REVERSE GENETICS OF THE MOUSE CENTRAL-NERVOUS-SYSTEM - TARGETED GENETIC-ANALYSIS OF NEUROPEPTIDE FUNCTION AND REVERSE GENETIC SCREENS FOR GENES INVOLVED IN HUMAN NEURODEGENERATIVE DISEASE

The development of gene targeting technology in mouse embryonic stem cells allows reverse genetics to be used to investigate the function of any cloned gene in the developing and adult brain. Promoter-trap, replacement and insertion vector strategies can be used to generate defined mutations in the chromosomal copy of a cloned gene in embryonic stem cells. These cells can be used to make chimaeric mice, some of which transmit the in vitro mutation via the germline to transgenic offspring. The phenotype of complete loss-of-function mutations (gene knock-outs) can be studied at molecular, cell biological, neurophysiological and behavioural levels, and allows inferences about gene function to be made. Precise small mutations can also be made using integrative vector or two-step replacement vector strategies, allowing specific questions to be asked about regulation and protein structure-function relationships. Reverse genetics can therefore be used as an alternative or additional approach to pharmacology for the study of molecular functions in the central nervous system. Reverse genetic studies of the involvement of particular molecules in neurological disease syndromes may be superior to pharmacological studies to the extent that the syndrome is determined by genetic predisposition. The general ways in which reverse genetics of the mouse can be used to ask questions about molecules in the central nervous system are illustrated by examples from ongoing work of this laboratory. Neuropeptides are an important class of transmitters in the brain, but only in very few cases have specific CNS functions been assigned to a particular neuropeptide. Targeted mutation of neuropeptide precursor and receptor genes offers a rapid way to learn about neuropeptide function. Complete loss-of-function mutations will provide information on any developmental roles of a neuropeptide and on overall behavioural and physiological effects of loss-of-function. More specific targeted mutations allow dissection of the individual roles of multiple neuropeptides that derive from a common precursor protein, and allow in vivo studies of the functional importance of particular amino acids. Experimental progress towards targeted mutation of the neurotensin receptor is described as an example. Recent technological improvements makes targeted mutation of a number of genes possible. This allows reverse screening to be undertaken for genes involved in particular neurobiological phenomena: genes are identified on the basis of molecular criteria (e.g. expression pattern), and gene-targeting used to check their relevance to a phenotype. Neurodegenerative disease is an important aspect of the human phenotype. In both Parkinson's disease and Alzheimer's disease particular neuronal cell-types or particular brain regions are much more susceptible than others. Reverse genetic screens have been established for molecules that might determine these susceptibilities. Subtractive hybridisation with biotinylated driver nucleic acid molecules is used, followed by removal of the biotinylated molecules and hybrid molecules with streptavidin coated magnetic beads, to generate subtracted cDNA libraries, e.g. for the dopaminergic-neurone rich area of mouse ventral midbrain. Candidate genes with localised expression patterns are identified by differential screening and differential display analysis followed by in situ hybridisation. The effects of targeted mutations in these genes on neuronal physiology and survival will be studied. Ongoing improvements in technology will make reverse genetics increasingly accessible, and technological solutions to all the problems of execution and interpretation that are commonly discussed can be envisaged. This, combined with a renaissance of standard mouse genetics as a fine physical map of the genome is established, will make mouse genetics a major contributing technology in neuroscience.