Transfection of primary human skin fibroblasts by electroporation

Primary human skin fibroblasts are an accessible source of phenotypically and karyotypically normal human cells, but are difficult to transfect with exogenous DNA. Here we demonstrate that both transient expression and stable transformation can be carried out by the method of electroporation. Highly efficient transient chloramphenicol acetyltransferase expression was shown after transfection with plasmid pRSVCAT. Stable transformation of human skin fibroblasts to G418 resistance was obtained after electroporation with neo-containing plasmids at an efficiency of approximately 1.4 x 10(-5)/micrograms DNA. The ability to easily transfect these cells with exogenous DNA may have important applications in the study of human genetic diseases and cancer.     

mRNA Transfection of Mouse and Human Neural Stem Cell Cultures

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.     

Electroporation-mediated transfection of mammalian cells with crude plasmid DNA preparations

We designed a simple and reproducible electroporation-mediated transfection procedure with which to screen mammalian expression vector-constructed cDNA libraries. Using a specific chamber composed of five parallel electrodes, the recipient cells can be electroporated separately with 40 plasmid DNA preparations in a single experiment. Over 300 crude plasmids prepared from E. coli (DH-5) carrying a pcD2neo-vector-derived cDNA library were tested. The efficiency of stable transfection by electroporation with crude plasmid DNA preparations was 10-times higher than with the CsCl-purified plasmid DNA. When the crude plasmids were digested with RNase, the efficiency of stable transfection markedly decreased, indicating that the contaminating bacterial RNA in the crude plasmid preparations has a strong carrier effect during the electroporation. Even when salmon sperm DNA or genomic DNA from the recipient cells was used as the carrier of the purified plasmid, the efficiency was not higher than that using the crude preparations. This procedure is useful not only for screening a number of cDNAs but also for routinely introducing biologically active foreign genes into cultured mammalian cells.     

Optimizing electroporation parameters for a variety of human hematopoietic cell lines

The parameters affecting electroporation of four human hematopoietic cell lines were investigated. The optimal conditions for electroporation are described for both transient and stable expression of foreign genes. A correlation exists between the levels of transient gene expression and stable transfection frequency. In addition, linear DNA yields higher stable transfection frequencies than supercoiled DNA. The cumulative results indicate that electroporation is a simple and useful method for obtaining transient and stable expression of foreign genes in human hematopoietic cells.     

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

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

In vivo plasmid DNA electroporation resulted in transfection of satellite cells and lasting transgene expression in regenerated muscle fibers

In vivo plasmid DNA electroporation resulted in elevated and lasting transgene expression in skeletal muscles. But the nature of the cells that contributed to sustained gene expression remains unknown. We followed the fate of plasmid DNA delivered with electroporation and systematically investigated the time course and location of transgene expression in muscle tissues both with GFP and luciferase. Furthermore, satellite cell activation after electroporation was confirmed by RT-PCR and immunohistochemistry analysis. The activated satellite cells were shown to be able to uptake the injected plasmid DNA and express transgene products as regenerated myocytes. We found that cells with longer gene expression durations were mostly regenerated muscle fibers. In contrast, expression in pre-existing muscle fibers was rather transient. We also presented in this study that immune response to transgene products might hamper the lasting gene expression. Based on these observations, we proposed that the underlying mechanism for prolonged transgene expression in the muscles after electroporation is related to the activation and transfection of myogenic satellite cells which subsequently developed into regenerated muscle fibers.     

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

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

Transfection of wild-type and 'Fanconi anemia-like' mouse lymphoma mutant cells by electroporation

An electroporation protocol for the successful transfection of mouse lymphoblastoid cells has been developed. Two cell lines, a normal and a mutant sensitive to DNA cross-linking agents, were used. The optimum conditions of electroporation in terms of uptake of the fluorescent dye lucifer yellow coupled with low toxicity were established. Subsequently, these conditions were used to achieve stable transfection by a plasmid expression vector. The plasmid integration patterns were determined by Southern blot analysis.     

Activated v-myc and v-ras oncogenes do not transform normal human lymphocytes.

