Infoscience

Thesis

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

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.

    Thèse École polytechnique fédérale de Lausanne EPFL, n° 3450 (2006)
    Section de chimie et génie chimique
    Faculté des sciences de base
    Institut interfacultaire de Bioingénierie (SV/STI)
    Laboratoire de biotechnologie cellulaire (SV/STI/SB)
    Jury: Otmane Boussif, Jeffrey Alan Hubbell, Kai Johnsson, Nicolas Mermod

    Public defense: 2006-2-10

    Reference

    Record created on 2006-01-23, modified on 2016-08-08

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