USE OF GENETICALLY ENGINEERED MICE AS MODELS FOR EXPLORING THE ROLE OF OXIDATIVE STRESS IN NEURODEGENERATIVE DISEASES

Julie K. Andersen

Division of Biogerontology, Andrus Gerontology Center, University of Southern California, Los Angeles, CA 90089-0191

Received 3/16/98 Accepted 3/20/98

2. INTRODUCTION

Until recently, the only animal models available to the neurobiologist for studying age-related neurodegenerative diseases in which genetics were believed to play either a primary or predisposing role were spontaneous genetic mutants. In the last two decades, however, techniques have been established for the introduction of selected mutations in vivo as a means of mimicking human disorders. The ability to create laboratory mouse strains containing targeted mutations has helped extend our understanding of many diseases including several of the neurodegenerative diseases associated with aging. Genetically engineered mouse strains have given us important information concerning various factors involved in degeneration of the nervous system during normal aging and in age-related disease states and have provided valuable animal models for testing new drug treatments.

Ectopic expression of novel genes can be achieved through a process called transgenics which involves the microinjection of cloned DNA into the pronuclei of fertilized mouse eggs (11). Depending on the site of chromosomal integration, the DNA can be transcribed and translated into a functional protein. The injected eggs are then introduced into pseudopregnant females and allowed to develop. If integration into the mouse's genomic DNA occurs at the one cell stage, the integrated DNA will be contained in all cells of the mouse's body including its germ line cells and therefore becomes a heritable complement of its genetic make-up. Normally, the DNA construct used for transgenic production is designed to contain the gene of interest expressed under the control of a regulatory promoter element which designates when (i.e. at what developmental times) and where (i.e. in what cell types) transgene expression will occur in the genetically engineered mouse.

A second type of genetic engineering called gene targeting (also called "gene knock-out") can be used to alter endogenous genes of interest in the mouse's DNA (12-13). First, the cloned gene fragment is altered in vitro often via insertion of a neomycin (neo) resistance gene into an exonic coding region of the gene. In addition, thymine kinase sequences from the herpes simplex virus (HSV-tk) are introduced at both ends of the linearized transgene. The altered gene is then introduced into pluripotent embryo-derived stem (ES) cells in culture by either direct injection or electroporation. Homologous recombination occurs between the altered transgene and the endogenous ES gene at the region of homology between the transgene and endogenous genomic target sequence. During homologous recombination the distal HSV-tk sequences are eliminated. Cells containing the homologously integrated copy of the gene can therefore then be selected by growth in media containing neomycin and gancyclovir. The selected cells are then introduced into mouse blastocysts where they can become any part of the tissue of the developing animal including the germ line cells. Resulting chimeric offspring are bred resulting in mice which, if the ES cells have become part of the germ line, carry the alteration in one copy of the gene in all cells (ie heterozygous mutants). These can be further bred to obtain mice in which the mutation is found in both copies of the gene (ie homozygous mutants).

These techniques have been used to create a myriad of genetically engineered animal models, some of which have been extremely important in allowing scientists to examine the role of oxidative stress in a number of neurodegenerative disease states. A number of these studies are described below.