Activated v-myc (pSV v-myc) and v-Ha-ras (GT10) oncogenes were introduced into normal human lymphocytes, NIH 3T3 fibroblasts, B-lymphoblastoid cells, and human epithelial cells, using a reconstituted Sendai virus envelope-mediated gene transfer technique. Efficient transfer of the plasmid in each cell type was demonstrable within 1.5 h of transfection by Southern blotting of extrachromosomal DNA extracts, which unexpectedly revealed that v-myc plasmid DNA was unstable in normal lymphocytes but not in the other cell types. The v-myc plasmid was stabilized when cotransfected into lymphocytes together with v-Ha-ras. The transfected v-Ha-ras plasmid was stable in all the cell types tested. v-myc plasmid expression was clearly detectable by 5 h in all cell types except human lymphocytes. Lymphocytes expressed v-myc when transfected together with v-Ha-ras. Transfected ras oncogene was efficiently expressed in all the cell types tested. Expression of the transfected genes increased at 24 and 48 h after transfection. Even though plasmid stability and expression were achieved in myc-ras-cotransfected lymphocytes, no effects on cellular DNA synthesis or immortalization were observed, in contrast to efficient transformation of NIH 3T3 fibroblasts by the same procedure. Our data suggest that efficient expression of transfected myc and ras oncogenes in normal quiescent human lymphocytes is not sufficient for the induction of cell growth and immortalization.     

Transient expression of the CD2 cell surface antigen as a sortable marker to monitor high frequency transfection of human primary B cells.

We have used the T cell surface molecule CD2 gene, expressed from the human cytomegalovirus promoter as a reporter to optimize a transfection system for human primary B cells. The CD2-encoding DNA was transfected into cells by electroporation and transient expression was monitored by flow cytometric analysis. By using our optimal electroporation conditions on activated primary B cells, more than 30% of the resulting viable cells expressed CD2 on the cell surface. Moreover, unactivated primary B cells could also be transfected using this system but subsequent expression of CD2 required cellular activation. Magnetic beads or plastic culture bottles coated with anti-CD2 antibodies have been used to selectively purify transfected cells. The high transfection efficiency combined with the ability to specifically purify transfected cells may allow future studies on specific genes transiently expressed in human primary B cells.     

Transfection of a DNA/protein complex into nuclei of mammalian cells using polyoma capsids and electroporation

We used empty capsids ofpolyoma virus to transfer DNA fragments and DNA/protein complexes into human cells. We encapsulated labeled and unlabeled single stranded DNA fragments by viral capsids. A complex of DNA with a DNA binding protein, recA, will also be taken up by the capsids, whereas the free protein is not incorporated. We further compared this gentle biological method of DNA transfection with a well-established physical method, electroporation. Electroporation also allows the transfer of DNA as well as protein into cells, although there is no proof that a DNA/protein complex can survive the procedure functionally. Whereas the viability of capsid transfected cells is unaffected (100%), electroporation reduces the viability to 90–95%. On the other hand, the amount of DNA found in the nucleus of electroporated cells is higher than for cells treated with loaded viral capsids.     

In Vitro Targeted Gene Electrotransfer to Endothelial Cells with Plasmid DNA Containing Human Endothelin-1 Promoter

Development of recombinant DNA technologies has allowed us to create new delivery systems that target specific cell types and that can be used in gene therapy. One of these targets is vascular endothelium because of its important role in tumor angiogenesis. For tumor endothelium-specific targeting, we prepared plasmid DNA encoding green fluorescent protein under the control of human endothelin-1 promoter (pENDO-EGFP), which is specific for endothelial cells. First we determined gene electrotransfer parameters for improved transfection of endothelial cells evaluating different osmolarity of electroporation buffer, voltages of applied electric pulses, and addition of fetal bovine serum immediately after electroporation to the cells for improved transfection and survival. Transfection efficacy of pENDO-EGFP in different endothelial and nonendothelial cell lines was determined next. Gene electrotransfer efficacy was evaluated using three different methods: fluorescence microscopy, fluorescence microplate reader, and flow cytometry. Our results showed that transfection efficacy was higher when cells were prepared in hypoosmolar compared to isoosmolar electroporation buffer. Furthermore, immediate addition of fetal bovine serum to the cells after pulsing also improved gene electrotransfer into target cells. We proved expression of EGFP under the control of human endothelin-1 promoter in endothelial cells, which was also significantly higher compared to nonendothelial cells. Taken together, we successfully constructed pENDO-EGFP, which was specifically expressed in endothelial cells using improved gene electrotransfer parameters.     

