Retargeting of pre-set regions on chromosome for high gene expression in mammalian cells

We have developed a system to hunt and reuse special gene integration sites that allow for high and stable gene expression. A vector, named pRGFP8, was constructed. The plasmid pRGFP8 contains a reporter gene, gfp2 and two extraneous DNA fragments. The gene gfp2 makes it possible to screen the high expression regions on the chromosome. The extraneous DNA fragments can help to create the unique loci on the chromosome and increase the gene targeting frequency by increasing the homology. After transfection into Chinese hamster ovary cells (CHO) cells, the linearized pRGFP8 can integrate into the chromosome of the host cells and form the unique sites. With FACS, 90 millions transfected cells were sorted and the cells with strongest GFP expression were isolated, and then 8 stable high expression GFP CHO cell lines were selected as candidates for the new host cell. Taking the unique site created by pRGFP8 on the chromosome in the new host cells as a targeting locus, the gfp2 gene was replaced with the gene of interest, human ifngamma, by transfecting the targeting plasmid pRIH-IFN. Then using FACS, the cells with the dimmest GFP fluorescence were selected. These cells showed they had strong abilities to produce the protein of interest, IFN-gamma. During the gene targeting experiment, we found there is positive correlation between the fluorescence density of the GFP CHO host cells and the specific production rate of IFN-gamma. This result shows that the strategy in our expression system is correct: the production of the interesting protein increases with the increase fluorescence of the GFP host cells. This system, the new host cell lines and the targeting vector, can be utilized for highly expressing the gene of interest. More importantly, by using FACS, we can fully screen all the transfected cells, which can reduce the chances of losing the best cells.

Effect of Gas Sparging in Mammalian Cell Bioreactors

One of the major problems in the operations of mammalian cell bioreactors is the detrimental effect of gas sparging. Since the most convenient way to oxygenate any bioreactor is by gas sparging, this adverse effect has often been one of the limiting oxygen transport problems in both laboratory and industrial mammalian cell bioreactors. When one examines the literature on the effect of gas sparging on the death of mammalian cells, a great deal of confusions has been reported. It is not clear from the published literature as to the leading cause for gas-sparged related cell death. These confusions prevent the rational design and operations of mammalian cell bioreactors. In our laboratory, we have attempted to address this problem both fundamentally as well as attempt to obtain a general understanding on the adverse effect of gas sparging. Our analyses first examined the fluid shear associated with the various sections that the gas bubbles encounter during entrance, passage through the bioreactor and the final exit of the gas bubbles. Our analyses showed that the major damage of the mammalian cells by gas bubbles is due to the burst of the bubbles when exiting the bioreactor. It was also our hypothesis that the entrained cells in the liquid boundary layer of the gas bubble upon bursting is the major cause for cell death. We have corroborated this hypothesis by correlating the liquid entrainment with the cell death rate using results from our laboratory as well as other studies. Pluonic F-68, a weak surfactant, has routinely been used in laboratory and industrial bioreactors. In the past, the protective effect of Pluronic F-68 has never been shown as to why it is effective. In our research, we have data using microphotography which clearly demonstrated and corroborated our entrainment hypothesis is the major reason for the effectiveness of Pluronic F-68 in protecting the cells from gas-sparged related cell death.