Abstract
A fibroblast-mediated gene delivery method was used for the endogenous expression of human granulocyte colony-stimulating factor (G-CSF) as a model for cytokine supplement therapy. Human G-CSF cDNA was inserted into the plasmid expression vector BMGNeo, which contains a partial sequence of bovine papilloma virus and a selectable marker gene. The recombinant plasmid (BMGNeo-GCSF) was transfected into NIH/3T3 fibroblasts by the calcium phosphate coprecipitation method, and the stably transformed cells were isolated by G418 selection. An appropriate clone producing a large amount of G-CSF was selected by enzyme immunoassay of the culture supernatants. Southern blot analysis suggested that the BMGNeo-GCSF plasmid replicated mainly as an episome, and Northern blot analysis demonstrated the high expression of human G- CSF mRNA in the cells. After the implantation of the G-CSF-producing fibroblasts into nude mice, prominent neutrophilia, about 30-fold the level of normal control, was observed within seven days. Moreover, the number of hematopoietic progenitor cells in spleen remarkably increased for all cell lineages in these mice. To regulate the in vivo expression of G-CSF, we designed a subcutaneous diffusion chamber apparatus that contains the G-CSF-producing fibroblasts. The leukocytosis (neutrophilia) induced in C3H mice after embedding the device quickly disappeared after ethanol treatment of the chamber. Furthermore, reinjection of the G-CSF-producing fibroblasts into the chamber caused a second neutrophilia.
