Cellular transfection of small interfering ribonucleic acids. Electroporation as an alternative to liposome-based delivery transfection methods

RNA interference has become an increasingly important tool for all aspects of molecular biology. Nevertheless, there is a technological challenge for performing gene knock-down with chemical transfection agents, as many relevant cell types are refractory to efficient transport of short interfering RNA (siRNA) into cytosol using these classical carrier-based methods. The aim of this project was to evaluate the potential of electroporation as a generic transfection method and to compare it with a standard and well-established chemical reversetransfection using cationic lipids as carriers for short-interfering RNAs. We have used an automated cell electroporation instrument from a swedish company, Cellectricon. The Cellaxess HT instrument is able to perform electroporation in 384 wells plates. We compared cell viability, transfection efficiency and extend of gene silencing caused by electroporation, with reverse-transfection using Lipofectamine RNAiMAX. Gene knock down efficiency has been measured at the protein function level by targeting ubiquitously expressed genes that are essential to cell survival. Cell viability and transfection efficiency are, then, opposed with this strategy. Results clearly showed a net improvement of "difficult-to-transfect" cell transfection but this improvement comes at the price of consuming up to ten fold more siRNAs. For "normal" cell line, lipofection remains the preferred methodology as it is more efficient and less expensive. Furthermore, the Cellaxess HT instrument has some serious drawbacks that pervert easy and reproducible result acquisition.

The use of RNA interference to increase PEI-mediated transient gene expression in mammalian cells

Martin Bertschinger

DNA uptake by polyethylenimine (PEI)-mediated transfection was investigated in Chinese hamster ovary (CHO DG44) cells. Rapid DNA uptake was observed with the maximum occurring during the first 45-60 min after addition of PEI-DNA complexes to cells. With longer incubation times, the aggregation of PEI-DNA particles reduced the rate of DNA uptake. At early times after transfection, the kinetics of DNA uptake were constant, suggesting that the limiting factor to PEI transfection was the rate of endocytosis. The most efficient transfection with PEI was observed at a density of 2 x 106 cells/ml and at a PEI:DNA ratio of 2:1 (w/w). DNA uptake at higher PEI:DNA ratios was also efficient, but the toxicity of PEI decreased the recombinant protein yield. The optimal PEI:DNA ratio was found to be related to the number of cells in the culture. Once in the cell, PEI:DNA particles must be disassembled to allow transcription of the recombinant gene. Here their disassembly was investigated using a simple in vitro assay. The particles were formed with either a linear (25 kDa or JetPEITM) or a branched PEI (2, 25, or 750 kDa) and then mixed with varying amounts of a competitor (RNA, DNA, or heparin). The amount of plasmid DNA released from particles was determined by agarose gel electrophoresis or spectroscopy. The stability of the particles to changes in pH, osmolarity, and the PEI:DNA ratio was also determined. Either the presence of heparin or high salt concentrations (1-2 M) yielded complete particle disassembly for all PEIs tested. The addition of RNA to particles formed with linear PEIs or branched 2 kDa PEI resulted in the rapid release of all the plasmid DNA, even at an RNA concentration of 50 µg/ml. However, the presence of RNA at concentrations up to 400 µg/ml induced only partial disassembly of particles formed with branched PEIs of 25 or 750 kDa. In the presence of competitor DNA, slow particle disassembly was observed with particles made with linear 25 kDa PEI, JetPEI, and branched 2 kDa PEI, but DNA release was not seen for particles made with branched PEIs of higher molecular weight. The presence of BSA only resulted in partial disassembly of PEI-DNA particles, and DNA release was not observed after the exposure of particles to acidic pH as low as 3. For all the PEIs tested, their stability increased as the PEI:DNA ratio increased. It was concluded from these studies that branched PEIs have a higher affinity for DNA than linear PEIs and that the intracellular disassembly of PEI-DNA particles may involve interactions between PEI and cellular RNA. In the second part of the work, RNA interference (RNAi) was used to enhance transient gene expression by knockdown of two different mRNAs, the E1A mRNA in human embryonic kidney 293 EBNA (HEK293E) cells and the ube2i mRNA in CHO DG44 cells and HEK293 cells. HEK293 cells are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNAi. The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1α (EF-1α) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to 3-fold. E1A mRNA depletion also resulted in a 2-fold increase in transient expression of a recombinant IgG in suspension cultures when the IgG light and heavy chain genes were driven by the EF-1α promoter. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells. Efficient RNAi-mediated depletion of ube2i mRNA in CHO DG44 cells was accomplished by co-expression of 2 shRNAs targeting two distinct sides in the ube2i mRNA, whereas knockdown with only one of the shRNAs at a time was insufficient for reducing ube2i mRNA levels. The reduction of the ube2i mRNA level in CHO DG44 cells was shown to enhance transient GFP expression 2-fold and IgG expression 2.5-fold. For these experiments, the GFP gene was under the control of the CMV IE promoter and the IgG light and heavy chain genes were under the control of the human EF-1α promoter. Thus, the enhancement of transient gene expression resulting from ube2i mRNA depletion was not promoter-dependent. These results demonstrated that sumoylation has a negative effect on transient gene expression in CHO DG44 and HEK293E cells.

