The use of zebrafish in forward genetic screens, in vertebrate biology, and in human disease modeling has been hampered by a general lack of control over transgene expression. The ability to regulate the temporal and spatial expression of transgenes within zebrafish will open up many new areas of research thus far inaccessible. We have successfully adapted to the zebrafish, inducible gene technology based on the highly specific interaction of inert small molecule inducers and ecdysone receptor (EcR)-based proteins that control the timing and level of transgene expression in eukaryotic cells. The functional receptor complex consists of an ecdysone receptor Iigand-binding domain (LBD) fused to a GAL4 DNA binding domain (DBD) and a truncated retinoid X receptor (RXR) protein fused to an acidic transactivation domain, such as VP16. Constitutive or regulated promoters drive expression of these receptor proteins providing spatial control. Temporal control is provided by the addition of an inducing ligand. Only in the presence of the inert ligand can the EcR-LBD protein heterodimerize with the transactivation domain fusion protein and bring about transcription of the target gene. This EcR-based gene switch shows extremely low levels of background reporter gene activity in the absence of inducer. In both transient and stable zebrafish transgenics, addition of an inducing ligand leads to a target gene's RNA within hours. Protein expression is seen within 6 hours after ligand addition, is proportional to ligand concentration, and increases in intensity over the ensuing 48 hours. The level of protein induction is directly related to the concentration of the inducer over a broad dynamic range and is rapidly reversed upon removal of the ligand. In the proposed studies, we will better characterize the inducer and receptor elements that allow transgenes to be independently and tightly regulated thereby achieving almost complete control over transgene expression in the zebrafish. Relevance to public health: The zebrafish has become an important model system for investigating many aspects of human disease initiation, growth and treatment. The ability to regulate zebrafish transgene expression would have wide-ranging implications for successful new inquiries into areas of developmental biology and human disease modeling that cannot be answered using currently available technology.
|Thakur, Prakash C; Davison, Jon M; Stuckenholz, Carsten et al. (2014) Dysregulated phosphatidylinositol signaling promotes endoplasmic-reticulum-stress-mediated intestinal mucosal injury and inflammation in zebrafish. Dis Model Mech 7:93-106|
|Stuckenholz, Carsten; Lu, Lili; Thakur, Prakash C et al. (2013) Sfrp5 modulates both Wnt and BMP signaling and regulates gastrointestinal organogenesis [corrected] in the zebrafish, Danio rerio. PLoS One 8:e62470|
|Thakur, Prakash C; Stuckenholz, Carsten; Rivera, Marcus R et al. (2011) Lack of de novo phosphatidylinositol synthesis leads to endoplasmic reticulum stress and hepatic steatosis in cdipt-deficient zebrafish. Hepatology 54:452-62|