Genetically Modified Striatal Astrocytes Grafted to the Brain: Pharmacological Control of an Inducible Promoter
Author(s) -
Edmund F. La Gamma,
Gary Weisinger,
Nicholas J. Lenn,
Robert E. Strecker
Publication year - 1992
Publication title -
neural plasticity
Language(s) - English
Resource type - Journals
eISSN - 2090-5904
pISSN - 1687-5443
DOI - 10.1155/np.1992.244
Subject(s) - neuroscience , striatum , biology , dopamine
In the late 1980s, several investigators applied the strategy of combining genetic engineering with brain grafting (for review see Gage et al., Neuron 1991; 6: 1). Disadvantages associated with current approaches include the use of non-brain donor tissue (fibroblasts), the use of tumor-derived cell lines, and the use of viral infection methods to introduce the gene of interest. We report here the use of the non-viral, calcium phosphate transfection method to introduce stably expressed genes into primary cultures of striatal astrocytes for subsequent use in brain grafting. Astrocytes may be the preferred cellular vehicle for gene replacement therapy in brain transplantation paradigms because they divide in culture, are brain-derived and regionally specific, possess a release mechanism , secrete growth factors, can migrate several millimeters from the transplant site, and have neurotransmitter receptors. In addition, we introduce and demonstrate the regulation of gene product expression by systemically administered drugs. Thus, by the incorporation of inducible promoters in the transfected gene sequence, drugs which act upon astrocyte neuro-transmitter receptors can regulate the level of transfected gene expression. Neonatal (day 2) rat striatal astrocytes were transfected with RSV-driven or enkephalin promoter-driven chloramphenicol acetyl trans-ferase (CAT) chimeric plasmids after 3 weeks in culture. In addition, the incorporation of the neomycin resistance gene combined with selective growth of transfected cells in G418-containing (300 /sg/ml) media for 3 additional weeks insured stable transfected gene incorporation from all surviving astrocytes. These cultured astrocytes expressed CAT enzymatic activity , which was measured both in cell lysates and in the conditioned media. As the cells in these cultures were 100% viable, the latter observation indicates that the astrocytes secreted CAT into the media. Following trans-plantation into the adult rat striatum, the transplanted astrocytes were identified histologi-cally by astrocyte-specific GFAP staining, as well as with antibody staining to CAT protein to specifically detect the transfected cells. To be certain that GFAP and CAT staining identified transplanted cells, CAT activity was also biochemically detected in tissue blocks containing the transplant at both 1 day and 3 weeks after transplantation.
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