EPFL2006

Transient gene expression for rapid protein production

Natalie Muller

Recombinant proteins are important for biomedical research and for the treatment of human disease. Therefore it is necessary to develop reproducible bioprocesses to rapidly produce proteins of adequate quality and quantity. Expression in mammalian cells is preferred if the proteins are to be properly folded and post-translationally modified. For the rapid production of milligram to gram quantities of a protein in mammalian cells, large-scale transient gene expression is an attractive option. The two most important components of this technology are the cell cultivation system and the gene delivery method. For this reason the goal of this thesis was to develop novel cost-efficient cell cultivation systems and gene delivery methods for the large-scale transfection of mammalian cells in chemically defined media free of any animal-derived components including serum. Cultivation of mammalian cells in suspension is essential for large-scale transient gene expression. A superior system for the cultivation of mammalian cells based on the agitation of cells in "square-shaped" glass bottles (square bottles) was developed. It is an inexpensive but efficient means to grow cells to a high density without instrumentation. The growth and viability of cultures of both human embryo kidney (HEK) 293E and Chinese hamster ovary (CHO) DG44 cells in agitated one-liter square bottles exceeded that in spinner flasks, reaching cell densities 1.5 times higher on average. Furthermore, an efficient and reproducible method to transfect cells in agitated square bottles was developed for both HEK 293E and CHO DG44 cells. A novel gene delivery method termed calfection that relies on the addition of DNA and CaCl2 directly to a suspension culture was developed. Calfection has a number of advantages over existing gene delivery methods such as calcium phosphate-DNA coprecipitation in that there are no time-dependent steps. The DNA/CaCl2 solution can be stored for long periods and is filterable. It is suitable for both large-scale transfections in bioreactors and for high-throughput transfections in microtiter plates. One drawback, however, is its dependence on the presence of serum in the culture medium. A second gene delivery method, serum-free transfection with polyethylenimine (polyfection), proved to be highly efficient for the transfection of both HEK 293E and CHO DG44 cells. A reproducible method for polyfection in microtiter plates, 50 ml centrifuge tubes, agitated square and round bottles, spinner flasks, and bioreactors was optimized for these two cell lines. Polyfection proved to be a simple and successful gene transfer method in both instrumented and non-instrumented cultivation systems. Importantly, it could be performed in serum-free chemically defined media. With HEK 293E cells, 80 mg/l of antibody was produced in agitated square bottles and with CHO DG44 cells, more than 2 g of antibody were produced in one week in a 150-liter bioreactor.

EPFL2005

Development of plasmid-based expression technology for rapid and sustained production of recombinant proteins in insect cells

Xiao Shen

Due to the high demand for heterogeneous protein production in insect cells, we have established efficient methods for plasmid-based transient gene expression (TGE) in various insect cell lines, including commonly used Spodoptera cell lines Sf9 and HighFive™ (H5) and the Diptera cell line Schneider S2 from Drosophila melanogaster, as a high-yield protein production platform. The cells were grown in suspension in orbitally shaken containers at working volumes of 5-300 mL. Rapid cell growth and high cell densities were obtained. Among the various transfection agents tested, polyethylenimine supported high transfection efficiencies in all three cell lines. Intensive optimizations of TGE processes for the three cell lines were executed. For example, various promoter and enhancer elements were compared. Using an optimal TGE process, transfection efficiencies (percentage of transfected cells) of over 85% were obtained for all three cell lines. Yields of the secreted protein tumor necrosis factor receptor-Fc fusion (TNFR-Fc) ranged from 100 – 200 mg/L for the three cell lines using a gene optimized for expression in insect cells. The molecular weight of the Sf9-derived dimeric TNFR-Fc was about 6 kDa less than the equivalent product synthesized by Chinese hamster ovary cells due to differences in glycosylation. In addition, several difficult-to-express proteins, that failed to be delivered at sufficient quantity using other expression systems, were successfully produced in S2 cells by TGE. A human serotonin receptor was expressed at 20-60 mg/L; empty and peptideloaded MHC II tetramer complexes were expressed at up to 150 mg/L; neutrophil serine proteases were expressed at levels over 50 mg/L; and protein tyrosine kinases were expressed at levels over 2 mg/L. These yields were obtained within 5 days of transfection at volumetric scales up to 300 mL. We also developed methods for the generation of stable polyclonal pools of recombinant S2 cells based on plasmid cotransfection in the context of the Piggybac transposon. After two weeks of genetic selection of recombinant cells, the recombinant pools remained stable for at least 100 days in the absence of selection with volumetric TNFR-Fc productivity over 40 mg/L in a 4-day batch culture. Our data highlight the use of plasmid-based expression systems in insect cells to meet the demand for rapid and sustained production of proteins in sufficient quantities for biomedical research and for the biotechnology industry.

EPFL2014