The goal of this research is the development of a model for gene transfer into the mammalian nervous system. In this model, genes for B-nerve growth factor (B-NGF) and B-galactosidase (B-gal) will be transferred into cultured neonatal rat, type 1 astrocytes. In turn these cells, and retroviral particles separately, will be introduced into specific regions of newborn rat brains. Replication defective retroviral vectors will be used containing cDNA coding sequences for mouse B-NGF or bacterial B-gal under transcriptional control of the retroviral LTR promoter, as well as a gene for neomycin resistance under the control the SV40 early promoter. Following gene transfer, recipient cells will be selected in neomycin and assessed for expression of B-NGF and B- gal. Levels of mature B-NGF will be determined by a two-site- radioimmunoassay and by immune precipitation using monoclonal and polyclonal antibodies to mouse B-NGF. Biological acitivity of B-NGF will be determined by a PC12 cell assay of neurite outgrowth. Expresson of B-gal will be assessed by histochemical staining for enzyme activity. Gene expression of B-NGF will be determined by hybridization to radioactivity labelled antisense RNA probes using total cellular RNA, and poly A+RNA by Northern and dot blot analysis. Levels of B-NGF and B-gal will be determined in uninfected cells, cells infected with the B-NGF retroviral vector, and cells infected with the B-gal retroviral vector. Changes in expression of the endogenous mRNA for B- NGF and receptor also will be evaluated in these experimental astrocyte cultures. Pending results of in vitro studies, retroviral vectors themselves and transfected astrocytes will be injected stereotactically into the CNS of newborn rats. Animals will be studies for cellular expression of B-gal and B-NGF by histocytochemistry and immunocytochemistry, respectively, as well as by in situ hybridization histochemistry. The survival and extent of migration of injected astrocytes will be determined over time. Evidence of NGF bioactivity will be evaluated in an experimental model in which unilateral sectioning of the fimbria-fornix leads to death of NGF dependent neurons in the nuclei of the basal forebrain. The experimental model is significant as it represents a model for some of the histologic changes in the human disease, Alzheimer disease.
| Hoffman, D; Breakefield, X O; Short, M P et al. (1993) Transplantation of a polymer-encapsulated cell line genetically engineered to release NGF. Exp Neurol 122:100-6 |
| Short, M P; Choi, B C; Lee, J K et al. (1990) Gene delivery to glioma cells in rat brain by grafting of a retrovirus packaging cell line. J Neurosci Res 27:427-39 |
| Short, M P; Rosenberg, M B; Ezzedine, Z D et al. (1990) Autocrine differentiation of PC12 cells mediated by retroviral vectors. Dev Neurosci 12:34-45 |
