epidermal-growth-factor and Retroviridae-Infections

Targeted cell ablation in animals is a powerful method for analyzing the physiological function of cell populations and generating various animal models of organ dysfunction. To achieve more specific and conditional ablation of target cells, we have developed a method termed Toxin Receptor mediated Cell Knockout (TRECK). A potential shortcoming of this method, however, is that overexpression of human heparin-binding epidermal growth factor-like growth factor (hHB-EGF) as a diphtheria toxin (DT) receptor in target cells or tissues may cause abnormalities in transgenic mice, since hHB-EGF is a member of the EGF growth factor family. To create novel DT receptors that are defective in growth factor activity and resistant to metalloprotease-cleavage, we mutated five amino acids in the extracellular EGF-like domain of hHB-EGF, which contains both DT-binding and protease-cleavage sites. Two of the resultant hHB-EGF mutants, I117A/L148V and I117V/L148V, possessed little growth factor activity but retained DT receptor activity. Furthermore, these mutants were resistant to metalloprotease-cleavage by 12-O-tetradecanoylphorbol-13-acetate stimulation, which is expected to enhance DT receptor activity. These novel DT receptors should be useful for the generation of transgenic mice by TRECK.

Topics: Amino Acid Sequence; Animals; DNA Mutational Analysis; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Mice; NIH 3T3 Cells; Receptors, Cell Surface; Retroviridae Infections

Cultured adult rodent hepatocytes are extensively used as a model system for gene transfer in vitro. In the present study, we examined the influence differentiation status and growth capacity of the hepatocytes on their infectivity in vitro by a retroviral vector. These parameters were initially studied in primary cultures of rat hepatocytes transduced with an ecotropic retroviral vector containing Escherichia coli beta-galactosidase. However, significant differences observed in the infectivity of hepatocytes from 12-day-old and adult rats led us to also examine hepatocytes from a transgenic mouse strain in which the SV40 large T antigen is fused to the regulatory sequences of the human anti-thrombin III gene. The large T antigen is expressed in the liver and these mice develop hepatoma within 7 months. A comparison of infectivity of hepatocytes from normal and transgenic mice of different ages indicated that in contrast to previous reports, hepatocytes which express differentiated functions during the first week of culture can still be efficiently infected by retroviral vectors. Optimal infection was observed between the second and fourth day of culture and does not appear to be due to transient cell dedifferentiation, but is more likely due to transient mitotic activity of mice cells since the role of growth factors seems crucial for infection. The peak of infection did not appear to correspond to transient cell dedifferentiation. We also found differences of infectivity between hepatocytes from normal and transgenic mice of different ages. Such differences are correlated with differences in in vitro BrdU incorporation, which was used to determine the proportion of dividing hepatocytes. These results indicate that the efficiency of infectivity of hepatocytes by recombinant retrovirus is probably related to their normal proliferative potential and not to some dedifferentiated stage. Hence these findings provide a model for efficient gene transfer in differentiated cells and suggest an approach for studies of liver-specific gene regulation and for somatic gene therapy of metabolic diseases as well.

Topics: alpha-Fetoproteins; Animals; Antigens, Polyomavirus Transforming; Cells, Cultured; Epidermal Growth Factor; Genetic Vectors; Liver; Liver Neoplasms, Experimental; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Transgenic; Rats; Rats, Wistar; Retroviridae; Retroviridae Infections; Transformation, Genetic

Topics: Antibodies, Monoclonal; Cell Line; Deltaretrovirus; Epidermal Growth Factor; Flow Cytometry; Humans; Leukemia; Lymphocyte Activation; Phosphorylation; Receptors, Immunologic; Receptors, Interleukin-2; Retroviridae Infections; T-Lymphocytes