Introduction of stable high-copy-number DNA into Chinese hamster ovary cells by electroporation

A new gene transfer protocol has been developed that introduces up to 800 copies of an expression vector into Chinese hamster ovary cells in a single step by electroporation. The DNA typically integrates in tandem repeats so that the restriction endonuclease site used to linearize the input DNA remains intact. This is likely due to ligation of vector DNA via cohesive ends prior to integration. This high-copy-number procedure is far more rapid than the conventional stepwise gene amplification method used to generate stable eukaryotic protein production cell lines. By employing the expression vector pJODtPA, in which the selectable marker dihydrofolate reductase (DHFR) and the human tissue plasminogen activator (tPA) casettes are separated by a spacer and an RNA polymerase II terminator, cell lines secreting as much as 24 pg/cell.day tPA were isolated following electroporation and a single methotrexate selection. Gene copies and expression levels are stable over long periods of growth. A single round of gene amplification was performed following the high-copy-number procedure to yield a clone having a tPA production level of 45 pg/cell.day.     

Optimization of transient gene expression in mammalian cells and potential for scale-up using flow electroporation

The goals of this study were to identify mammalian cell lines which could be efficiently transiently-transfected and scaled-up for protein production. The transfection efficiencies of eight cell lines (NSO, NSO-TAg, CV-1, COS-7, CHO, CHO-TAg, HEK 293, and 293-EBNA) were measured using electroporation for DNA delivery and green fluorescent protein (Evans, 1996) as the reporter gene. In addition, we have evaluated the effects of stable expression of viral proteins, cell cycle manipulation, and butyrate post-treatment in small scale experiments. The cell lines varied widely in their GFP transfection efficiencies. Stable expression of simian virus 40 large T-antigen or Epstein Barr nuclear antigen failed to significantly increase transfection efficiency above that seen in the parental lines. Aphidicolin (a DNA polymerase inhibitor), which blocked cells from S or G2/M, brought about an increase in transfection efficiency in two cell lines. The primary effect of butyrate (a histone deacetylase inhibitor) post-treatment was an increased intensity of the fluorescent signal of green fluorescent protein, as measured by flow cytometry (1.0 to 4.2-fold, depending on the cell line). The combined use of aphidicolin pretreatment followed by butyrate treatment post- electroporation yielded increases in fluorescence intensities ranging from 0.9 to 6.8-fold. Based on their high transfection efficiencies in small scale experiments, rapid growth, and ability to grow in suspension culture, CHO, CHO-TAg, and 293-EBNA were selected to assess the feasibility of using flow electroporation for large-scale transfections. Using secreted placental alkaline phosphatase as a reporter, 293-EBNA cells produced the highest protein levels in both the presence and absence of butyrate. These data indicate that flow electroporation provides an efficient method of DNA delivery into large numbers of cells for mammalian protein production. This revised version was published online in August 2006 with corrections to the Cover Date.     

Optimization of electroporation for transfection of human fibroblast cell lines with origin-defective SV40 DNA: development of human transformed fibroblast cell lines with mucopolysaccharidoses (I-VII)

To simplify the process of transfection of human fibroblasts and to acquire a suitable number of transformants, we investigated experimental conditions of electric pulse-induced transfection of human fibroblasts using origin-defective simian virus 40 DNA (SV40 (ori-) DNA). Voltage, pulse duration, number of pulses and the concentration of SV40 (ori-) DNA led to the formation of 10 to 30 foci/25 cm2 6 weeks after transfection, using 2 to 3 x 10(6) cells and a square wave pulse generator. Optimal condition was determined to be 2 or 3 pulses at a voltage of 1500 to 2000 V/0.4 cm with 30 microseconds pulse width, using 2 micrograms of linearized SV40 (ori-) DNA. With this approach we developed human transformed fibroblasts cell lines with all types of mucopolysaccharidoses. The transformed fibroblasts grew rapidly and the saturation density exceeded that of the parental cells. All the transformed cell clones expressed T antigen, and deficiency in specific enzymes was conserved. A point mutation which occurred in the human beta-glucuronidase gene in a patient with mucopolysaccharidosis type VII was also conserved.     

