The long term objective is to understand the molecular mechanism of imprinting, the marking of chromosomes as being of maternal or paternal origin. The study of imprinting should help answer basic questions about inheritance and gene regulation during development. I propose to examine the feasibility of using embryonic stem (ES) cells undergoing X chromosome inactivation as a model system for imprinting. ES cells, derived from mouse embryos, can grow in tissue culture, and by simple changes in growth conditions, the cells can also be induced to undergo differentiation in vitro. ES cells are known to form two layers as they differentiate into embryoid bodies, an endoderm-like outer layer, and an ectoderm-like inner layer. I propose to use ES lines which are heterozygous for a number of X-linked genes, and an allele-specific assay for specific RNAs to characterize X-inactivation in these cells. I propose 1) to characterize X-inactivation in the inner ectoderm layer and the endoderm layer, 2) to investigate large scale methods of separating the two layers for biochemical analysis, and 3) to analyze whether DNA methylation patterns found in mouse embryos are preserved in ES cells. In addition, I propose to study the distribution of a transcript, termed Xist, recently discovered to be synthesized only from the inactive X chromosome. I propose to use in situ hybridization and PCR-based assays to learn more about the role of Xist in cases where the paternal X chromosome is preferentially inactivated. %%% The mystery of how a chromosome is marked as being either from the mother or the father should begin to be solved by the development of this system and the results of these experiments.
