NSF Award
9405373
Abstract 9405373 The broad long-term objective is to elucidate the mechanism and significance of the epigenetic phenomenon of genomic imprinting in mammalian development. Genomic imprints effect differential expression of certain genes according to parental origin, i.e., in the same cell, the two alleles at one locus are expressed unequally. These genes are important developmentally, as lethalities and developmental anomalies are coincident with their abnormal expression. The specific aims are to (i) develop a highly sensitive allele-specific expression assay for each of the four known imprinted genes, (ii) utilize these assays to determine the allele-specific expression pattern of imprinted genes throughout pre-implantation and post-implantation development, and (iii) determine the nature of the 'leaky' expression of imprinted alleles in ES cells; such alleles have been shown to be completely silent in other situations. The experimental design and methods to be employed, in accordance with the specific aims, are: (i) Develop a Single Nucleotide Primer Extension, SNuPE, assay for each of the four known imprinted genes so as to be able to determine allele-specific expression of each. This assay is PCR-based, and relies on a single known nucleotide difference in maternal versus paternal transcripts. It can detect quantitatively the ratio of maternal versus paternal RNA molecules to one part in a thousand in a small number of cells. To find the necessary nucleotide differences, DNA from the laboratory domesticus and feral castaneus subspecies will be sequenced and compared. (ii) These two subspecies will be crossed to produce Fl embryos, and the allele-specific SNuPE expression assays performed on tissues microsurgically isolated from pre-implantation, early post-implantation, and later stages of embryonic development. (iii) Fl ES cell lines will be derived and differentiated in chimeras to determine if the leaky expression is reversible in vivo. The degree of activity of specific alleles will be determined by the SNuPE assays. %%% Genes exist which display differential expression depending upon the parental origin of the gene. Such genes are identified as imprinted. They are known to be important in organismal development in that developmental anomalies and lethalities are coincident with their abnormal expression but mechanisms for imprinting are not known. This work will expand our understanding of the significance and mechanism(s) of the genetic phenomena of imprinting. ***
