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
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.
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
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.
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.
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.
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
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
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.
Nucleofection permits efficient transfection even with difficult cell types such as primary and non-dividing cells, and is used to deliver various nucleic acids including DNA, mRNA, and siRNA. Unlike DNA and siRNA, mRNA is subject to rapid degradation, which necessitates instant early translation following mRNA delivery. We examined factors important in translation following nucleofection and observed rapid phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) following nucleofection, which occurred in the absence of delivered nucleic acid. We studied the involvement of 3 ubiquitous kinases capable of phosphorylating eIF2α in mammalian cells and identified that nucleofection-mediated phosphorylation of eIF2α was dependent on general control non-derepressible 2 (GCN2) and protein kinase RNA-activated (PKR)-like ER kinase (PERK) but not PKR. A reduction in translation due to eIF2α phosphorylation was observed post nucleofection demonstrating functional significance. Understanding the impact of nucleofection on translational machinery has important implications for therapeutics currently under development based on the delivery of mRNA, DNA, and siRNA. Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies.
Nucleofection; mRNA therapy; eIF2α; PERK; GCN2
Germline stem cells (GSCs) can be used for large-animal transgenesis, in which GSCs that are genetically manipulated in vitro are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objectives of this study were to explore a non-viral approach for transgene delivery into goat GSCs and to investigate the efficiency of nucleofection in producing transgenic sperm. Four recipient goats received fractionated irradiation at 8 weeks of age to deplete endogenous GSCs. Germ-cell transplantations were performed 8-9 weeks post-irradiation. Donor cells were collected from testes of 9 week-old goats, enriched for GSCs by Staput velocity sedimentation, and transfected by nucleofection with a transgene construct harboring the human growth hormone gene under the control of the goat beta-casein promoter (GBC) and a chicken beta-globin insulator (CBGI) sequence upstream of the promoter. For each recipient, transfected cells from 10 nucleofection reactions were pooled, mixed with non-transfected cells to a total of 1.5×108 cells in 3ml, and transplanted into one testis (n = 4 recipients) by ultrasound-guided cannulation of the rete testis. The second testis of each recipient was removed. Semen was collected starting at 9 months after transplantation for a period of over a year (a total of 62 ejaculates from 4 recipients). Nested genomic PCR for hGH and CBGI sequences demonstrated that 31.3%±12.6% of ejaculates were positive for both hGH and CBGI. This study provides proof-of-concept that non-viral transfection (nucleofection) of primary goat germ cells followed by germ cell transplantation results in transgene transmission to sperm in recipient goats.
Transgenic animals; germline stem cells; spermatogenesis; nucleofection; germ cell transplantation
The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.
Here, we report the development of a fully defined xeno-free medium (RegES), capable of supporting the expansion of human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and adipose stem cells (ASC). We describe the use of the xeno-free medium in the derivation and long-term (>80 passages) culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS), while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.
Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific applications relating to establishment, expansion and differentiation of various stem cell types.
Neural stem cells (NSCs) have the ability to proliferate and differentiate into various types of cells that compose the nervous system. To study functions of genes in stem cell biology, genes or siRNAs need to be transfected. However, it is difficult to transfect ectopic genes into NSCs. Thus to identify the suitable method to achieve high transfection efficiency, we compared lipid transfection, electroporation, nucleofection and retroviral transduction. Among the methods that we tested, we found that nucleofection and retroviral transduction showed significantly increased transfection efficiency. In addition, with retroviral transduction of Ngn2 that is known to induce neurogenesis in various types of cells, we observed facilitated final cell division in rat NSCs. These data suggest that nucleofection and retroviral transduction provide high efficiency of gene delivery system to study functions of genes in rat NSCs.
Electroporation; Lipid-Mediated transfection; Neural stem cells; Nucleofection; Retrovirus
The mouse retina constitutes an important research model for studies aiming to unravel the cellular and molecular mechanisms underlying ocular diseases. The accessibility of this tissue and its feasibility to directly obtain neurons from it has increased the number of studies culturing mouse retina, mainly retinal cell suspensions. However, to address many questions concerning retinal diseases and protein function, the organotypic structure must be maintained, so it becomes important to devise methods to transfect and culture whole retinas without disturbing their cellular structure. Moreover, the postmitotic stage of retinal neurons makes them reluctant to commonly used transfection techniques. For this purpose some published methods employ in vivo virus-based transfection techniques or biolistics, methods that present some constraints. Here we report for the first time a method to transfect P15-P20 whole murine retinas via nucleofection, where nucleic acids are directly delivered to the cell nuclei, allowing in vitro transfection of postmitotic cells. A detailed protocol for successful retina extraction, organotypic culture, nucleofection, histological procedures and imaging is described. In our hands the A-33 nucleofector program shows the highest transfection efficiency. Whole flat-mount retinas and cryosections from transfected retinas were imaged by epifluorescence and confocal microscopy, showing that not only cells located in the outermost retinal layers, but also those in inner retinal layers are transfected. In conclusion, we present a novel method to successfully transfect postnatal whole murine retina via nucleofection, showing that retina can be successfully nucleofected after some optimization steps.
