Constructing a p53-fused dual luciferase reporter and verifying its function

Abstract

To construct a p53-fused dual luciferase reporter and to test whether this reporter can mimic wild-type p53 activities in a high-throughput screen. A restriction endonuclease site was added to each terminus and the stop codon of the wild-type full-length p53 open reading frame (ORF) was removed by PCR. A restriction endonuclease site was added to each terminus and the start codon of the firefly luciferase ORF was removed by PCR. The two modified ORFs were inserted upstream of the IRES-induced renilla luciferase ORF in a CMV-derived vector. The p53 fusion protein was expressed in cells to test its MDM2-mediated degradation, subcellular localization, and induction of p53-responsive promoter. The p53-fused dual luciferase reporter was successfully constructed. After transfection into the host cells, the reporter expressing the p53 fusion protein that was degraded by oncoprotein MDM2, was mainly located inside the nucleus, and induced the p53-responsive promoter, respectively. The p53-fused dual luciferase reporter (p53FL/IRES/RL) can identify modulators of P53 protein level in a high-throughput screen of genetic or chemical libraries.