The broad, long-term objective is to understand the significance and molecular mechanism of the epigenetic phenomenon of genetic imprinting in mammalian development. Genetic imprinting in the mouse apparently effects differential expression of certain genes according to parental origin, and the normal level of expression of at least some of these genes is critical for normal embryogenesis. It is being realized that genetic imprinting underlies the etiology of a number of important inherited human syndromes, as well as cancers, and there is evidence that homologous genes may be imprinted in human and mouse.
The specific aims are to (i) determine the role of the distal region of chromosome 7 in androgenetic and parthenogenetic inviability. The importance of possible aberrant expression of the distal 7 gene, H19, will also be addressed, (ii) define in detail the distribution of androgenetic (two paternal genomes) and parthenogenetic (two maternal genomes) cells in chimeras in relation to their differentiative and proliferative capacity, and to the pathology they induce. This will provide basic information which might lead to the elucidation of the aberrant gene expression that must exist, (iii) test whether imprinted genes might act as developmental switch genes in hematopoiesis, and (iv) identify and determine the function of a significant number of developmentally important imprinted genes. The experimental design will make use of unique embryonic stem (ES) cell lines in which patterns of genetic imprinting are abnormal. These include cell lines with extreme maternal/paternal genetic imbalance, i.e. androgenetic and parthenogenetic, and those with partial imbalance, i.e. carrying paternal or maternal duplication of the distal region of chromosome 7. These cells are presumed to possess differential or mutually exclusive expression of certain genes. The methods to be employed in accordance with the specific aims will be (i) derivation and analysis of the developmental capacity in chimeras of ES cells with paternal and maternal duplication of distal 7. The role of H19 will be examined by manipulating its expression in androgenetic and parthenogenetic ES cells, then determining the fate of these altered cells in chimeras, (ii) production of chimeras using androgenetic and parthenogenetic ES cells or cleavage-stage eggs carrying a cell-- autonomous marker that can be visualized by DNA-DNA in situ hybridization in histological sections, (iii) analysis of the reconstitution of hematopoietic lineages by androgenetic and parthenogenetic hematopoietic stem cells, and (iv) differential (+/-) screening of androgenetic and parthenogenetic cDNA libraries employing subtraction technology.
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