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Author
Casalé Cabanes, Núria
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Abstract
Pharmaceutical biomolecules are increasingly important in the development of new therapies to treat different diseases. These molecules, usually proteins, are produced on a large scale mainly in mammalian cell cultures. For this it is necessary to genetically modify these cell lines and obtain a stable line that expresses the protein of interest. Selection markers are essential in the process, as they allow differentiation and separation of the population of cells that has been modified from that which has not.
One of the main problems of large-scale crops is cell death or apoptosis, which occurs easily if there are alterations in the system, significantly affecting production and the costs of the process. Therefore, one of the ways to increase productivity is the use of anti-apoptotic genes (such as bcl-2 or BHRF1) that block the gene signaling cascade that leads to cell death. The BHRF1 gene is an antiapoptotic gene originating from the Epstein Bar virus (EBV) that has been used to modify hybridoma cell cultures, demonstrating that it prevents cell death when the culture is exposed to adverse conditions and which, in addition, has a direct effect on the production of the crop, making it more resistant in general.
The aim of this project is to study if the anti-apoptotic capacity of BHRF1 can be used as a selection marker in mammalian cell lines. Thus, only the cells incorporating the gene would be able to survive suboptimal conditions and, in addition, would have improved properties when it comes to resistance and production.
In this work, three bicistronic plasmids have been constructed, GFPneo3, GFP-BHRF1 and BHRF1puro3. These vectors contain resistance genes to neomycin and puromycin, antibiotics commonly used as selection markers, combined with the GFP reporter gene or the BHRF1 gene, which is to be studied as a possible selection marker. The plasmids have been sequenced to verify that they do not contain mutations and have been introduced into the HEK293(GS-) cell line, one of the main lines used in the production of recombinant proteins. It has been shown that the genes are expressed, as it has been possible to detect the fluorescence expressed by GFP. The expression of these genes in their different combinations would make it possible to compare the selection using antibiotics such as puromycin and neomycin with the selection using BHRF1, in addition to characterizing the effect of the gene on the cell line. Due to lack of time, the final goal of the project could not be met. Nevertheless, this work lays down the necessary bases and plasmids to carry out an in-depth study of BHRF1 as a selection marker in various cell lines and its influence on crop productivity.
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