infection. The Adenoviral (Ad) vectors were added to B cell cultures with or without calcium phosphate precipitation. For transfection and nucleofection, naked plasmid DNA was utilized. Nucleofection technology represents a modified electroporation technique for effective transfer of nucleic acids to the nucleus and thus enhances the efficiency of transfer particularly for primary cells. Efficiency of ex vivo gene transfer was determined by flow cytometry using GFP, CD19, and a vital dye as markers. Nucleofection yielded the highest level of gene transfer with 60–65% of B cells being GFP+. Efficiencies were 30–35% for retrovirus, 20% for Ad5/11, 15% for Ad5/35, and 5% for lipofectamine-mediated transfection. Calcium phosphate precipitation increased efficiencies for Ad vectors to 30% (Ad5/11) and 25% (Ad5/35). Lipofectamin caused the greatest cell death at 80%, followed by nucleofection (35%), and viral vector (10–15% in each case). For all methods, gene transfer efficiencies were nearly identical for B cells from C57BL/6 or C3H/HeOuJ mice. In conclusion, recent advances in gene transfer technologies provide alternatives to retroviral vectors for primary B cells. If stable gene transfer is desired, non-integrating vector systems may be combined with transposon- or phage integrase-based systems or future site-specific systems to achieve integration into the host B cell genome.
B cells; Transfection; Ex vivo gene transfer; Plasmid DNA; Adenovirus; Retrovirus
Nucleofection is an emerging technology for delivery of nucleic acids into both the cytoplasm and nucleus of eukaryotic cells with high efficiency. This makes it an ideal technology for gene delivery and siRNA applications. A 96-well format has recently been made available for high-throughput nucleofection, however conditions must be optimized for delivery into each specific cell type. Screening each 96-well plate can be expensive, and descriptions of methods and outcomes to determine the best conditions are lacking in the literature. Here we employ simple methods, including cell counting, microscopy, viability and cytotoxicity assays to describe the minimal experimental methods required to optimize nucleofection conditions for a given cell line.
We comprehensively measured and analyzed the outcomes of the 96-well nucleofection of pmaxGFP plasmids encoding green fluorescent protein (GFP) into the A-549 human lung epithelial cell line. Fluorescent microscopy and a plate reader were used to respectively observe and quantify green fluorescence in both whole and lysed cells. Cell viability was determined by direct counting/permeability assays, and by both absorbance and fluorescence-based plate reader cytotoxicity assays. Finally, an optimal nucleofection condition was used to deliver siRNA and gene specific knock-down was demonstrated.
GFP fluorescence among conditions ranged from non-existent to bright, based upon the fluorescent microscopy and plate reader results. Correlation between direct counting of cells and plate-based cytotoxicity assays were from R = .81 to R = .88, depending on the assay. Correlation between the GFP fluorescence of lysed and unlysed cells was high, ranging from R = .91 to R = .97. Finally, delivery of a pooled sample of siRNAs targeting the gene relA using an optimized nucleofection condition resulted in a 70–95% knock down of the gene over 48 h with 90–97% cell viability.
Our results show the optimal 96-well nucleofection conditions for the widely-used human cell line, A-549. We describe simple, effective methods for determining optimal conditions with high confidence, providing a useful road map for other laboratories planning optimization of specific cell lines or primary cells. Our analysis of outcomes suggests the need to only measure unlysed, whole-cell fluorescence and cell metabolic activity using a plate reader cytotoxicity assay to determine the best conditions for 96-well nucleofection.
In times of rapidly increasing numbers of immunological approaches entering the clinics, antigen delivery becomes a pivotal process. The genuine way of rendering antigen presenting cells (APC) antigen specific, largely influences the outcome of the immune response. Short peptides bear the demerit of HLA restriction, whereas the proper way of delivery for long peptide sequences is currently a matter of debate. Electroporation is a reliable method for antigen delivery, especially using nucleic acids. The nucleofection process is based on this approach with the twist of further ensuring delivery also into the nucleus. Beside the form of antigen, the type of APC used for immune response induction may be crucial. Dendritic cells (DC) are by far the most commonly used APC; however B cells have entered this field as well and have gained wide acceptance.
