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An indispensable collection of novel techniques that have proven most useful for studying the physiological properties of p53 both in vitro and in vivo. The techniques provide proven solutions to problems in studying the purification, target identification, gene expression, quantitation, interaction, signaling, transactivation, and transrepression of p53. The methods are also useful for delineating the functions of other proteins that may act as tumor or growth suppressors. Each technique includes step-by-step instructions, troubleshooting notes, a theoretical review, and discussion of associated problems that might arise during the course of investigation.
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Methods in Molecular Biology TM VOLUME 234 p53 Protocols Edited by Sumitra Deb Swati Palit Deb Adenovirus Expressing p53 1 1 Adenovirus Expressing p53 Matthew Holmes, Elizabeth Rosenberg, and Kristoffer Valerie 1. Introduction Mutation in the p53 gene is the most frequently found genetic abnormality in human tumors, resulting in increased resistance to chemo- and radiotherapy (1,2). The underlying rationale for p53-mediated cancer gene therapy is to restore the ability of p53 mutant tumors to undergo apoptosis. When the wildtype p53 gene is delivered into tumor cells of various origins by adenoviral vector, the cells become more sensitive to cisplatin and radiation therapy (3). In addition, adenovirus expressing p53 is a great laboratory tool for the determination of function, for conveniently producing large quantities of p53 protein, and for determining the effect of p53 on cellular gene expression using microarray technology (4). The technology for making recombinant adenovirus was developed in the early 1980s by several groups (5,6). Although the principles for making recombinant adenovirus remain the same today, sophisticated and clever wa