Gene trap studies of the mouse genome and embryonic development

grantor: University of TorontoIn recent years, gene trap insertional mutagenesis strategy has been developed and successfully used to disrupt and study developmentally regulated genes in the mouse embryos. Using a gene trap approach, two independent mouse embryonic stem (ES) cell clones were identified and investigated for developmental expression pattern, disrupted gene sequences and phenotypic effects. The strategy made use of a promoterless 'lacZ' reporter gene to disrupt and mark genomic loci and to determine the expression pattern of genes. I193 and R140 gene trapped ES cell clones were isolated in a retinoic acid (RA) response screen and were then characterized by myself as described in this dissertation. The RA-induced Dr repetitive element and RA-induced 'Aquarius ' gene were cloned from the I193 clone. Dr elements are highly similar in sequence, show peculiar structural features and are expressed in a spatially restricted manner. The expression of Dr transcripts seems to be part of an ES cell differentiation program induced by RA. 'Aquarius' is a novel gene with weak similarity to RNA-dependent RNA polymerases (RRP) of the murine hepatitis viruses and it contains an RRP motif. During embryonic development, 'Aquarius' is expressed in mesoderm, neural crest and neural crest-derived tissues, and in neuroepithelium. Mice homozygous for the 'Aquarius' gene trapped allele are viable, are normal in size and weight, and breed normally. The gene trapped locus, designated 'myocyte maintenance' ('mym'), was identified and characterized in the R140 clone. ' mym' is expressed in heart, limbs, gut, meninges and choroid plexus. The disrupted 'mym' locus was transmitted through the mouse germ line on to different genetic backgrounds. Homozygous mutant embryos died during midgestation due to heart failure. Myocardial wall and trabeculae were found to be diminished in thickness and size, and were hypoplastic. In mutant myocytes the organization of actin-myosin filaments and cell junctions were disrupted and the expression of myosin light chain isoforms was deregulated. ' mym' is required for the maintenance and organization of heart myocytes during midgestation development. The results presented in this thesis indicate the facility of gene trapping to identify and study developmentally regulated mammalian genes and genetic elements.Ph.D