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 p21FLuc knock-in reporter strain will facilitate imaging studies of p53-dependent and -independent stress responses within the physiological context of the whole animal.

Basal p21 controls population heterogeneity in cycling and quiescent cell cycle states

“…S1E). The single-cell dynamics, which indicated that p21 decreases at the start of S phase and increases at G2, were in line with previous studies that used synchronized populations to show the cell cycle-dependent regulation of p21 (23)(24)(25). Finally, at intermediate growth factor stimulation [5 ng/mL EGF, 1.25% (vol/vol) serum], ectopic p21 restored the heterogeneity in cell cycle activity exhibited by WT cells (Fig.…”

Illuminating Cancer Systems with Genetically Engineered Mouse Models and Coupled Luciferase ReportersIn Vivo

“…Additionally, due to overall low photon output, luciferase reporters traditionally have been limited to macroscopic imaging analysis. However, recent advances in low-light microscopy technologies have permitted the interrogation of live cells and live bioluminescent tissues ex vivo at high magnifications (37-39), a notable advance that extends the capacity of BLI.…”

zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish

“…Luciferase-based imaging has been widely used in mice for monitoring gene expression, cell proliferation and migration, and bacterial and viral infections (Contag and Bachmann, 2002;Luker and Luker, 2008;Liu et al, 2011;Tinkum et al, 2011;Martínez-Corral et al, 2012;Maguire et al, 2013). Detection of bioluminescent signals generated from the luciferase-luciferin reaction is sensitive, virtually free of background noise, and can reveal biological information even from deep tissues.…”