title: 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.
authors.affiliation: Department of Cell Biology and Physiology, Washington University, St. Louis, Missouri 63130, USA.
authors.forename: Kelsey L
authors.initials: KL
authors.lastname: Tinkum
authors.forename: Luciano
authors.initials: L
authors.lastname: Marpegan
authors.forename: Lynn S
authors.initials: LS
authors.lastname: White
authors.forename: Jinwu
authors.initials: J
authors.lastname: Sun
authors.forename: Erik D
authors.initials: ED
authors.lastname: Herzog
authors.forename: David
authors.initials: D
authors.lastname: Piwnica-Worms
authors.forename: Helen
authors.initials: H
authors.lastname: Piwnica-Worms
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics* / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D055879:Gene Knock-In Techniques; D022781:Hepatocytes / Q000378:metabolism; D049409:Luciferases, Firefly / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011839:Radiation, Ionizing; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D049409:Luciferases, Firefly; D048049:Extracellular Signal-Regulated MAP Kinases