Ligand Regulated Site Specific Recombination in Mammalian Cells

Abstract

Chapter 1 of this thesis summarizes the current knowledge about the nuclear receptor superfamily of transcription factors. Chapter 2 describes the materials and methods used to carry out the research presented in the next 4 chapters. In chapter 3, I demonstrate that the FLP site specific DNA recombinase can be regulated by steroid ligands. The kinetics of recombination mediated by FLP-steroid receptor ligand binding domain fusion proteins (FLP-LBD) are described in this chapter and the parameters affecting these kinetics are discussed. In chapter 4 the effect of synthetic steroid ligands on the kinetics of FLP-LBD mediated recombination are explored using a combination of site directed mutagenesis and dose response experiments. In chapter 5, the generation of EBDs with altered ligand specificities by amino acid changes at 2 positions in the EBD is described. Finally, in chapter 6, the capacity of a number of nuclear receptor superfamily members to conditionally repress FLP is analysed