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Establishment of immortal differentiated diploid hepatocyte lines from transgenic mice and their use for studying viral and chemical agents

Objective

Immortal, differentiated hepatocyte lines will be generated from transgenic mouse strains bearing metallithionein (MT-I)-driven SV40 T-antigen constructs for the development of in vitro systems for the identification of potentially mutagenic agents. The lines will also be employed for transfecting Hepatitis B virus (HBV) DNA to study HBV gene expression and replication in vitro.
There is great demand for stable hepatocyte culture systems in areas such as pharmacology, toxicology, virology, carcinogenesis, and biotechnology. At present hepatocytes in primary culture maintain their differentiated functions only for a few days which limits their usefulness in long term studies. Therefore, hepatocytes lines were derived from transgenic mice that bear an immortalizing transgene which is expressed in the liver prior to the appearance of tumor cells in the animal. Resulting hepatocyte lines are immortal, non tumorigenic, and stably maintain their differentiated fuctions for extended periods. A new hepatocyte line has been derived from an adult mouse and is in the process of being adapted to multiply in serum free medium in response to epidermal growth factor (EGF) and insulin. To characterize the control of the cell cycle of adult line BGK 202 it was demonstrated that cells express the kinases cdc2, CDK2, and cyclins. Sequential phosphorylation processes were shown that indicated that cdc2 clearly participates in the control of G2/M but not in G1/S transition. Attempts are being made to obtain stably transfected cells using the complete hepatitis B virus (HBV) genome. Fetal transgenic mouse cell line FMH 202 was employed as target for assaying indirectly acting mutagens in attempts to set up a short term toxicity assay for the identification of indirectly acting mutagens. It was shown that the fetal line lends itself for such tests, however further validation is needed for standardizing the assay systems for routine use.

Novel differentiated liver cell lines were established for immediate use in biotechnology, virology, biochemistry, and toxicology. The cells appear to be useful for short term toxicological test systems in vitro.
There is great demand for stable hepatocyte culture systems in areas such as pharmacology, toxicology, virology, carcinogenesis and biotechnology. At present, hepatocytes in primary culture maintain differentiated functions for a few days, which limits their usefulness in long-term studies. Also, hepatoma cell lines are available which exhibit only a fraction of hepatic functions. Thus, the goal of this research project is to obtain diploid, immortal hepatocyte lines derived from adult transgenic mice expressing an immortalizing transgene (SV40 virus T- and t-antigens) in the liver. Resulting cell lines will be analyzed for their ability to express hepatic functions including drug metabolizing enzyme systems which are essential for the metabolic activation of indirectly acting mutagens.
Attempts will be made to employ such hepatocyte lines for the establishment of an in vitro test system for the identification of potentially mutagenic agents in chemically defined culture medium. Also, the cell lines will be employed to evaluate the roles of Hepatitis B Virus (HBV), growth factors & of plasma membrane constituents in mediating contact inhibition phenomena, and in controlling cell growth and the progression of hepatocytes toward malignancy in culture. For this purpose, basic investigations are required to define mechanisms that control the balance between growth and differentiation of hepatocytes in culture. Furthermore, generated hepatocyte lines will be of broad interest for studies in toxicology and, after appropriate genetic manipulations, and for the production of pharmaceutically important proteins & glycoproteins in large scale.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

FRAUNHOFER-GESELLSCHAFT ZUR FOERDERUNG DER ANGEWANDTEN FORSCHUNG E.V.
Address
Nikolai-fuchs-strasse 1
30625 Hannover
Germany

Participants (1)

Institut National de la Santé et de la Recherche Médicale
France
Address
Rue Henri Le Guilloux
35033 Rennes