A Live Zebrafish-Based Screening System for Human Nuclear Receptor Ligand and Cofactor Discovery
(a) Schematic diagram of the multi-component ligand trap (LT) construct. Upon heat pulse, the zebrafish hsp70 promoter directs ubiquitous expression of the GAL4 DNA-binding domain (DBD) fused in-frame to a human nuclear receptor ligand-binding domain (LBD) and an affinity tag cassette (FSH-tag). Upon binding of this fusion protein to the GAL4 UAS (upstream activating sequence) response elements, in the presence of active hormone and cofactors, expression of the reporter gene (nuclear enhanced Green Fluorescent Protein (eGFP)) occurs. Expression of nuclear GFP is used to monitor receptor ligand sensor activity in a cell- and tissue autonomous manner in live zebrafish. The second component makes use of the tags to co-purify bound hormones or cofactors. I = insulator elements; pA = polyadenylation signal; NLS = nuclear localization signal. (b) Western blots of GAL4-NR fusion proteins. Embryos (F2; 72 hpf) were heat pulsed for 30 min at 37°C and recovered for 1 h at room temperature. 10 embryos were pooled and lysed in 50 µl of FSH buffer (see material and methods) followed by adding SDS buffer and boiling. Proteins were detected using the FLAG-M2 antibody. (c) Time course of fusion protein expression. TRβ embryos (F2; 72 hpf) were heat induced as in 1b) and recovered for the times indicated. Each sample contained 10 embryos.