Important note: Information in this article was accurate in 1992. The state of the art may have changed since the publication date.
RETROVIRAL GENE TRANSFER OF THE HUMAN BASIC FIBROBLAST GROWTH FACTOR (HBFGF) GENE IN HUMAN STROMA (MEETING ABSTRACT)
Proc Annu Meet Am Soc Clin Oncol; 11:A241 1992. Unique Identifier : AIDSLINE ICDB/92680972 Wieder R; Shirke S; Wilson EL; Gabrilove JL; Memorial Sloan-Kettering Cancer Center, New York, NY 10021
Abstract:
Exogenous bFGF stimulates stromal growth, delays senescence and stimulates myelopoiesis. This project was undertaken to determine the effects of constitutive bFGF expression by stromal cells on their growth, senescence and support of hematopoiesis. Moloney leukemia virus-based vector N2, coding for bacterial neomycin phosphotransferase, was used as a backbone to construct vectors containing independently promoted hbFGF cDNA. Vectors were packaged by transfecting helper-free ecotropic packaging line G+P-env86 and using conditioned media to transduce amphotropic cell line G+P-AM12. Clones were selected and supernatant viral titers were determined on NIH3T3 cells. A titering method was developed for bFGF content in cell lysates using an ELISA with a polyclonal rabbit antihuman antiserum. Viral supernatants that conferred high bFGF levels in transduced fibroblast populations were used to transduce human stromal cells. Normal long-term bone marrow cultures were established and light-density cells were demidepleted weekly prior to transduction. Transduction experiments were carried out using viral supernatants with 8 ug/ml polybrene for 4 hr. Selection was begun 2 days later with 200 ug/ml G418. bFGF content of stromal populations ranged from 0.8 ng bFGF/5 x 10(4) cells in the untransduced and N2-transduced cells to values up to 2.5 ng bFGF/5 x 10(4) cells. The bFGF content of untransduced cells increased with increased passage. The viral supernatant that yielded the highest bFGF levels (AP4) was used to transduce other stromal populations. The kinetics of growth of AP4-transduced cells were greater than N2-transduced cells by a metabolic colorimetric assay (as measured by Cell Titer 96). Studies are ongoing to correlate bFGF content of stromal populations with kinetics of growth number of generations to reach senescence and support of hematopoiesis in vitro and in vivo in nude mice.
Keywords: Fibroblast Growth Factor, Basic/*GENETICS Gene Products, env Genetic Vectors Human Moloney Leukemia Virus/*GENETICS Transduction, Genetic *Transfection ABSTRACT 921030
M92A1040
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Important note: Information in this article was accurate in 1992. The state of the art may have changed since the publication date.
