The utilization of hepatocytes for cellular gene therapy would be greatly added by the ability to culture and passage primary hepatocytes in vitro. A period of in vitro growth would allow introduction of critical genes and an analysis of their expression prior to the transplantation into the recipient. In addition, the identification of cell types with prolonged proliferative capacities, both in vitro and in vivo, will be essential to sustained correction which are important for hepatocyte cell proliferation in vitro. We will utilize our present protocol which gives survival for over two weeks and continued expression of liver specific functions as baseline conditions. Factors from a variety of sources will be tested for their ability to increase DNA synthesis or prolong survival. The second goal will be to use cellular transplantation as an assay system for cells capable of proliferation and hepatospecific gene expression in vivo. The general strategy for this goal will be to isolate cells from transgenic animals carrying the chloramphenicol acetyltransferase (CAT) gene as a marker. Various cell fractions from the liver and the pancreas marked by CAT will be reintroduced into a recipient animal by transplantation. The donor cells which repopulate the recipient liver will then be assessed for their proliferative capacity and their ability to express a variety of hepatic gene products. Isolation of, or enrich for, cells of the liver which have prolonged growth potential will greatly enhance the feasibility of cellular transplantation as a gene therapy modality.
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