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K Tani, K Ozawa, H Ogura, T Takahashi, A Okano, K Watari, T Matsudaira, K Tajika, H Karasuyama and S Nagata
Department of Hematology-Oncology, University of Tokyo, Japan.
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.
This article has been cited by other articles:
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| Copyright © 1989 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
