Construction of Promoter Elements for Strong, Moderate, and Weak Gene Expression in Drosophila melanogaster.

BACKGROUND/OBJECTIVES: Transcriptional promoters play an essential role in regulating protein expression. Promoters with weak activity generally lead to low levels of expression, resulting in fewer proteins being produced. At the same time, strong promoters are commonly used in studies using transgenic organisms as model systems. This approach can have various negative consequences for the organism, as many regulatory proteins need to be expressed in small quantities, and excessive expression can have harmful effects on cells and organisms. Therefore, it is important to select the right promoter when creating transgenic organisms for research and practical applications. METHODS: In this study, we used the Drosophila melanogaster genome as a source of natural promoter sequences for RNA polymerase II. These sequences were extracted and used to create a set of promoters that are suitable for practical application. The promoters were tested in a model system using fluorescent reporter genes in S2 cells and transgenic lines of Drosophila. RESULTS: We assessed the expression levels of fluorescent reporter genes to rank the tested promoters from strongest to weakest. Six individual promoters of different sizes were established and compared. Additionally, we designed and tested three pairs of bidirectional promoters that could be used to simultaneously express two proteins. CONCLUSIONS: Based on our findings, we grouped the tested promoters into three categories: strong, moderate, and weak. These promoters can be utilized in transgenic model systems for protein production at different levels, from high to low. Bidirectional promoters, constructed "head-to-head", meaning oppositely directed with the minimum distance between them, represent a novel tool for the co-expression of proteins.