Retina; Mice; Nucleofection; Organotypic culture
Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.
Mesenchymal stem cells (MSCs) are multipotent nonhematopoietic cells with the ability to differentiate into various specific cell types, thus holding great promise for regenerative medicine. Early clinical trials have proven that MSC-based therapy is safe, with possible efficacy in various diseased states. Moreover, genetic modification of MSCs to improve their function can be safely achieved using electrogene transfer. We previously achieved transfection efficiencies of up to 32% with preserved viability in rat MSCs. In this study, we further improved the transfection efficiency and transgene expression in human MSCs (hMSCs), while preserving the cells viability and ability to differentiate into osteoblasts and adipocytes by increasing the plasmid concentration and altering the osmotic pressure of the electrotransfer buffer. Using a square-wave electric pulse generator, we achieved a transfection efficiency of more than 80%, with around 70% viability and a detectable transgene expression of up to 30 days. Moreover, we demonstrated that this transfection efficiency can be reproduced reliably on two different sources of hMSCs: the bone marrow and adipose tissue. We also showed that there was no significant donor variability in terms of their transfection efficiency and viability. The cell confluency before electrotransfer had no significant effect on the transfection efficiency and viability. Cryopreservation of transfected cells maintained their transgene expression and viability upon thawing. In summary, we are reporting a robust, safe, and efficient protocol of electrotransfer for hMSCs with several practical suggestions for an optimal use of genetically engineered hMSCs for clinical application.
Liew and colleagues employ square-wave electric pulses to achieve efficient gene transfer in human mesenchymal stem cells (hMSCs). Robust gene transfer is reproducible across donors and with both bone marrow- and adipose-derived hMSCs. Transfected cells exhibit high viability, normal differentiation capacity, and durable transgene expression.
Human adult mesenchymal stem cells (hMSC) are under active investigation as cellular carriers for gene therapy. hMSC possess natural tropism toward tumors, however, the targeting of hMSC to specific cell populations within tumors is unexplored. In the case of glioblastoma multiforme (GBM), at least half of the tumors express EGFRvIII on the cell surface, an ideal target for antibody-mediated gene/drug delivery. In this study, we investigated the feasibility of genetically modifying hMSC to express a single-chain antibody (scFv) to EGFRvIII on their surface. Nucleofection was used to transfect hMSC with cDNA encoding scFv EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSC was selected based on antibiotic resistance and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSC. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly, human MSC expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells in vitro and at least 7-fold increased retention in human U87-EGFRvIII expressing tumors in vivo. In summary, we provide the first conclusive evidence of genetic modification of hMSC with a single-chain antibody against an antigen expressed on the surface of tumor cells, thereby opening up a new venue for enhanced delivery of gene therapy applications in the context of malignant brain cancer.
mesenchymal stem cells; single-chain antibody; surface; targeting; brain tumors; EGFRvIII
Nonviral gene delivery to human mesenchymal stem/stromal cells (MSC) can be considered a very promising strategy to improve their intrinsic features, amplifying the therapeutic potential of these cells for clinical applications. In this work, we performed a comprehensive comparison of liposome-mediated gene transfer efficiencies to MSC derived from different human sources—bone marrow (BM MSC), adipose tissue-derived cells (ASC), and umbilical cord matrix (UCM MSC). The results obtained using a green fluorescent protein (GFP)-encoding plasmid indicated that MSC isolated from BM and UCM are more amenable to genetic modification when compared to ASC as they exhibited superior levels of viable, GFP+ cells 48 hr post-transfection, 58±7.1% and 54±3.8%, respectively, versus 33±4.7%. For all cell sources, high cell recoveries (≈50%) and viabilities (>85%) were achieved, and the transgene expression was maintained for 10 days. Levels of plasmid DNA uptake, as well as kinetics of transgene expression and cellular division, were also determined. Importantly, modified cells were found to retain their characteristic immunophenotypic profile and multilineage differentiation capacity. By using the lipofection protocol optimized herein, we were able to maximize transfection efficiencies to human MSC (maximum of 74% total GFP+ cells) and show that lipofection is a promising transfection strategy for MSC genetic modification, especially when a transient expression of a therapeutic gene is required. Importantly, we also clearly demonstrated that intrinsic features of MSC from different sources should be taken into consideration when developing and optimizing strategies for MSC engineering with a therapeutic gene.
Boura and colleagues compare liposome-mediated gene transfer efficiencies to human mesenchymal stem cells (MSC) derived from bone marrow (BM MSC), adipose tissue (ASC), or umbilical cord matrix (UCM MSC). MSC isolated from BM and UCM were more amenable to genetic modification when compared to ASC. Modified cells from all sources maintained transgene expression for 10 days and retained their characteristic immunophenotypic profile and multilineage differentiation capacity.