Construction of reporter gene vectors with an oestrogen responsive promoter. 3ERE, TATA and Gal4ff were inserted into the BamHI and NotI sites of the plasmid pBR322 vector containing a Tol2 element. Xeno-oestrogens bind to and activate the ER. The homodimer complex binds to specific DNA sequences called the oestrogen response element (ERE) within regulatory regions of oestrogen responsive genes. Transcription is induced and messenger RNA is translated into protein. GAL4ff activates “UAS (Upstream Activated Sequence) – GFP (Green Fluorescent Protein), in this “two-step” activation process (Step 1: Oestrogen induces synthesis of GAL4ff protein, step 2: GAL4ff protein induces synthesis of GFP).