In vivo protein labeling for structural and functional investigation of the 5-HT3A neurotransmitter receptor

In this thesis, two different fluorescent labeling techniques for in vivo investigations on the 5-HT3 receptor (5-HT3R) functions are presented. This plasma membrane protein contains five subunits surrounding an ion channel that opens after binding of a 5-HT3-specific neurotransmitter. The first technique, described in the first part of this report, focuses on receptor labeling via genetic fusion to spectral variants of the green fluorescent protein. I found that the resulting chimeras containing one fluorescent protein per subunit exhibit preserved ligand binding and channel activity, opening a wide range of biological research applications. Among these, I present the possibility to follow the 5-HT3R trafficking and localization during its entire life cycle by multicolor imaging in live cells, starting with its cytoplasmic biogenesis and ending with its ligand-induced internalization. The intracellular subunit assembly is shown to occur in the endoplasmic reticulum, and the importance of the cytoskeleton microtubules for proper membrane targeting is unraveled. The utility of bioluminescent 5-HT3R contructs was also demonstrated in another approach using the chemical disruption of cellular actin filaments to produce vesicular fractions of cells, in the order of 0.1 to a few micrometers in diameter. These so-called native vesicles, containing the labeled receptors in their membrane, were shown to be suitable for measurements using fluorescence confocal microscopy of ligand binding and of ion influx, opening new possibilities for miniaturized bioanalytics. In a third approach, I demonstrated that after detergent-solubilization of the receptor, the green fluorescent protein (GFP) inserted into the receptor sequence permitted to monitor ligand binding via fluorescence resonance energy transfer (FRET). Furthermore, I could observe a spatial reorientation of the receptor GFPs upon binding an agonist to the receptor. In the second part of this thesis, I adapted the mis-acylated suppressor tRNA technology to mammalian cells, permitting the introduction of unnatural amino acids at specific positions in the protein of interest. I achieved an efficiency of amino acid incorporation using in vitro aminoacylated suppressor tRNA in the order of 15% in CHO cells. A novel methodology for the quantification of background natural nonsense codon readthrough in different cell lines was also developed, permitting to select the most suitable codon-anticodon pair for this suppressor tRNA technique in various cell lines. Finally, I present a general strategy to increase the aforementioned artificial incorporation efficiency by down-regulating the competing eukaryotic release factor 1 (eRF1) using small interfering RNAs.

Establishment of recombinant mammalian cell lines

Madiha Derouazi

Recombinant proteins are gaining in importance for therapeutic applications. The proteins are expressed in stable cell lines, within which recombinant DNA has incorporated into the host cells genome. Identification and isolation of extremely high producers from transfected cell populations is a tedious and time-consuming effort. We proposed two different approaches for the establishment of recombinant cell lines: affinity cell sorting and microinjection. Even with the help of single cell sorting and analysis systems (flow cytometry) rarely more than thousand different cell clones, stably expressing a desired gene can be evaluated. We are considering a new approach in which polyclonal cell populations will be established which co-express a protein of interest and an epitope-tagged cell surface receptor. A microbead fixed antibody specific to the epitope tag is hoped to assist in the isolation of rare, but highly productive cells from large suspensions of transfected cells. For this reason we have also developed an efficient method for the bulk transfection of CHO cells. On the side of vector-development for this approach, we have chosen to investigate the utility of a mouse serotonin 5-HT3 receptor as a cell surface marker. This receptor can be expressed at very high levels (up to 1 x 107 receptors per cell) in heterologous systems. The receptor is detectable on the cell surface by means of fluorescent ligands, which allows the determination of transfection efficiencies in suspension cultures. A Flag-tag was fused to the amino terminal end of the receptor without adverse effects on receptor function or ligand binding. Only 5 % of receptor DNA in the transfection cocktail was sufficient to detect positive cells in a population. The specific interaction of the epitope-tagged receptor with the cognate antibody fixed to microbeads was tested in comparison to non-transfected control cells using laser scanning confocal microscopy. A 80% enrichment of cells expressing GFP and the 5-HT3 receptor was achieved in 30 minutes. We have investigated microinjection as a gene transfer method for the establishment of recombinant mammalian cell lines. The conditions for the injection of plasmid DNA into either the nucleus or the cytoplasm of CHO-DG44 cells were established using pMYK-EGFP, an expression vector with the enhanced green fluorescent protein (EGFP) gene under the control of the murine CMV promoter and the puromycin resistance gene for selection. To estimate plasmid copy number during microinjection, it was first necessary to determine the injection volume by fluorescence quantification of injected FITC-dextran. For microinjection into the cytoplasm the volume ranged from 200 to 350 fl and for nuclear microinjection it was about 180 fl. We then investigated the impact of DNA concentration and injection pressure on transient EGFP transient expression following microinjection into the cytoplasm or nucleus. As a general rule, we observed that concentration of the DNA solution injected into the cells was the most important parameter regardless of the pressure or the volume of injection. Recombinant cell lines expressing intracellular EGFP were established following microinjection into the nucleus and the cytoplasm with linear and circular DNA. Selection with puromycin, clonal expansion, and cell banking were completed within three to four weeks post-microinjection. In parallel, recombinant cell lines were established after calcium-phosphate transfection with linear and circular DNA. The number of plasmid copies integrated was determined by Southern blot and the number and localization of integration sites was determined by fluorescence in-situ hybridization (FISH). Growth of the cells and stability of EGFP expression were investigated by cultivation over a two-month period. This approach presents several advantages over other DNA delivery methods such as control of the quantity and localization of DNA delivered into the cell and speed of recovery of recombinant cell lines. This approach may be relevant for the establishment of recombinant cell lines from cells that are not easily transfected by classical methods.

