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PhD thesis: a PhD thesis.
Correlation between cell cycle and calcium phosphate transient transfection of CHO cells
Ecole Polytechnique Fédérale de Lausanne
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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.
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