lactoferrin and Cell-Transformation--Viral

The expression of human lactoferrin in the mammary gland is an attractive approach to diminish its current production cost. Previous attempts to produce lactorferrin in the milk of transgenic animals resulted in very high cost and uncertain results. In this paper, we have directly infused replication-defective adenovirus encoding human lactoferrin cDNA into the mammary gland of goats via the teat canal. In this way, we obtained a high level of expressed human lactoferrin up to 2g/L in the milk of goats. The milk serum was collected from the infected mammary gland 48 h post-infection and subjected to a 10% SDS-PAGE and Western blotting. A approximately 80-kDa protein was visualized after viral vector infection. Our results demonstrate that intraductal injection of recombinant replication-defective adenovirus vectors may provide a very useful tool for large-scale production of recombinant proteins of biopharmaceutical interest.

Topics: Adenoviridae; Animals; Blotting, Western; Cell Culture Techniques; Cell Line; Cell Transformation, Viral; Cells, Cultured; Collagenases; DNA Replication; DNA, Complementary; DNA, Viral; Electrophoresis, Polyacrylamide Gel; Epithelial Cells; Female; Gene Expression; Genetic Vectors; Goats; Humans; Lactoferrin; Mammary Glands, Animal; Milk; Molecular Weight; Polymerase Chain Reaction; Recombinant Proteins; Transfection

32D C13(G) is an interleukin 3(IL3)-dependent non-tumorigenic murine hematopoietic cell line which undergoes terminal differentiation into granulocytes when exposed to granulocytic colony stimulating factor (G-CSF). Infections of 32D C13(G) cells with either Kirsten rat sarcoma virus or Balb murine sarcoma virus, both containing a v-ras oncogene, generates clones that can permanently grow in G-CSF without differentiation. 32D-Ki-ras cells show a heterogeneous morphology ranging from the promyelocytic to the myelocytic stage of differentiation, and express high levels of both myeloperoxidase (MPO) and lactoferrin (LF) mRNA. 32D-Ha-ras cells show a more immature phenotype and express MPO but no LF mRNA. The apparent differentiation block of both 32D Ki-ras and 32D Ha ras can be reversed by treatment with the chemical inducers retinoic acid, sodium butyrate or dimethylsulphoxide, which leads to terminal differentiation into granulocytes. When 32D-Ki-ras and 32D-Ha-ras cells are cultured in medium containing IL-3 they become adherent and express some monocyte-macrophage markers. Upon prolonged exposure to IL3, 32D-Ki-ras, but not 32D-Ha-ras, resume suspension growth. Both 32D-Ki-ras and 32D-Ha-ras rapidly die if grown in chemically defined medium in the absence of any growth factor and are non-tumorigenic in immunosuppressed mice. These findings indicate that ras activation may interfere with the normal response to growth and differentiation factors in cells of the granulocytic lineage. These alterations may represent a critical, although non-sufficient, step in leukemogenesis.

Topics: Butyrates; Butyric Acid; Cell Differentiation; Cell Division; Cell Transformation, Viral; Colony-Stimulating Factors; Dimethyl Sulfoxide; Gene Expression Regulation; Genes, ras; Granulocyte Colony-Stimulating Factor; Granulocytes; Harvey murine sarcoma virus; Hematopoiesis; Hematopoietic Stem Cells; Interleukin-3; Kirsten murine sarcoma virus; Lactoferrin; Peroxidase; Sarcoma Viruses, Murine; Tretinoin