Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis.

Simian Cos-1 cells were transfected electrically with the plasmid pCH110 carrying the beta-galactosidase gene. The efficiency of transfection was determined by a transient expression of this gene. When the plasmid was introduced into a cell suspension 2 s after pulse application, the transfection efficiency was shown to be less than 1% as compared with a prepulse addition of DNA. Addition of DNAase to suspension immediately after a pulse did not decrease transfection efficiency, thus the time of DNA translocation was estimated to be less than 3 s. The use of electric treatment medium, in which the postpulse colloid-osmotic cell swelling was prevented, did not affect the transfection efficiency. These results contradict both assumptions of free DNA diffusion into cell through the long-lived pores and of involvement of osmotic effects in DNA translocation. Transfection of cells in monolayer on a porous film allowed creation of the spatial asymmetry of cell-plasmid interaction along the direction of electric field applied. A pulse with a polarity inducing DNA electrophoresis toward the cells resulted in the 10-fold excess of transfection efficiency compared with a pulse with reverse polarity. Ficoll (10%) which increases medium viscosity or Mg2+ ions (10 mM) which decrease the effective charge of DNA, both reduced transfection efficiency 2-3-fold. These results prove a significant role of DNA electrophoresis in the phenomenon considered. The permeability of cell membranes for an indifferent dye was shown to increase noticeably if the cells were pulsed in the presence of DNA. This indicates a possible interaction of DNA translocated with the pores in an electric field, that results in pore expansion.     

Enhancement of transient gene expression by fed-batch culture of HEK 293 EBNA1 cells in suspension

Enhanced green fluorescence protein (GFP) and erythropoietin (EPO) were used as reporters to assess and improve transient gene expression in HEK 293 EBNA1 cells. The production of EPO only lasted 3 days and reached 18.1 mg/l in suspension cultures in 1 l batch bioreactors. However, GFP expression examined in well-plate experiments persisted for 12 days in transfected cells but decreased rapidly within the next 15 days. These results suggest that the retaining of a plasmid in cells may not be a limiting factor for protein expression in large-scale transient transfection. To improve cell maintenance and protein expression, a fed-batch culture was performed using an enriched medium, a mixture of equal volumes of 293 SFM II medium and a 5 × amino acid solution prepared based on DMEM/F12 medium formula. EPO reached 33.6 mg/l, representing 86% increase over that of the batch culture. Moreover, the total amount of EPO produced was increased by 165% in view of the volume increase in the fed-batch culture. The serum-free medium used in this work enables cells growing well and transfection without medium change. Thus, the process reported here is simple and easy to scale up.     

Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes.

A method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.     

High yield electroextraction of proteins from yeast by a flow process

High yields of intracellular enzymes from yeast can be obtained by application of a series of electric field pulses with a flow process. Up to 80-90% of the total activity can be liberated without any further or previous treatment of cells. The method is based on electroinduced changes in the cell envelope leading to a leakage of part of the intracellular proteins without formation of debris and permits treatment of large volumes. Field parameters require a limited electrical power. Treatment of at least 20% wet weight suspensions is possible. The optimal field conditions must be adjusted to the suspension concentration. Maximal yield is obtained within 4h at 30 degrees C for enzymes from Saccharomyces cerevisiae such as hexokinase, 3-phosphoglycerate kinase, and glyceraldehyde-3-phosphate dehydrogenase. The extraction of beta-D-galactosidase from Kluyveromyces lactis lasts 10h but can be accelerated by adding dithiothreitol in the postpulse medium. The specific activities of the electroextracted enzymes are higher than those obtained by mechanical disintegration or enzymatic lysis.     

Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single‐cell bioluminescence imaging

The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single‐cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single‐cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay. Copyright © 2013 John Wiley & Sons, Ltd.     

Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.     

