New Conditional Gene Targeting Methods : For Studying Neurotrophic Mechanisms in Selected Neuronal Populations

Abstract: Powerful techniques to manipulate the mouse genome have had a great impact on our understanding of biology. The major drawback with conventional transgenic methods is that the genetic alteration will be present in every cell of the animal, from conception and onwards. Many genes are normally expressed in several different organs and at different times and an effect observed in the transgenic mouse could therefore be derived from various tissues or be a developmental consequence. Furthermore, some mutations will result in a lethal phenotype and prevent investigations on the adult gene function. This is a particular problem when studying the brain since it is not yet fully developed at birth. Consequently, a conditional methodology is required, where precise chromosomal alterations can be achieved in restricted tissues at chosen times in the living animal. This issue has been successfully tackled during the last decade. Conditional gene targeting is today a fact.This thesis demonstrates that it is possible to use an internal ribosomal entry sequence for tissue-specific direction of conditional gene targeting tools. General cassettes were constructed and shown to work in cells. Moreover, a knock-in transgenic mouse expressing the Cre recombinase in catecholaminergic neurons was made. This strain will offer possibilities to study genetic mechanisms in dopamine and noradrenaline producing cells and to build mouse models for common human diseases involving such neurons.Furthermore, another transgenic mouse was created having the Cre recombinase under tight tetracycline-regulated control. This strain can mediate inducible genetic recombination in brain neurons and be completely silenced by feeding the mice the antibiotic doxycycline. Moreover, by altering the time-point of doxycycline administration, chromosomal manipulations can be achieved in different neuronal patterns. This transgene is a general tool that can be combined with any mouse having floxed genes and also with lines producing tetracycline transactivators in other locations.Finally, constructs aiming at conditional manipulation of the nerve growth factor gene and targeting of sensory neuronal populations are described.