IngentaConnect Expression of the reporter genes lacZ and EGFP in human glioblast...

Abstract:

Objective: Glioblastoma has become a model target for viral gene therapy approaches. To circumvent some of the inherent problems of viral vectors, we examined the feasibility of Listeria monocytogenes for foreign gene expression in glioblastoma cells.

Methods: The internalin-dependent uptake of L. monocytogenes into human glioma cell lines and into 22 cell cultures obtained during neurosurgery was studied by in vitro infection assays. Internalization rates of wild-type L. monocytogenes were compared with a deletion mutant and to four strains with plasmid-based recomplementation of the internalin A/B operon. For gene transfer experiments, plasmids encoding lacZ or EGFP under control of the cytomegalovirus promotor were transfected into Listeriae. Incubation times in cell culture were studied regarding infection of cells, intracellular listerial replication and reporter gene translation. Gene transfer efficiency was determined by β-galactosidase activity and by fluorescence microscopy.

Results: The internalin-defective strain (ΔinlAB2) showed less uptake compared with the wild type. An increased invasion rate was observed in strain ΔinlAB2 + inlB_PinlA overexpressing internalin B; complementation of the entire operon (ΔinlAB2 + inlAB_PinlA) revealed a synergistic effect. For gene transfer experiments into glioblastoma cells, the pathogenic strain 4bL312 was employed, and efficiencies up to 5% were achieved.

Discussion: Internalins A and B are major determinants for listerial uptake into neuroepithelial tumors. The L. monocytogenes vector system enables foreign gene expression in glioblastoma cells. Ongoing research deals with optimization of transfection efficiencies, with the use of less pathogenic substrains and the cloning of suicide gene vectors.

Keywords: ASTROCYTOMA; GENE THERAPY; GLIOBLASTOMA; LISTERIA MONOCYTOGENES; MENINGIOMA

Document Type: Research article

DOI: 10.1179/174313209X403896

Affiliations: 1: Department of Neurosurgery, Justus-Liebig-University, Klinikstrasse 29, D-35385 Giessen, Germany. ulf.nestler@neuro.med.uni-giessen.de 2: Department of Neurosurgery, Institute for Medical Microbiology, Justus-Liebig-University, Giessen, Germany 3: Department of Neurosurgery, Justus-Liebig-University, Giessen, Germany 4: Institute for Medical Microbiology, Justus-Liebig-University, Giessen, Germany

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Expression of the reporter genes lacZ and EGFP in human glioblastoma cells using Listeria monocytogenes as vector for gene transfer