Epi-CHO, an episomal expression system for recombinant protein production in CHO cells

This study describes the development of a transient expression system for CHO cells based on autonomous replication and retention of transfected plasmid DNA. A transient expression system that allows extrachromosomal amplification of plasmids permits more plasmid copies to persist in the transfected cell throughout the production phase leading to a significant increase in transgene expression. The expression system, named Epi-CHO comprises (1) a CHO-K1 cell line stably transfected with the Polyomavirus (Py) large T (LT) antigen gene (PyLT) and (2) a DNA expression vector, pPyEBV encoding the Py origin (PyOri) for autonomous plasmid amplification and encoding Epstein-Barr Virus (EBV) nuclear antigen-1 (EBNA-1) and OriP for plasmid retention. The CHO-K1 cell line expressing PyLT, named CHO-T was adapted to suspension growth in serum-free media to facilitate large-scale transient transfection and recombinant gene expression. Enhanced green fluorescent protein (EGFP) and human growth hormone (hGH) were used as reporter proteins to demonstrate transgene expression and productivity. Transfection of suspension-growing CHO-T cells with the vector pPyEBV encoding hGH resulted in a final concentration of 75 mg L(-1) of hGH in culture supernatants 11 days following transfection.     

Purification of transiently transfected cells by magnetic affinity cell sorting

A method was developed to purify transiently transfected HeLa cells or African green monkey kidney CV-1 cells by magnetic affinity cell sorting. Monolayer cultures were transfected with mammalian expression vectors coding for either of two novel cell surface antigens, the Tac subunit of the human IL-2 receptor or vesicular stomatitis virus G protein. During the transient expression phase, cell populations were placed in suspension and mixed with monoclonal-antibody-coated magnetic particles in the presence of a sorting solution designed to minimize nonspecific cell/cell and cell/particle interactions. Transfected cells expressing the vector-encoded cell surface antigen were then isolated by application of a magnetic field. Reconstruction experiments indicated that IL-2 receptor-positive cells were bound about 100-fold more efficiently than receptor-negative cells. In transient transfection experiments, populations of greater than 90% antigen-positive cells were reproducibly obtained.     

Use of flow cytometry to rapidly optimize the transfection of animal cells

Plasmid transfection is the first step in the generation of stably transformed animal cells and is also a useful tool for analyzing transient gene expression. Maximizing the transfection efficiency and expression level from the introduced plasmid is critical to the success of these processes. By means of lipid-mediated transfection, a plasmid vector expressing the green fluorescence reporter protein has been coupled with flow cytometry to conveniently investigate those parameters that impact the efficacy of transfection of lepidopteran insect cells. The key feature of this technique is the rapid and simultaneous quantification of transfection efficiency and heterologous protein expression level per cell. Using this technique, we developed an optimized transfection protocol for insect cells by investigating the following parameters: lipid incubation time, lipid/DNA mixture incubation time, lipid and DNA concentration, incubation vessel and transfection duration. Following optimization, transfection efficiencies of 37%-40% were obtained for Bombyx mori Bm5 and Spodoptera frugiperda Sf-21 cells.     

Transient gene expression in mammalian cells grown in serum-free suspension culture

In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI) to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method). The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression levels. The precise ratio is dependent on the DNA concentration. For example, using 1 μg/ml DNA by the indirect method, the ratio of optimal PEI:DNA was about 10–13:1. However, the ratio increases to 33:1 for 0.1–0.2 μg/ml DNA. By testing several different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared to 1 μg/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1 (CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production of milligram amounts of recombinant proteins in 2–5 l spinner culture scale within 3–5 days. Fermentor scale experiments, however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

DNA transfection of mouse lymphoid cells by the combination of DEAE-dextran-mediated DNA uptake and osmotic shock procedure

Several mouse lymphoid cell lines were efficiently transfected with plasmid DNA by a novel method combining DEAE-dextran-mediated DNA uptake and osmotic shock procedure. The cells were first incubated with DNA-DEAE-dextran complex, treated with hypertonic Tris-HCl buffer containing 0.5 M sucrose and 10% poly(ethylene glycol), and then exposed to hypotonic RPMI 1640 medium. This transfection protocol exhibited maximal frequencies of 0.3% and 3.10(-5) for transient gene expression and stable transformation in P3-NSI/1-Ag4-1 cells, respectively.     

