The Ahr-locus encodes the structural gene for the AH-receptor (AHR), a ligand activated transcription factor that mediates many of the biological responses of 2,3,7,8-trachlorodibenzo-p-dioxin (TCDD) and related polychlorinated biphenyls (PCBs). One of the directions of our research is to develop new models of the AHR signalling pathway and to use these models to identify the molecular basis of species dependent responses to TCDD (NIEHS R29-ES05703). In this proposal, we describe experiments that represent a new direction for our laboratory that should provide information valuable in characterizing the risks that compounds like TCDD and PCBs pose to the environment. Over the last two years, we have cloned the AHR cDNA and found that it contains a basic-region/helix-loop-helix domain similar to that found in its dimerization partner ARNT. More recently, our mutagenesis studies have provided a functional map of the AHR and have allowed us to localize those domains required for DNA binding, agonist binding, dimerization and transcriptional activation. In addition, we have cloned and characterized the AHR's structural gene, identified many of the 5' regulatory elements that control its expression and mapped its chromosomal location in mice and humans. In this application, we propose to use this new information and the related molecular probes to identify those developmental stages most sensitive to TCDD-toxicity and to construct transgenic mouse lines with mutant and controllable Ahr- and Arnt-loci. One of our highest priorities will be directed towards development of a targeting vector for use in """"""""knocking- out"""""""" the AHR and ARNT in embryonic stem cells (Bs cells). By injecting these recombinant Es cells into blastocysts, we propose to generate transgenic mice which are heterozygous and homozygous for a null mutation at the Ahr- and Amt-loci. We propose that the mouse strains gene rated under this project will prove valuable in the following ways, 1) to determine the phenotype of mice lacking an AHR or ARNT, 2) to identify the effects of TCDD and PCB mixtures that are not mediated by the AHR or ARNT, 3) as prototype strains for subsequent in vivo mutation studies of the Ahr- and Arnt-loci (e.g. humanizing the murine model), and 4) determining the role of altered gene expression in TCDD toxicity.
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