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Background: In previous studies we have found that the tetracycline (Tet)-regulatable system functions best in recombinant adenoviral (Ad) vectors when the Tet transactivators and the Tet-regulatable element (TRE) are incorporated into separate viral vectors. However, such a dual vector system is disadvantaged by the need to use relatively high titres that may elicit an immune response. Therefore, to develop a system that could be used at low titres while mediating strong, tightly regulatable gene expression in the central nervous system (CNS), we incorporated the woodchuck hepatitis virus post-transcriptional enhancer (WPRE) into a neuron-specific Tet-regulatable Ad system., Methods: The WPRE was incorporated into Ad vectors encoding the Tet-Off (tTA) transactivator driven by the synapsin-1 and CMV promoters and encoding the TRE driving EGFP expression (TRE)-EGFP., Results: The addition of the WPRE to the neuron-specific Tet-regulatable system mediated a greater than three-fold increase in transgene expression in primary hippocampal neurons with no loss of gene regulation. The results also showed that the addition of the WPRE enhanced transgene expression in the CNS without the loss of neuron specificity and without affecting the ability to regulate transgene expression., Conclusions: We have further developed a tetracycline-regulatable neuron-specific expression system such that it can now be used at low titres with no loss of transgene expression or ability to regulate transgene expression. It should therefore be of significant value to studies investigating neuronal gene function and to those seeking to develop effective neuronal gene therapy strategies., (Copyright (c) 2004 John Wiley & Sons, Ltd.)