Demethylation using the epigenetic modifier, 5-azacytidine, increases the efficiency of transient transfection of macrophages

This study was aimed at developing a method for high-efficiency transient transfection of macrophages. Seven methods were evaluated for transient transfection of murine macrophage RAW 264.7 cells. The highest transfection efficiency was achieved with DEAE-dextran, although the proportion of cells expressing the reporter gene did not exceed 20%. It was subsequently found that the cytomegalovirus plasmid promoter in these cells becomes methylated. When cells were treated with the methylation inhibitor 5-azacytidine, methylation of the plasmid promoter was abolished and a dose-dependent stimulation of reporter gene expression was observed with expression achieved in more than 80% of cells. Treatment of cells with 5-azacytidine also caused increased efficiency of transfection of macrophages with plasmids driven by RSV, SV40, and EF-1alpha promoters and transient transfection of human HepG2 cells. Inhibition of methylation also increased the amount and activity of sterol 27-hydroxylase (CYP27A1) detected in RAW 264.7 cells transfected with a CYP27A1 expression plasmid. Treatment of cells with 5-azacytidine alone did not affect either cholesterol efflux from nontransfected cells or expression of ABCA1 and CYP27A1. However, transfection with CYP27A1 led to a 2- to 4-fold increase of cholesterol efflux. We conclude that treatment with 5-azacytidine can be used for high-efficiency transient transfection of macrophages.     

Analysis with flow cytometry of green fluorescent protein expression in leukemic cells.

BACKGROUND: The measurement of DNA content with propidium iodide (PI) in cells transfected with expression vectors encoding the green fluorescent protein (GFP) is a useful tool in studying a variety of biological functions of proteins within cells. The purpose of this study was to determine conditions of formaldehyde fixation that permit intracellular GFP fluorescence and adequate DNA histograms to be generated following transient transfection of cells with a GFP-encoding plasmid. Cell cycle analysis was also performed in GFP-positive cells. METHODS: The murine myeloid leukemic cell line, 32Dcl3, was used as the model system. Cells were transfected with a GFP-encoding plasmid (pEGFPC1). Following fixation in different formaldehyde concentrations and permeabilization with 70% ethanol, cells were stained with PI and analyzed by flow cytometry for GFP fluorescence and DNA content. Transfected cells were also analyzed for GFP fluorescence and DNA content following release from nocodazole block. RESULTS: Fixing cells in 0.51-1.75% formaldehyde concentrations prior to ethanol permeabilization resulted in 14-19% of transfected cells being GFP-positive, with acceptable coefficients of variation on the G(1) peak of DNA histograms. Analysis of cells synchronized to and released from the G(2)-M phase by nocodazole suggested that GFP-positive cells, when compared to GFP-negative cells, did not appear to progress out of G(2)-M following release from nocodazole block. Simultaneous detection of GFP fluorescence and DNA content by PI staining is possible following transient transfection of cells with a single expression vector encoding GFP. Our results demonstrate that GFP expression can be detected, using flow cytometry to perform cell cycle analysis in murine leukemic cells.     

Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.

The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed.     

Detection by flow cytometry of protoplast fusion and transient expression of transferred heterologous CD4 sequences in COS-7 cells

Transfer and expression of a plasmid containing the gene encoding the human T-cell antigen CD4 by protoplast fusion was measured by flow cytometry (FCM). Protoplasts were prelabeled with fluorescein isothiocyanate (FITC) and fused to COS-7 cells. Nonspecific protoplast adsorption to the plasma membrane was differentiated from successful protoplast fusion by the addition of an antibody directed against fluorescein to quench extracellular protoplast fluorescence. Transfection efficiencies were defined as both percent CD4 expressing cells and CD4 expression levels on a single cell basis in the transient immunofluorescence assay. Cell sorting studies indicated that intracellular protoplast-associated fluorescence immediately after fusion exhibited a good correlation with transient CD4 transfection efficiencies as measured by indirect immunofluorescence. Reconstruction experiments comparing CD4 transfer efficiencies of protoplast fusion and calcium phosphate transfection showed that fusion resulted in a higher percentage of CD4 expressing transfectants, while calcium phosphate transfection yielded higher CD4 expression levels on a single cell basis. Thus, FCM appears to be useful as a new tool for sensitive detection of transient expression of heterologous reporter genes in COS-7 cells.     

High level transient gene expression in human lymphoid cells by SV40 large T antigen boost.

