Methods for assaying gene imprinting and methylated cpg islands

Abstract

Genomic imprinting is a parent of origin-dependent gene silencing that involves marking of alleles in the germline and differential expression in somatic cells of the offspring. Imprinted genes and abnormal imprinting have been implicated in development, human disease, and embryonic stem cell transplantation. We have established a model system for genomic imprinting using pluripotent 8.5 d.p.c. mouse embryonic germ (EG) cell lines derived from an interspecific cross. We find that allele-specific imprinted gene expression has been lost in these cells. However, partial restoration of allele-specific silencing can occur for some imprinted genes after in vitro differentiation of EG cells into somatic cell lineages, indicating the presence of a gametic memory that is separable from allele-specific gene silencing. We have also generated a library containing most methylated CpG islands. A subset of these clones was analyzed and revealed a subdivision of methylated CpG islands into 4 distinct subtypes: CpG islands belonging to high copy number repeat families; unique CpG islands methylated in all tissues; unique methylated CpG islands that are unmethylated in the paternal germline; and unique CpG islands methylated in tumors. This approach identifies a methylome of methylated CpG islands throughout the genome.

Description

[0001] This application claims the benefit of application Ser. Nos. 60 / 206,158 and 60 / 206,161 filed May 22, 2000.[0002] This invention was made using funds from the U.S. government under a grant from the National Institutes of Health numbered CA65145. The U.S. government therefore retains certain rights in the invention.BACKGROUND OF THE INVENTION [0003] Genomic imprinting is a parental origin-specific gene silencing that leads to differential expression of the two alleles of a gene in mammalian cells. Imprinting has attracted intense interest for several reasons: (i) Imprinting is by definition reversible and may be regulated over a large genomic domain (1). (ii) Imprinted genes and the imprinting mechanism itself are important in human birth defects and cancer (2). (iii) It has been suggested that imprinting cannot be reprogrammed without passage through the germline and thus constitutes a barrier to human embryonic stem cell transplantation (3). [0004] Experimental studies of the...

AI Technical Summary

Benefits of technology

[0009] One embodiment of the invention provides a method of forming embryonic germ cells useful as a model system for studying imprinting. A male and a female mammal of the same species are mated to form a pregnant female mammal. The male and the female mammals are sufficiently genetically divergent such that at least 50% of genes in resulting offspring have at least one sequence difference between alleles of said genes. An embryo is obtained from the pregnant female mammal at a stage of embryonic development between when 2-3 somites become visualizable and when gonads are recognizable. The embryo is dissected and cells of the embryo are dissociated. The dissociated cells are cultured to provide embryonic germ cell lines.

Problems solved by technology

Experimental studies of the timing and mechanism of genomic imprinting have been hampered by the fact that imprinting requires passage through the germline, analysis of which poses a difficult experimental target Thus, there is a need in the art for an experimental model system which allows direct examination of allele-specific gene silencing in the dynamic process of genomic imprinting.

However, the resulting sequences were mainly dispersed repeats, ribosomal DNA and other repeated sequences with no characterization of unique, methylated CpG island.

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