A novel strategy for conditional gene knockout based on ΦC31 integrase and Gal4/UAS system in Drosophila

Abstract

The Gal4/upstream activating sequences (UAS) system offers great advantages in target gene expression by separating the responder line and diverse tissue-specific expressed Gal4 driver lines in Drosophila. The bipartite system is commonly used in gain-of-function analysis, and by combining with the RNA interference technology, it can also be applied in loss-of-function analysis. However, the off-target effect caused by this strategy has not been well solved so far. Furthermore, it can only partially knockdown a specific gene expression. In this study, a novel conditional gene knockout method that combined the use of ϕC31 integrase and Gal4/UAS system was described. The target gene was preliminarily flanked by ϕC31 integrase recognition sites attB and attP, followed by conditional expressed Gal4 lines to drive the recombinase that were under UAS control to achieve spatial and temporal gene deletion. We found the strategy performed well in Drosophila, and the efficiency was higher than 82% in gene knockout by self-excision. Our strategy takes advantage of exiting Gal4 library to drive the recombinase, rather than conventionally used method which the recombinase was droved directly by specific promoters, thereby providing a more flexible and versatile tool for gene function analysis in Drosophila.