High level transient gene expression in lymphoid cells has always been challenging because of the difficulty to efficiently transfect such cells. This has precluded any attempt to clone cDNA encoding proteins by means of their specific biological function in lymphoid cells. We have developed a very efficient transient eukaryotic expression system analogous to the well-known expression system in COS cells. Firefly luciferase and human CD2 genes were used as reporter genes and cloned into the eukaryotic shuttle vector pCDM8 which contains the strong cytomegalovirus promoter and the SV40 origin of replication for autonomous plasmid replication in permissive host cells that express the large SV40 T Antigen. Co-transfection of the reporter plasmids together with an SV40 T Ag expressing plasmid resulted in the several fold amplification of either the Luc activity or the cell surface expression of the CD2 marker in a transient assay. The level of amplification was dependent on the strength of the promoter used to drive the SV40 T Ag expression and was correlated with the extent of autonomous replication of the reporter plasmid in transfected cells. This highly efficient transient gene expression by SV40 T Ag boost was suitable to several human cell lines, making this system of general interest for expression cloning strategies or other gene transfer application that need high level expression.     

Optimization of electroporation for transfection of mammalian cell lines

Electroporation can be a highly efficient method for introducing DNA molecules into cultured cells for transient expression of genes or for permanent genetic modification. However, effective transformation by electroporation requires careful optimization of electric field strength and pulse characteristics. We have used the transient expression of the firefly luciferase gene as a rapid and sensitive indicator of gene expression to describe the effects on transfection efficiency of altering electroporation field strength and shape. Using the luciferase assay, we investigated the correlation of cell viability with optimal transfection efficiency and determined the optimal parameters for a number of phenotypically distinct mammalian cell lines derived from the nervous and immune systems. The efficiency of electroporation under optimal conditions was compared with that obtained using DEAE-dextran or calcium phosphate-mediated transformation. Transfection by electroporation using square wave pulses, as opposed to exponentially decaying pulses, was found to be significantly increased by repetitive pulses. These methods improve the ability to obtain high efficiency gene transfer into many mammalian cell types.     

Optimizing the generation of stable neuronal cell lines via pre-transfection restriction enzyme digestion of plasmid DNA

Transfection of mammalian cell lines is a widely used technique that requires significant optimization, including transfection method or product used, DNA vector, cell density, media composition and incubation time. Generation and isolation of stable transfectants from the large pool of untransfected or only transiently transfected cells can be laborious and time-consuming. Transfection of DNA is usually performed with a non-linearized plasmid, since it is assumed that cutting the plasmid beforehand leads to a lower efficiency of transfection or the degradation of linearized DNA by cytosolic nucleases. However, the transfected circular plasmid will be linearized by a random cut within the cell and it might be possible that sensitive parts of the plasmid such as the resistance gene or the gene of interest are destroyed upon linearization. On the other hand, linearizing a plasmid before transfection by a single, defined cut with a selected restriction enzyme in a non-coding area of the gene has the advantage of ensuring the integrity of all necessary gene elements of the plasmid. In this study, we have compared these different methods in order to increase both transient and stable transfection efficiency in mammalian cells. We report that linearization of plasmid DNA prior to transfection can increase both the efficiency of stable clone generation and target gene expression, but is dependant on the site of linearization within the vector.     

A Double-Pulse Approach For Electrotransfection

Gene transfer and expression can be obtained by delivering calibrated electric pulses on cells in the presence of plasmids coding for the activity of interest. The electric treatment affects the plasma membrane and induces the formation of a transient complex between nucleic acids and the plasma membrane. It results in a delivery of the plasmid in the cytoplasm. Expression is only obtained if the plasmid is translocated inside the nucleus. This is a key limit in the process. We previously showed that delivery of a high-field short-duration electric pulse was inducing a structural alteration of the nuclear envelope. This study investigates if the double-pulse approach (first pulse to transfer the plasmid to the cytoplasm, and second pulse to induce the structural alteration of the envelope) was a way to enhance the protein expression using the green fluorescent protein as a reporter. We observed that not only the double-pulse approach induced the transfection of a lower number of cells but moreover, these transfected cells were less fluorescent than the cells treated only with the first pulse.