Bioluminescence Imaging Captures the Expression and Dynamics of Endogenous p21 Promoter Activity in Living Mice and Intact Cells

Abstract

To interrogate endogenous p21^WAF1/CIP1 (p21) promoter activity under basal conditions and in response to various forms of stress, knock-in imaging reporter mice in which expression of firefly luciferase (FLuc) was placed under the control of the endogenous p21 promoter within the Cdkn1a gene locus were generated. Bioluminescence imaging (BLI) of p21 promoter activity was performed noninvasively and repetitively in mice and in cells derived from these mice. We demonstrated that expression of FLuc accurately reported endogenous p21 expression at baseline and under conditions of genotoxic stress and that photon flux correlated with mRNA abundance and, therefore, bioluminescence provided a direct readout of p21 promoter activity in vivo. BLI confirmed that p53 was required for activation of the p21 promoter in vivo in response to ionizing radiation. Interestingly, imaging of reporter cells demonstrated that p53 prevents the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway from activating p21 expression when quiescent cells are stimulated with serum to reenter the cell cycle. In addition, low-light BLI identified p21 expression in specific regions of individual organs that had not been observed previously. This inducible p21^FLuc knock-in reporter strain will facilitate imaging studies of p53-dependent and -independent stress responses within the physiological context of the whole animal.

ACKNOWLEDGMENTS

We thank Jiling Song, Sofia Origanti, and Courtney Robinson for technical support during the early stages of this project. We thank Mike White for performing blastocyst injections, Chris Ryan and Emily Powell for editorial assistance, and Reece Goiffon for assistance with statistics. Shin-ichiro Imai and Cindy Brace are thanked for help in isolating and culturing primary hepatocytes. Raleigh Kladney is thanked for help with mouse perfusions. Jason Weber and Jason T. Forys are thanked for advice on experiments related to serum starving of MEFs. We thank the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital for the use of the Embryonic Stem Cell Core and electroporation services. The RNAi Consortium of the Broad Institute and the Children's Discovery Institute are thanked for providing shRNAs.

This study was supported in part by grant P50 CA94056 to the Molecular Imaging Center at Washington University, by P30 NS057105 to Washington University, by NIH grant MH63140 to E.D.H., and by a DOD Prostate Cancer Research Program Training Award Grant (PC101951) to K.L.T. H.P.-W. is a Research Professor of the American Cancer Society. The Siteman Cancer Center is supported in part by an NCI Cancer Center Support Grant (P30 CA91842).