The goal of this research is to apply an innovative technology for gene knockout to identify and understand developmental effects of drugs and other chemicals in the premier non-mammalian model for developmental toxicology and pharmacology (zebrafish, Danio rerio). Many drug-metabolizing genes are regulated via the pregnane X receptor (PXR, NR1I2). The transcriptional targets of PXR are not known in zebrafish, nor are any potential differences between developmental and adult PXR gene regulation. We hypothesize that PXR regulates a broad suite of genes essential to drug and pollutant metabolism in zebrafish. Developing a full understanding of the regulatory roles played by PXR is critical to obtaining a mechanistic foundation for understanding chemical effects in this vital animal model. To address the role of PXR in zebrafish, we propose to use a new genetic engineering technology, transcription activator-like effectors (TALE) nucleases (TALENs), to knock out PXR functioning in zebrafish. Engineered gene knockout model organisms offer potent systems for studying drug metabolism and disposition, and zebrafish provide access to early, sensitive stages of development, with transparent embryos allowing direct observation of all developmental stages. Targeted nuclease activity will be directed using the high-affinity binding of FokI-TALE fusion proteins to genomic DNA, followed by targeted FokI restriction cleavage, resulting in DNA double strand breaks and error-prone repair. Using this knockout line we will determine the genes regulated by PXR in developing zebrafish. We will also use known zebrafish PXR ligands, including pregnenolone-16?-carbonitrile to induce transcription. An important challenge in model organism research is the establishment of direct correlations with human effects. In vitro studies using isolated ligand binding domains (LBDs) reveal differences in ligand binding between human and zebrafish but do not adequately reflect the complexity of in vivo cellular interactions. To address the differences in ligand specificity between human PXR and zebrafish PXR, we will create 'humanized PXR'zebrafish using chimeric PXR constructs containing a human ligand binding domain in the zebrafish genomic context. We will take advantage of the homologous recombination induced by the TALENs-produced double strand break repair to insert the chimeric DNA into the proper genomic context. We will determine the response of embryos from humanized PXR zebrafish to hyperforin and pregnenolone sulfate, compounds for which in vitro data indicate significant species differences in PXR activation between humans and zebrafish. This project uses an emerging and highly innovative technology (TALENs) that has only been applied in a handful of studies in animals and will produce one of the first "humanized" zebrafish, and the first humanized zebrafish for pharmacological and toxicological research. This research will provide essential information and tools for other researchers who may study mechanisms of chemical effect in the zebrafish.
The pregnane X receptor (PXR) is a nuclear receptor that regulates many drug-metabolizing genes, and thus can determine the persistence and action of many drugs and other toxicants and carcinogens. To address the transcriptional role of PXR in the powerful zebrafish (Danio rerio) development model, we propose to use a new genetic engineering technology, transcription activator-like effectors (TALE) nucleases (TALENs), to knock out PXR functioning in zebrafish. We will also develop a 'humanized PXR'zebrafish using chimeric PXR constructs that will be a better model for drug and teratogen screening than wild-type zebrafish, with PXR activation that more closely resembles that of human PXR.