Transfection using plasmid preparations

There is provided a method for plasmid DNA extraction to reproducibly yield relatively crude preparation which is suitable for transfection into mammalian cells. The inventors have determined that the high level of purification previously thought to be necessary in order to successfully allow transfection of the plasmid DNA into cells is not required. The inventors have developed a method of producing a transfectable plasmid preparation that can be performed on a large scale that avoids the use of specialized equipment and reagents.

Correlation between cell cycle and calcium phosphate transient transfection of CHO cells

Frédéric Grosjean

Calcium phosphate transfection is a widely used method to produce recombinant proteins in mammalian cells. However, the mechanisms involved in plasmid DNA transfer to the nucleus of the transfected cells remain poorly understood and result in great variation of transfection efficiency. The role of the cell cycle in transfection by the calcium phosphate method was studied using Chinese Hamster Ovary (CHO) cells as a model system. Adherent cell cultures were synchronized at the G1/S boundary using mimosine. At various time points after the removal of the drug the cells were transfected with reporter plasmids that expressed either the enhanced green fluorescent protein (EGFP) or a red fluorescent protein (DsRedExpress). Fluorescent protein expression was monitored by fluorometry and live imaging. Cell cycle was monitored by flow cytometry. It was shown that the cell cycle had major implications on at least two levels. The correlation between the percentage of cells in S phase at the time of transfection complex addition and the resulting reporter gene expression demonstrated that efficient transfer of exogenous DNA in mammalian cells was dependent on the cell cycle. A faster transfection protocol, developed within this work, showed that the optimal timing for the transfection complex addition was different depending on the transfection set-ups used. Further investigations demonstrated that mitosis played a key role, probably due to the nuclear membrane disruption, to provide access to the nuclear environment for the plasmid DNA molecules. The glycerol shock performed at the end of the transfection was also shown to play an important role for efficient transfer of reporter gene in nuclei. This strong osmotic shock resulted in a cellular volume decrease of approximately 55%. As no intracellular plasmids were lost during the process, this resulted in a significant increase in intracellular plasmid concentration. Glycerol shock has to be performed while cells proceed through mitosis. The nuclear membrane disruption and the increasing intracellular plasmid concentration probably facilitate the access to the nuclear environment. The synergy of glycerol shock and cells proceeding through mitosis was a requirement for efficient reporter gene transfer in the nuclei. If one of those two was omitted, or if they did not happened at the same time, very low reporter gene expression levels were achieved. As it was found that plasmid DNA was driving reporter gene expression even after a delay of 45 minutes post cellular uptake, it was possible to efficiently target additional sub-populations of cells as they proceeded through mitosis with additional glycerol shocks. Those repetitive glycerol shocks resulted in an increase of positive cells among the transfected populations. In conclusion, the results obtained in this work permitted to define a model explaining the importance of the cell cycle in the calcium phosphate transfection, highlighting mitosis and glycerol shock as key events in plasmid DNA molecules transfer to the nucleus.

EPFL2003

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

Gene Expression in Synovial Membrane Cells After Intraarticular Delivery of Plasmid-Linked Superparamagnetic Iron Oxide Particles—A Preliminary Study in Sheep

Alke Fink, Heinrich Hofmann, Benedikt Steitz

This study evaluated in vivo gene deliver y and subsequent gene expression within cells of the synovium in the presence of static and pulsating magnetic field application following intraar ticular injection of super paramagnetic iron oxide nanopar ticles linked to plasmids containing reporter genes encoding for fluorescent proteins. Plasmids encoding genes for either green fluorescent protein or red fluorescent protein were bound to super paramagnetic nanopar ticles coated with polyethyleneimine. Larger (200–250 nm) and smaller (50 nm) nanopar ticles were compared to evaluate the effects of size on transfection efficiency as well as any associated intraar ticular reaction. Comparisons between groups were evaluated at 24, 72, and 120 h time periods. Inflammator y response was mild to moderate for all injected par ticles, but was present in the majority of synovial membrane samples evaluated. Larger par ticles tended to be associated with more inflammation than smaller ones. Never theless, intraar ticular application of both experimental and control nanoparticles were well tolerated clinically. Gene expression as determined by obser vation of either green or red intracellular fluorescence was difficult to assess by both epifluorescent light, and confocal microscopy. An insufficient concentration of nanopar ticles in relation to joint volume likely resulted in a limited number of samples with positive evidence of iron staining and with suspected positive evidence of cells expressing fluorescent proteins. Our results indicate that intraar ticular administration of functionalized super paramagnetic iron oxide nanopar ticles resulted in a mild to moderate synovitis and there was in conclusive evidence of gene expression. Fur ther research is warranted to determine the best and most effective repor ter assay for assessment of the in vivo gene deliver y into the joints. In addition, the best suited concentration and size of nanopar ticles, which will optimize gene delivery and expression, while minimizing intraarticular inflammation, needs to be determined.

2006