Department of Molecular and Cell Genetics; Tottori University
In order to develop an in vitro assay system for the investigation of human imprinted loci, we have constructed mouse A9 hybrids retaining a single human chromosome of known parental origin, via microcell-mediated chromosome transfer. Normal human fibroblasts transfected with a selectable marker were fused to the mouse A9 cells. The human chromosomes with the marker in the hybrids were independently transferred into A9 cells by microcell fusion. More than 700 microcell hybrid clones were isolated and karyotyped. Parental origins of the transferred human chromosomes were determined using microsatellite repeat polymorphisms. There are two major imprinted clusters on human chromosome llpl5 and 15qll-ql3 regions. Expression and methylation studies revealed that the appropriate imprinting status of genes on these was maintained in the A9 hybrids. Using this system, we have identified new imprinted genes and studied molecular mechanism of its regulation. Our strategy in the CREST project will be introduced, and other members in our group will present the details in this workshop.