Developmental Genetics Programme; The Babraham Institute
Genomic imprinting is essential for normal mammalian development, yet the full role of imprinting in embryogenesis, postnatal physiology, behavior and disease etiology remains to be determined. In order to assess the complete extent of imprinting and determine the mechanisms by which genes are imprinted, it is important to undertake a systematic screen to identify as many genes as possible that are subject to imprinted expression control.
Allelic methylation differences are characteristic of the imprinted genes identified to date, and this feature may be used to identify novel imprinted genes. Methylation sensitive representation difference analysis (Me-RDA) is a screen that has been developed to identify regions of imprinted methylation. This technique combines restriction digestion using a methylation sensitive restriction enzyme with PCR and subtractive hybridization, in order to isolate fragments that are unmethylated specifically on one allele, and methylated on the other allele. The success of Me-RDA has been demonstrated in its application to mouse distal chromosome 2 and the identification of the Nesp and Gnasxl imprinted transcripts.
In order to perform a genome wide screen for regions of imprinted methylation, we are now applying Me-RDA to parthenogenetic and androgenetic mouse embryos. A prerequisite for using these monoparental embryos in Me-RDA is that the embryos maintain the anticipated pattern of parental allele specific methylation at imprinted loci. Restriction digestion and PCR on DNA from these embryos, in comparison to normally fertilized embryos, has been used to confirm that their methylation follows the expected pattern for the imprinted loci examined.
The application of Me-RDA to parthenogenetic and fertilized mouse embryos will permit the identification of regions of maternal specific methylation. To date this screen has isolated clones from the Peg1/Mest, Peg3/Pwl, Peg5/Nnat and Gnasxl loci. Candidate novel imprinted loci isolated from this screen are currently being tested in order to determine their imprinting status.