E-Book Overview
This readily reproducible collection of cutting-edge methods for the study of eicosanoids concentrates on studying the regulation of expression and function of enzymes, particularly cyclooxygenase (and its two isoforms), phospholipase A2, and lipoxygenases involved in the synthesis of established eicosanoids. Additional chapters are devoted to the characterization and distribution of the thromboxane A2 receptor in tissues and the biological roles of novel eicosanoids. Eicosanoid Protocols offers new and established researchers powerful, state-of-the-art tools to probe the regulation and function of eicosanoids.
E-Book Content
Methods in Molecular Biology
TM
VOLUME 120
Eicosanoid Protocols Edited by
Elias A. Lianos
HUMANA PRESS
Foreign Gene Expression
1
1 Use of a Retroviral Vector for Expression of a Foreign Gene Brent W. Miller and Aubrey R. Morrison 1. Introduction Expression of foreign proteins in both cultured and intact mammalian cells has become essential to evaluating cellular biology and physiology including the proteins involved in the control of eicosanoid production. Many transient expression vectors are commercially available and are in widespread use. However, some cell types, such as the renal mesangial cell, have been difficult to transform with these vectors and require a different strategy to obtain stable expression of the exogenous gene. Viral vectors, including retroviruses, adenoviruses, and the Sinbis virus have been developed over the last decade for foreign gene expression in mammalian cells. This chapter will discuss the use of a replication-incompetent retroviral vector in cell culture to express an exogenous protein. The molecular biology of retroviruses and retroviral vectors has been exhaustively reviewed elsewhere (1–3). The sophistication of these vectors and their use con