MINISYMPOSIUM VII. Conditional Gene Disruption in the Mouse: Concepts and Applications.
11:00 AM-12:30 PM
Grand Ballroom V
Chair: Adashi, Eli1, 1
(M21) GENETIC DISSECTION OF NUCLEAR HORMONE RECEPTOR FUNCTIONS IN MICE.
Greiner, Erich1, Tronche, Francois2, Kellendonk, Christoph3, Reichardt, Holger4, Umland, Thorsten5, Schütz, Günther1, 1 German Cancer Research Center, Heidelberg, Germany2 Institut de Biologie, College de France, Paris, France3 Howard Hughes Medical Institute Research Laboratories, New York, NY4 Universität Würzburg, Würzburg, Germany5 Schering AG, Berlin, Germany
ABSTRACT- Nuclear hormone receptors function to transduce hormonal signals into transcriptional responses by controlling the activity of target genes directly. These target genes comprise a genetic network whose coordinate activity defines the physiological response to hormonal signals. Dissecting nuclear hormone receptor functions in vivo by gene targeting and transgenic strategies represents an invaluable and powerful approach to increase our knowledge of these genetic networks and their physiological functions. Genetic engineering in embryonic stem cells and mice has been revolutionized by the use of site-specific recombination systems consisting of two elements: the recombinase enzyme and a short DNA element specifically recognized by the recombinase. By employing site-specific recombination systems (e.g., the Cre-loxP system from bacteriophage P1 and the Flp-FRT system from the budding yeast Saccharomyces cerevisiae) "clean" germ line mutations following a single targeting event can be generated. In addition, genes can be (in)activated in a conditional manner in the living mouse. Besides gene targeting approaches, transgenic mice with stable genomic integrations of large DNA fragments derived from yeast and bacterial artificial chromosomes provide a means of analyzing gene function in vivo, because the expression of transgenes from large gemonic fragments display copy number dependence and position independence. Experimental strategies to genetically manipulate nuclear hormone receptor functions in vivo will be presented. Genetic manipulations dissecting the glucocorticoid receptor function will illustrate the described experimental strategies including loss-of-function mutations, target gene mutations induced in a temporally and spatially restricted fashion, function-selective mutations, and overexpression of the glucocorticoid receptor. Mice with a disrupted GR gene (GR null mutation) die shortly after birth due to respiratory failure indicating an important role of GR in lung function. To separate DNA-dependent from DNA-independent functions of the GR a point mutation that prevents dimerization and subsequent dimeric DNA binding was introduced into the D-loop of the receptor. Mice carrying this mutation (GRdim) survive and distinguish between GR functions dependent on DNA binding and those mediated by protein-protein interaction. Since mice with a disrupted GR gene die after birth, cell-specific mutations have been generated with the Cre-loxP system. The GR gene was inactivated in liver, thymus, monocytes/macrophages, and brain. Besides gene targeting approaches, transgenic mice with stable genomic integrations of two additional GR alleles derived from a yeast artificial chromosomes will be presented, which will allow investigation of the effects of an increased gene dosage of the GR.
KEY WORDS: nuclear hormone receptor, gene targeting, site-specific recombination, allelic series