EPFL2005

100-liter transient transfection

Madiha Derouazi, Philippe Girard, Martin Jordan, Florian Maria Wurm

This is the first report of two successful 100 l scale transienttransfections in a standard stirred bioreactor. More than half a gram of a monoclonal antibody (IgG) were produced in less than 10 days using a technology called large-scale transient gene expression(LS-TGE). Suspension adapted HEK 293 EBNA SF cells were transfectedwithin a 150 l (nominal) bioreactor by a modified calcium phosphateco-precipitation method with more than 75 mg of plasmid DNA per run.A mixture of three different plasmids, one encoding for the heavychain of a human recombinant immunoglobulin, the other for the corresponding light chain and a third one for the green fluorescent protein (GFP, 2-4% of DNA in transfection cocktail)were co-transfected. The GFP vector was chosen to monitor transfection efficiency. Expression of GFP could be registered asearly as 20 h after DNA addition, using fluorescence microscopy. We demonstrate that transient transfection can be done at the100 l scale, thus providing a new tool to produce hundreds of milligrams or even gram amounts of recombinant protein. Akey advantage of LS-TGE resides in its speed. In the presentedcases, the entire production process for the synthesis of halfa gram of a recombinant antibody, including DNA preparationand necessary expansion of cells prior to transfection, wasexecuted in less than a month. Having an established transfection/expression process allows to run productioncampaigns for any given protein, within one facility, with onesingle host cell line and therefore only one single seed train. Without any need to create and maintain stable cell lines, expression of new r-proteins is not only faster and more economical but also more flexible.

2002

Study of transposon-mediated cell pool and cell line generation in CHO cells

Sowmya Balasubramanian

The goal of this thesis was to evaluate the use of artificial transposon systems for the generation of recombinant cell pools and cell lines with Chinese hamster ovary (CHO) cells as the production host. Transposons are naturally occurring genetic elements present in the genome of most organisms. Several transposons of metazoan origin have been engineered to facilitate the integration of one or more recombinant genes (transgenes) into the genome of the host cell. In this thesis, three of the most commonly used transposons, piggyBac (PB), Tol2, and Sleeping Beauty (SB), were used for transgene delivery into CHO-DG44 cells. The transposon systems described here involves co-transfection of the host cells with a helper plasmid for the transient expression of the transposase, and a donor plasmid coding for the transgene and a gene for the selection, both positioned between two transposon repeat sequences that are required for DNA transposition. Through the optimization of various selection parameters we showed that PB-mediated cell pools can be generated with selection duration of as little as 5 days in the presence of puromycin. All three transposon systems, PB, Tol2, and SB, resulted in cell pools with similar volumetric TNFR:Fc productivities that were about 9 times higher than those generated by conventional plasmid transfection. Transposon-mediated cell pools had 10 - 12 transgene integrations per cell. However, we demonstrated that some the integration events occurred via DNA recombination rather than transposition. We also isolated clonal cell lines from cell pools. As expected, the average volumetric TNFR:Fc productivity of transposon-derived cell lines was higher than that of cell lines generated by conventional transfection. In 14-day fed-batch cultures, protein levels up to 900 mg/L and 1.5 g/L were obtained from transposon-mediated cell pools and cell lines, respectively. The stability over time of the volumetric productivity of cell pools was determined by maintaining the cells in culture for 3 months in the absence of selection. In general, the productivity decreased to 50 % its initial level over the first 7 weeks in culture and then remained constant for the following 5 weeks. In contrast, the volumetric protein yield from transposon-mediated cell lines remained constant for up to 4 months in the absence of selection. We also showed that the three transposon systems could be used for cell pool generation with CHO-K1 and CHO-S with similar volumetric productivities as observed with CHO-DG44 cells. Finally, we utilized the PB transposon system for generating cell pools co-expressing up to 4 different transgenes, enhanced green fluorescent protein (EGFP), secreted alkaline phosphatase (SEAP), and the light and heavy chains of an IgG1 monoclonal antibody, by simultaneous transfection of all four transgenes. We showed that PB-mediated cell pools had increased volumetric productivity of each of the proteins compared to those generated by conventional co-transfection. The use of the PB transposon system increased the percentage of cells in the pools that were co-expressing all four proteins as compared to the results with cell pools generated by conventional transfection. In conclusion, the transfection of CHO cells with the PB, Tol2 or SB transposon system is a simple, efficient, and reproducible approach to the generation of cell pools and cell lines for the rapid production of recombinant proteins.