E-Book Overview
This comprehensive collection of the major cross-disciplinary methods and protocols used in current opioid research covers topics ranging from molecular and genetic techniques, to behavioral analyses of animal models and to clinical practices. The well-practiced authors describe their best molecular techniques for the cloning and expression of opioid receptors, and for the quantitative characterization of their signaling pathways, as well as for mapping the distribution and detecting the expression levels of opioid receptors, opioid peptides, and their messages in both brain tissues and individual cells. Also included are methods for the creation of in vitro and in vivo animal models to study opioid functions, as well as the clinical applications in the treatment of pain and opioid addiction.
E-Book Content
M E T H O D S I N M O L E C U L A R M E D I C I N E TM
Opioid Research Methods and Protocols Edited by
Zhizhong Z. Pan
Molecular Cloning of Opioid Receptors
3
1 Molecular Cloning of Opioid Receptors by cDNA Library Screening Ying-Xian Pan 1. Introduction In order to obtain cDNA clones encoding opioid receptors, one conventional strategy is to screen a cDNA library by using either a nucleic acid probe or an antibody probe. Many opioid receptor cDNA clones have been identified by the cDNA library screening (1–16). Different types of cDNA libraries made from a variety of tissues or cells are available from various companies such as Strategene, ClonTech, and Invitrogen. cDNA libraries are commonly constructed in bacteriophage λ vectors, which are advantageous in their highly efficient and reproducible packaging systems in vitro. However, cDNA expression libraries are usually made in mammalian expression plasmid vectors, which can be screened by expression cloning with a specific radiolabeled ligand or an antibody probe in a mammal