In this study, we compared B cells to DC with regard to nucleofection efficiency and intensity of resulting antigen expression. APC were transfected either with plasmid DNA containing the reporter gene green fluorescent protein (GFP) or directly with in vitro-transcribed (IVT) GPF mRNA as a surrogate antigen. Out of nearly 100 different nucleofection programs tested, the top five for each cell type were identified and validated using cells from cancer patients. Flow cytometric analyses of transfected cells determining GFP expression and viability revealed a reverse correlation of efficiency and viability. Finally, donor dependant variances were analyzed.
In summary, nucleofection of both DC and B cells is feasible with plasmid DNA and IVT mRNA. And no differences with regard to nucleofectability were observed between the two cell types. Using IVT mRNA omits the danger of genomic integration and plasmid DNA constructs permit a more potent and longer lasting antigen expression.
Electronic supplementary material
The online version of this article (doi:10.1186/s13104-015-1446-8) contains supplementary material, which is available to authorized users.
Antigen presenting cells; Dendritic cells; B cells; Nucleofection; Electroporation; IVT mRNA; Technical optimization
Inhibition of CC chemokine ligand 20 (CCL20), which is expressed by human keratinocytes after proinflammatory cytokine stimulation, may reduce migration of recipient Langerhans cells into tissue-engineered allogeneic skin grafts and minimize immune rejection by the recipient. Here, we screened CCL20 gene knockout clones in the human immortalized skin keratinocyte line HaCaT and tested multiple transfection methods for optimal efficiency.
The CCL20 gene was PCR amplified from HaCaT genomic DNA. Both the short arm (1,969 bp) and long arm (2,356 bp) of human CCL20 were cloned into ploxP-targeting vectors at either side of the neomycin resistance cassette, respectively. The resulting ploxP-hCCL20-targeting vector was linearized and electroporated into HaCaT. The positive HaCaT clones were screened under the pressure of both G418 and GANC, and identified by PCR and Southern blot. The ploxP-hCCL20-EGFP fluorescent expression vector was also constructed and transfected into 293FT and HaCaT cells by jetPEI liposome and nucleofection electroporation for evaluating the transfect efficiency under fluorescent microscope.
The replacement targeting vector ploxP-hCCL20 (11.9 kb) for exon 2 of the human CCL20 gene was successfully constructed and transfected into HaCaT cells. The selected HaCaT clones did not show any evidence of CCL20 gene knockout by either PCR or Southern blot analysis. We also successfully constructed a fluorescent expression vector ploxP-hCCL20-EGFP (13.3 kb) to assess possible reasons for gene-targeting failure. Transfection efficiencies of ploxP-hCCL20-EGFP into 293FT and HaCaT cell lines by jetPEI liposome were 75.1±3.4% and 1.3±0.2%, respectively. The transfection efficiency of ploxP-hCCL20-EGFP into HaCaT cells using nucleofection electroporation was 0.3±0.1% (P=0.000), but the positive control vector pmaxGFP (3,490 bp) using the same method was 38.3±2.8%.
Overall low transfection efficiencies of ploxP-hCCL20-EGFP into HaCaT cells, regardless of transfection method, may either be due to the high molecular weight of the vector or to the fact that this particular cell line may be inherently difficult to transfect.
Chemokine CCL20; Gene Targeting; Keratinocytes; Transfection
Background. Hemophilia is a rare recessive X-linked disease characterized by a deficiency of coagulation factor VIII or factor IX. Its current treatment is merely palliative. Advanced therapies are likely to become the treatment of choice for the disease as they could provide a curative treatment.
