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The gene therapy strategy using the hsvl-thymidine kinase gene (TK) and ganciclovir (GCV) injections that has been used for treating human glioblastomas has not been as effective as expected after the first animal experiments. A better understanding of the different steps involved in this treatment, like gene transfer, gene expression, and sensitivity of the recipient cells, is needed. After proposing sensitivity criteria for the TK/GCV system and for the bystander effect, based on the levels of GCV that can be reached in vivo, we studied seven human glioblastoma cell lines (U87, U118, U251, SNB19, SNB75, SF295, SF539) for their sensitivity to the TK/GCV system. We also studied their in vitro bystander effect and their in vitro transfectability using LipofectAMINE as a transfection enhancer. Among six human glioblastoma cell lines stably transfected with the TK gene, five were sensitive to TK/GCV, and two had a good in vitro bystander effect. The in vitro transfectability of the cell lines tested was low (or = 1%) compared to that of an established animal cell line, C6 rat glioma, in which 20-30% of the cells can be transfected routinely. According to this in vitro analysis, most of the glioblastoma cell lines should be sensitive to the TK/GCV system, but there is an urgent need for agents to increase transfection efficiency.