pair fragment does not contain all of the required positive regulatory elements that govern expression of the β4 gene, as there are regions of the brain that express relatively high levels of the β4 gene but in which no transgene activity was detected. In some cases, as described above, this may be due to the absence of specific regulatory elements in the transgene used in the present study. For example, the CNR4 element identified by the Deneris group and shown to drive transgene expression in the pineal gland, adrenal gland and specific regions of the brain, was not part of the transgene construct. It is also possible that other, yet to be identified positive regulatory elements are also required for appropriate β4 gene expression. In addition to the β4 promoter activity that corresponds to endogenous β4 mRNA expression, we also detected promoter activity in brain regions that have not been reported to express the β4 subunit. These observations likely underscore the importance of negative regulatory elements in β4 gene expression and may be a consequence of the absence of a cell-type-specific repressor in the transgene used in this study. We previously demonstrated in vitro that this repressor, referred to as
