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A strong cohort of established investigators from a variety of disciplines detail their best techniques for advancing NPY research. Among the many readily reproducible methods described are those for cloning, for the production of transgenic and knockout models, and for the use of antisense technology to define the receptors and the signal transduction pathways mediating NPY actions. A range of the major qualitative and quantitative techniques used to study tissue mRNA distribution are also included, as well as methods for radioligand binding. Highly practical and state-of-the-art, Neuropeptide Y Protocols constitutes a gold-standard collection of proven techniques for advancing today's understanding of this significant neuropeptide, as well as for developing the promising NPY-based therapeutics now emerging for many important disorders.
E-Book Content
Methods in Molecular Biology TM VOLUME 153 Neuropeptide Y Protocols Edited by Ambikaipakan Balasubramaniam HUMANA PRESS cDNA Cloning 3 1 Cloning Neuropeptide Tyrosine cDNA Carolyn A. Worby 1. Introduction The nervous system is composed of a diverse population of cells selectively expressing different genes which ultimately determine the type of neurotransmitters and neuropeptides produced and released. Neuropeptide tyrosine (NPY) is one of the most abundant and widely expressed peptides in the mammalian nervous system (1). It was originally isolated by virtue of a chemical assay that detects the presence of a carboxyl-terminal amide in proteins (2); a hallmark of potential hormonal function. Its widespread expression in the central and peripheral nervous systems, in addition to the carboxyl-terminal amide, suggested that NPY could be a critical neurotransmitter in a variety of neuronal processes. We were therefore interested in obtaining the cDNA for huma