Gene transfer in astrocytes: Comparison between different delivering methods and expression of the HIV-1 protein Nef

To identify a good system to introduce foreign genes into normal and tumora l astrocytes, we studied the efficiency of two chemical methods, calcium ph osphate precipitation and lipofection, and of a viral-mediated transfer by a vector derived from the highly attenuated modified vaccinia virus Ankara (MVA), Using the beta-galactosidase (beta-gal) gene (lacZ) as reporter, we searched for optimal experimental conditions to obtain an efficient gene tr ansfer into human embryonic and neonatal rat astrocytes and into a human as trocytoma cell line (U373 MG). The beta-gal protein production was evaluate d by cytochemical staining and enzymatic activity assay. Among chemical met hods, lipofection was the most efficient system to transfect astrocytes in providing up to 60% of beta-gal-positive cells in all the cell types analyz ed. MVA infection also proved to be an efficient system to introduce hetero logous genes into human embryonic astrocytes that appeared 80-100% positive 48-96 hr after an infection at a multiplicity of 1-10, In contrast, only a limited infection was observed with rat astrocytes, human astrocytoma cell s, and human leptomeningeal cells. A recombinant MVA vector expressing the human immunodeficiency virus-1 (HIV-1) regulatory protein Nef was used to t ransfect human embryonic astrocytes, and the resulting Nef expression was c ompared with that detected after lipofection in the same cells. By Western blot analysis, Nef expression was observed in human astrocytes 24-96 hr aft er infection and was similar to that present in stably HIV-1-infected astro cytoma cells, Lipofection resulted in lower Nef expression, In spite of the se promising results, the negative effects of MVA infection on cell viabili ty and the possibility that a productive infection occurs in human embryoni c astrocytes limit the use of this vector for gene delivery in developmenta lly immature human glial cells. (C) 1999 Wiley-Liss, Inc.