Department of Molecular and Cell Genetics, School of Life Sciences; Faculty of Medicine, Tottori University
We have developed a system, for the analysis of genomic imprinting in vitro and the detection of human new imprinted genes, which centers on the construction of mouse A9 cells containing single human chromosomes of known parental origin. This system relies on the maintenance of the epigenetic regulation of human loci in mouse hybrids following transfer from human fibroblasts.
Human fibroblasts were transfected with markers for either Blasticidin S or neomycin resistance, followed by cell fusion with A9 cells and microcell mediated chromosome transfer of human chromosomes to A9 cells to generate hybrid clones. A9 hybrids containing a single intact human chromosome 11 were identified, the parental origin of which was determined by polymorphic analysis using STS markers. Analysis of known imprinted genes demonstrated that the imprinting status of human loci was maintained in A9 hybrids. Human H19 and KvLQT1 were expressed exclusively from the maternal allele in A9 hybrids, and appropriate paternal specific DNA methylation of H19 was also exhibited. Additionally, the appropriate imprinted expression of SNRPN, IPW and NECDIN on human chromosome 15 was maintained in A9 hybrids containing human chromosome 15, as was the methylation status of SNRPN (1,2).
These A9 hybrids provide a unique resource for the identification of new imprinted genes within the 11p15.5, 15q11-q13 and other imprinted domains. Allele specific expression of human expressed sequence tags (ESTs) can be determined by RT-PCR analysis of A9 hybrids, without the prior requirement for the identification of expressed polymorphisms. Currently, 225 human expressed sequence tags (ESTs) have been mapped to 11p15.5, from 11pter to the marker D11S1750. We analyzed the expression of 100 of these ESTs in A9 hybrids containing maternal or paternal human chromosome 11. Two ESTs with maternal specific expression and one EST with paternal specific expression were identified. Expressed polymorphism has been identified for one of these clones which has enabled us to confirm that it is imprinted in vivo, in human fibroblasts from several families. We have also screened the expression of 90 ESTs from the 15q11-q13 imprinted region, and have identified 5 candidates with allele specific gene expression in A9 hybrids.
This demonstrates the power of this novel approach for the identification of imprinted genes within imprinted domains on human chromosome. We are now screening human chromosomes 1, 6, 7, 10, 14, 18 and 19, and also testing a possible usefulness of a cDNA microarray system using the hybrid cells for the identification of new imprinted genes.
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Mitsuya, K., Meguro, M., Sui, H., Schulz, T.C., Kugoh, H., Hamada, H. and Oshimura, M. (1998) Epigenetic reprogramming of the human H19 gene in mouse embryonic cells does not erase the primary parental imprint. Genes to Cells 3:245-255.