Methods. The present study looks into the use of a safe non-viral transfection method based on nucleofection to express and secrete human clotting factor IX (hFIX) where human adipose tissue derived mesenchymal stem cells were used as target cells in vitro studies and NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice were used to analyze factor IX expression in vivo studies. Previously, acute liver injury was induced by an injected intraperitoneal dose of 500 mg/kg body weight of acetaminophen.
Results. Nucleofection showed a percentage of positive cells ranging between 30.7% and 41.9% and a cell viability rate of 29.8%, and cells were shown to secrete amounts of hFIX between 36.8 and 71.9 ng/mL. hFIX levels in the blood of NSG mice injected with ASCs transfected with this vector, were 2.7 ng/mL 48 h after injection. Expression and secretion of hFIX were achieved both in vitro cell culture media and in vivo in the plasma of mice treated with the transfected ASCs. Such cells are capable of eventually migrating to a previously damaged target tissue (the liver) where they secrete hFIX, releasing it to the bloodstream over a period of at least five days from administration.
Conclusions. The results obtained in the present study may form a preliminary basis for the establishment of a future ex vivo non-viral gene/cellular safe therapy protocol that may eventually contribute to advancing the treatment of hemophilia.
Advanced therapies; Hemophilia B; Factor IX; Non-viral; Gene therapy; Nucleofection; Human mesenchymal adipose-derived stem cells
Mesenchymal stem cells (MSCs) have attracted significant attention as effective delivery vehicles for targeting brain disorders. We have shown that MSCs derived from human adipose tissue (hAMSCs) and genetically modified with viruses display high anti-glioma tropism and can deliver bone morphogenetic protein 4 (BMP4), a therapeutic agent that reduces the clonogenic ability of glioma stem cells. Virus-safety concerns have led to the development of biodegradable nanoparticles that can transfect human cells. We identify nanoparticles for engineering hAMCSs to secrete BMP4 both in vitro and in vivo to treat brain cancer. hAMSCs were transfected using poly (beta-amino ester-PBAE)-based nanoparticles. Transfection efficacy and toxicity were evaluated to determine the optimal formulation to transfect hAMSCs with a BMP4-expressing plasmid. In vitro functional assays evaluated the effect of the transfection on hAMSC phenotype. In vivo imaging and histological analyses tracked engineered hAMSCs (NP-AMSCs) in the brain after local and systemic administration to rodents with orthotopic human tumors. Optimized nanoparticles transfected hAMSCs with 75 ± 2% efficacy and 71 ± 7% viability which was significantly superior (p < 0.0001) to LipofectamineTM 2000(16 ± 3% transfection, 54 ± 5% viability) the leading commercial product. PCR and Western blot analysis confirmed BMP4 production from NP-hAMSCs (52.5 ng protein/106 NP-hAMSCs). NP-hAMSCs were significantly better in migration and invasion (p = 0.005) than hAMSCs transduced with lentivirus. Flow cytometry showed that NP-hAMSCs retain multipotency. Intranasal, intravenous, and local delivery of luciferase- and GFP-expressing NP-hAMSCs in a rodent model of human glioma showed transgene expression 5 days after injection and NP-hAMSC migration towards the brain and within the tumor. We demonstrate that PBAE-nanoparticle formulations effectively transfect hAMSCs to produce BMP4. NP-AMSCs migrate towards the brain and penetrate the tumor in a model of human glioma after local and systemic administration. NP-engineered BMP4-secreting hAMSCs can be a safe and effective alternative to viral transduction for stem cell-based anti-glioma gene therapies.
