International Mammalian Genome Society

The 15th International Mouse Genome Conference (2001)


POSTER 236 - CONSTRUCTION OF ES CELL LINES WHICH FACILITATE EXPRESSION GENE TRAP MUTAGENESIS STRATEGIES IN CARDIOMYOCYTES

Dr. Barry Rosen
INSERM U470
Biochimie
Universite de Nice
Parc Valrose
Nice, 06108
France

Co-Authors: 1)Ranc F, 2)Stanford WL
Institutions: 1)INSERM U470, 2) The Gene Trap Laboratory, Samuel Lunenfeld Research Institute

Gene trap mutagenesis is an efficient strategy for simultaneously identifying, mutating, and reporter tagging random loci in the mouse genome.  The ability of ES cells to differentiate into a various lineages, can be utilized as a tool to pre-screen gene trap events by their reporter expression patterns in vitro. We are developing expression screening methods suitable for large numbers of gene trap events representing the entire transcribed genome. We are generating ES cell lines in which the puromycin resistance gene has been knocked into the cardiomyocyte specific nkx2.5 locus by gene targeting.  Nkx2.5 is homogeneously expressed in precardiac mesoderm and all populations of both embryonic and adult cardiomyocytes, and the knock-in should allow selection of pure populations of these cells.  Duplicate sets of embryoid bodies will be generated from individual gene trap events, and one duplicate subjected to puro selection to specifically enrich in cardiomyocyte lineage cell populations.  Gene trap events displaying enhanced reporter expression in pure cardiomyocyte populations compared to mixed populations of differentiated cells are then identified and will be used to generate mutant mice.  This strategy will be developed in a high throughput format to eventually allow the evaluation of thousands of individual gene trap events and thus the generation of many novel and informative cardiac mutations and phenotypes


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