The study presents improvements in methods for targeted gene expression in Drosophila using GAL4, LexA, and Split GAL4 transcription factors, along with the GAL80 repressor. The researchers optimized core promoters, UTRs, operator sequences, and activation domains to enhance the reliability and strength of gene expression. They used site-specific integration to compare different constructs at the same genomic location and characterized PhiC31 integration sites for their ability to support transgene expression in the nervous system. The optimized reagents allow for more precise and complex genetic manipulations. The ability to express a gene of interest in a spatially restricted manner in a transgenic animal has greatly contributed to the use of Drosophila in various biological studies. In conjunction with RNA interference and proteins engineered to alter or report cell function, directed gene expression enables precise manipulation of single cells or cell types and their visualization. This is essential in the nervous system, where understanding function requires probing the structure and activity of individual, identified cells. The researchers previously described an approach for identifying a large set of enhancers that can each reproducibly drive expression of a reporter gene in a distinct, small subset of cells in the adult central nervous system. They found limitations in current methods using yeast GAL4 and bacterial LexA transcription factors. In this article, they report efforts to systematically improve these widely used methods. The GAL4 transcriptional activator from Saccharomyces cerevisiae functions in Drosophila, and the GAL4/UAS binary expression system has become a powerful tool for directed gene expression. The researchers evaluated factors affecting the pattern and strength of GAL4-driven expression, including codon usage, transcriptional terminator, and activation domain. They also showed how varying the number of UAS sites or including introns or post-transcriptional regulatory elements in the UTRs influences the expression level of the target gene and the perdurance of its product. The repressor LexA is a regulator of the SOS response to DNA damage in Escherichia coli. Fusing a C-terminal activation domain derived from GAL4 or VP16 to LexA allows it to drive in vivo transcription of reporter transgenes in Drosophila whose promoters contain LexAop motifs. Use of LexA/LexAop as a complementary binary system in conjunction with GAL4/UAS has proven useful in various applications. However, the published LexA drivers tend to be less effective than GAL4 as transcriptional activators. The DNA-binding and transcription-activating functions of GAL4 are accomplished through different functional domains of the protein that can be separated into distinct polypeptides. When association of the separated domains is promoted by protein–protein interactions, the activity of GAL4 as a transcription factor is restored. This is the basis of the two-hybrid method for screening for protein–protein interactions. Another strategy for refining expression patterns in GAL4 lines is targeted suppression of GAL4 activity in a subset of cellsThe study presents improvements in methods for targeted gene expression in Drosophila using GAL4, LexA, and Split GAL4 transcription factors, along with the GAL80 repressor. The researchers optimized core promoters, UTRs, operator sequences, and activation domains to enhance the reliability and strength of gene expression. They used site-specific integration to compare different constructs at the same genomic location and characterized PhiC31 integration sites for their ability to support transgene expression in the nervous system. The optimized reagents allow for more precise and complex genetic manipulations. The ability to express a gene of interest in a spatially restricted manner in a transgenic animal has greatly contributed to the use of Drosophila in various biological studies. In conjunction with RNA interference and proteins engineered to alter or report cell function, directed gene expression enables precise manipulation of single cells or cell types and their visualization. This is essential in the nervous system, where understanding function requires probing the structure and activity of individual, identified cells. The researchers previously described an approach for identifying a large set of enhancers that can each reproducibly drive expression of a reporter gene in a distinct, small subset of cells in the adult central nervous system. They found limitations in current methods using yeast GAL4 and bacterial LexA transcription factors. In this article, they report efforts to systematically improve these widely used methods. The GAL4 transcriptional activator from Saccharomyces cerevisiae functions in Drosophila, and the GAL4/UAS binary expression system has become a powerful tool for directed gene expression. The researchers evaluated factors affecting the pattern and strength of GAL4-driven expression, including codon usage, transcriptional terminator, and activation domain. They also showed how varying the number of UAS sites or including introns or post-transcriptional regulatory elements in the UTRs influences the expression level of the target gene and the perdurance of its product. The repressor LexA is a regulator of the SOS response to DNA damage in Escherichia coli. Fusing a C-terminal activation domain derived from GAL4 or VP16 to LexA allows it to drive in vivo transcription of reporter transgenes in Drosophila whose promoters contain LexAop motifs. Use of LexA/LexAop as a complementary binary system in conjunction with GAL4/UAS has proven useful in various applications. However, the published LexA drivers tend to be less effective than GAL4 as transcriptional activators. The DNA-binding and transcription-activating functions of GAL4 are accomplished through different functional domains of the protein that can be separated into distinct polypeptides. When association of the separated domains is promoted by protein–protein interactions, the activity of GAL4 as a transcription factor is restored. This is the basis of the two-hybrid method for screening for protein–protein interactions. Another strategy for refining expression patterns in GAL4 lines is targeted suppression of GAL4 activity in a subset of cells