International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)

B2. The Identification of Novel Imprinted Loci Using Methylation Sensitive Representation Difference Analysis (Me-RDA)

Rachel J. Smith, Wendy Dean and Gavin Kelsey,
Developmental Genetics Programme, The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK

A number of mammalian genes are expressed from only one allele, depending on the parent of origin, a mode of regulation that is termed genomic imprinting. Appropriate expression of imprinted genes is essential for normal development and defects in imprinted gene regulation result in a number of human disorders and cancer. However, it is likely that only a fraction of imprinted genes has been identified. In order to fully assess the role of imprinted genes in development and disease, and to determine common mechanisms that may underlie their regulation, it is essential to identify further genes subject to imprinted expression control.

We have developed a screen for imprinted genes based on allelic methylation differences. Regions of parental allele specific methylation (differentially methylated regions - DMRs) are a consistent feature of imprinted genes identified to date, and may be important in their control. Methylation sensitive representation difference analysis (Me-RDA) combines restriction digestion using a methylation sensitive enzyme such as HpaII, with subtraction hybridisation and PCR, to recover fragments from DMRs that are hypomethylated on one parental allele. By the use of appropriate DNAs of monoparental origin, this screen may be applied to specific subchromosomal regions (Kelsey et al, 1999. Genomics 62:129-138), or to systematically screen the entire genome.

We have performed genome wide screens for imprinted genes in the mouse, by the application of Me-RDA to DNAs from parthenogentic (Pg) and androgenetic (Ag) embryos, which contain, respectively, chromosomes of exclusively maternal or paternal origin. Over 250 cloned difference products from the Pg screen have been analysed, of which 57.7% show differences between Pg and normally fertilised DNAs; of 146 clones picked from the Ag screen, 12.3% correspond to methylation differences. These screens have sucessfully isolated clones from the Peg1/MEST, Peg3/Pw1/Ocat, Peg5/Nnat, Gnasxl, Snrpn, U2af1-rs1, Zac1, H19, Meg3/Gtl2, and Nesp imprinted loci. Novel regions of allele specific methylation have been identified, including two domains of imprinted methylation on mouse chromosome 15, a chromosome on which imprinting has not previously been described. The transcripts associated with these novel DMRs are currently being examined to determine their imprinting status. As we are able to isolate multiple independent fragments from these DMRs, Me-RDA could offer a comprehensive screen for imprinted loci in the mouse genome.

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