Chapter 2 Use of gene transfer approaches to study regulation of neuropeptide gene expression

Abstract

This chapter discusses gene transfer techniques to study the regulation and expression of the human proenkephalin gene and several other opioid genes. A mouse anterior pituitary tumor-derived corticotrophic cell line has been transfected with the human proenkephalin gene and the characteristics of several of the isolated clones are discussed in the chapter. It also discusses the expression of proenkephalin in three transient systems: (1) proenkephalin fusion genes, (2) Xenopus oocytes, and (3) vaccinia virus. Gene transfer experiments can be divided into two major categories—namely, stable transfections and transient assay systems. To study stably transfected cell lines, a clone of eukaryotic cells that has integrated the foreign gene into its chromosomal DNA is isolated. The major advantage of this system is the ability to isolate stable cell lines, which can be grown indefinitely in culture and used to study a variety of regulatory phenomena. The gene of interest can be introduced into eukaryotic cells by several different methods, including (a) chemical methods, such as the calcium phosphate precipitation method, (b) physical methods, such as microinjection or electroporation, (c) viral mediated DNA transfer, and (d) the fusion of DNA-containing membranous vesicles, such as liposomes, the envelopes of Sendai virus particles, or protoplasts to cells.