High-efficiency genetic modification of human embryonic stem (hES) cells would enable manipulation of gene activity, routine gene targeting, and development of new human disease models and treatments. Chemical transfection, nucleofection, and electroporation of hES cells result in low transfection efficiencies. Viral transduction is efficient but has significant drawbacks. Here we describe techniques to transiently and stably express transgenes in hES cells with high efficiency using a widely available vector system. The technique combines nucleofection of single hES cells with improved methods to select hES cells at clonal density. As validation, we reduced Oct4 and Nanog expression using siRNAs and shRNA vectors in hES cells. Furthermore, we derived many hES cell clones with either stably reduced alkaline phosphatase activity or stably overexpressed green fluorescent protein. These clones retained stem cell characteristics (normal karyotype, stem cell marker expression, self-renewal, and pluripotency). These studies will accelerate efforts to interrogate gene function and define the parameters that control growth and differentiation of hES cells.
Human embryonic stem cells; Nucleofection; Transfection; Transgene expression; RNA interference; Green fluorescent protein; Oct4; Nanog
The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in 'hanging drops' and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.
Neuroscience; Issue 81; Cellular Biology; Cell Migration Assays; Transfection; Neurogenesis; subventricular zone (SVZ); neural stem cells; rostral migratory stream (RMS); neuroblast; 3D migration assay; nucleofection
There is considerable interest in using goats as models for genetically engineering dairy animals and also for using stem cells as therapeutics for bone and cartilage repair. Mesenchymal stem cells (MSCs) have been isolated and characterized from various species, but are poorly characterized in goats.
Goat MSCs isolated from bone marrow (BM-MSCs) and adipose tissue (ASCs) have the ability to undergo osteogenic, adipogenic and chondrogenic differentiation. Cytochemical staining and gene expression analysis show that ASCs have a greater capacity for adipogenic differentiation compared to BM-MSCs and fibroblasts. Different methods of inducing adipogenesis also affect the extent and profile of adipogenic differentiation in MSCs. Goat fibroblasts were not capable of osteogenesis, hence distinguishing them from the MSCs. Goat MSCs and fibroblasts express CD90, CD105, CD73 but not CD45, and exhibit cytoplasmic localization of OCT4 protein. Goat MSCs can be stably transfected by Nucleofection, but, as evidenced by colony-forming efficiency (CFE), yield significantly different levels of progenitor cells that are robust enough to proliferate into colonies of integrants following G418 selection. BM-MSCs expanded over increasing passages in vitro maintained karyotypic stability up to 20 passages in culture, exhibited an increase in adipogenic differentiation and CFE, but showed altered morphology and amenability to genetic modification by selection.
Our findings provide characterization information on goat MSCs, and show that there can be significant differences between MSCs isolated from different tissues and from within the same tissue. Fibroblasts do not exhibit trilineage differentiation potential at the same capacity as MSCs, making it a more reliable method for distinguishing MSCs from fibroblasts, compared to cell surface marker expression.
Electronic supplementary material
The online version of this article (doi:10.1186/2049-1891-6-1) contains supplementary material, which is available to authorized users.
Adipose; Bone marrow; Characterization; Differentiation; Goat; Mesenchymal stem cells
Cell lines from Atlantic salmon kidney have made it possible to culture and study infectious salmon anemia virus (ISAV), an aquatic orthomyxovirus affecting farmed Atlantic salmon. However, transfection of these cells using calcium phosphate precipitation or lipid-based reagents shows very low transfection efficiency. The Amaxa Nucleofector technology™ is an electroporation technique that has been shown to be efficient for gene transfer into primary cells and hard to transfect cell lines.
Here we demonstrate, enhanced transfection of the head kidney cell line, TO, from Atlantic salmon using nucleofection and subsequent flow cytometry. Depending on the plasmid promoter, TO cells could be transfected transiently with an efficiency ranging from 11.6% to 90.8% with good viability, using Amaxa's cell line nucleofector solution T and program T-20. A kill curve was performed to investigate the most potent antibiotic for selection of transformed cells, and we found that blasticidin and puromycin were the most efficient for selection of TO cells.
The results show that nucleofection is an efficient way of gene transfer into Atlantic salmon cells and that stably transfected cells can be selected with blasticidin or puromycin.
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.
PCR amplified gene; Cell viability; Nucleofection; Plasmid; Transfection efficiency