Electroporation of human embryonic stem cells: Small and macromolecule loading and DNA transfection

Cryopreservation, directed differentiation, and genetic manipulation of human embryonic stem cells (hESCs) all require the transport of exogenous small molecules, proteins, or DNA into the cell. The absence of standard small and macromolecule loading techniques in hESCs as well as the inadequacies of current DNA transfection techniques have led us to develop electroporation as an efficient loading and transfection methodology. The electroporation parameters of pulse voltage, duration, and number have been explored and evaluated in terms of cell viability, molecular loading, and transfection efficiency on a per cell basis. Small molecule loading was assessed using propidium iodide (PI) and the disaccharide trehalose. Additionally, protein loading was investigated using a glutathione-S-transferase green fluorescent protein (GST-GFP) conjugate, and DNA transfection optimization was performed by constitutive expression of GFP from a plasmid. The optimum pulse voltage must balance cell viability, which decreases as voltage increases, and loading efficiency, which increases at higher voltages. Short pulse times of 0.05 ms facilitated PI and trehalose loading, whereas 0.5 ms or more was required for GST-GFP loading and DNA transfection. Multiple pulses increased per cell loading of all molecules, though there was a dramatic loss of viability with GST-GFP loading and DNA transfection, likely resulting from the longer pulse duration required to load these molecules.     

An improved method of electroporation for introducing biologically active foreign genes into cultured mammalian cells

We have developed a modified, reproducible, and efficient method for introducing cloned genes into mammalian cells by using an electric field followed by treatment with sodium butyrate. Transfection frequencies with plasmid pSV2-neo, consisting of an antibiotic (G418) resistance gene and simian virus 40 (SV40) early promoter, by electroporation were higher than those by calcium phosphate DNA precipitation. Treatment with sodium butyrate following electroporation significantly increased the frequency of transfection in various types of cell lines and primary cultured cells including human skin fibroblasts. Treatment with sodium butyrate also increased the transient expression of the gene for chloramphenicol acetyltransferase (acetyl-CoA; chloramphenicol O3-acetyltransferase, CAT, EC 2.3.1.28) when the gene was introduced into BALB/c 3T3 cells by electroporation. Electroporation combined with sodium butyrate treatment is an improved method for stable and transient biochemical transformation of foreign genes in cultured mammalian cells.     

DNA对电穿孔转染效率的影响

以外源红细胞生成素cDNA的表达产物为指标,研究了运载DNA和重组表达质粒的构象对电穿孔转染CHO细胞的效率的影响.结果250mg/L的运载DNA可使外源基因表达水平提高3倍;线性化质粒DNA比超螺旋DNA更适合于用电穿孔方法获得永久表达.这一结果提示,运载DNA的存在和质粒DNA的线性化对提高电穿孔转染CHO细胞的效率是必须的.     

The induction of growth arrest in fibroblasts by SV40 T antigen

DNA tumor viruses such as SV40, Ras and papillomaviruses are the most commonly used agents in immortalization of non-hematopoietic cells, but the results are quite different. Some of them even lead instead to a senescence-like state. To verify the potential of SV40 T antigen-mediated immortalization or properties and functions of it to regulate cell growth, human dermal fibroblasts were cultured and then transfected with eukaryotic expressing plasmid psv3-neo which containing SV40 T DNA. We found that expression of oncogenic SV40 T in human dermal fibroblasts resulted in growth, arrest, earlier than the occurrence of control cell senescence, although telomerase was positive and cells grew faster than control ones in early stage following transfection. These observations suggest that SV40 T antigen can activate growth arrest in human dermal fibroblasts under normal growth condition instead of always prolonging the lifespan of fibroblasts. Moreover, high rate of cell division in early stage after transfection may be associated with the expression of telomerase activity.     

High-efficiency gene transfection by in situ electroporation of cultured cells

It is demonstrated in this study that high-efficiency gene transfection can be obtained by directly electroporating cultured mammalian cells in their attached state using a pulsed radio-frequency (RF) electric field. A plasmid DNA containing the reporter gene beta-gal was introduced into COS-M6 cells and CV-1 cells using this in situ electroporation method. At the optimal electric field strength (1.2 kV/cm), we found that over 80% of the M6 cells took up and expressed the beta-gal gene with a cell survival rate of about 50%. In contrast, the transfection efficiency was less than 20% when the M6 cells were electroporated in suspension. It was shown that CV-1 cells could also be electroporated highly efficiently using the in situ method. Furthermore, we have measured the time required to express the beta-gal gene after the plasmid DNA was introduced. We found that the percentage of cells expressing beta-gal reached a peak value about 10 h after electroporation. This time-course was the same for both attached and suspended cells, suggesting that the observed difference in transfection efficiency was mainly the result of effects of the detachment treatment on the electroporation process rather than on the gene expression.