Establishment of Methylation at the Imprinted Mouse Gene Rasgrf1 during Spermatogenesis

Posted May 26th, 2010 at 2:37 pm.

Charlotte Rahn-Lee

Mentor: Dr. Tamara Davis

I am researching the establishment of methylation at the imprinted gene Rasgrf1 in mice. Imprinting is a phenomenon that occurs at a small number of mammalian genes by which one parent’s allele is expressed and the other is silent. Imprinted genes require the cell to distinguish between the maternal and the paternal alleles, a distinction which cannot be made by the gene’s sequence. Differential methylation, a phenomenon where one parent’s allele is methylated and the other is not, is thought to play an important role in this process. However, differentially methylated genes must change their methylation pattern in developing gametes so that the next generation will receive alleles that reflect the sex of the parent, rather than the sex of the grandparent, from which they were inherited.

Rasgrf1 is an imprinted gene found in mice. It has a differentially methylated domain 5′ of the promoter which is paternally methylated. The gene is expressed paternally in brain, heart, and stomach tissues, but biallelically expressed in other tissues, such as lung. In somatic cells one of the two alleles is methylated, but both alleles must be methylated in sperm. We do not know when this transformation takes place. In my project I will examine when during spermatogenesis methylation of the unmethylated allele is established.

I will look for methylation of DNA in developing sperm of F1 hybrids of Mus musculus domesticus (BL/6) and Mus musculus castaneus (CAST) during different stages of development. First I will need to sequence the differentially methylated domain of this gene in the two strains of mice to find polymorphisms I can use to distinguish the parental origin of the alleles in the F1 hybrids. Then bisulfite mutagenesis can be performed on DNA from different developmental stages of male F1 hybrids. Bisulfite mutagenesis is a process in which the sequence of unmethylated DNA is systematically altered, while the sequence of methylated DNA is protected against mutagenesis. The resulting DNA will be sequenced to determine which alleles are methylated at each stage. This will give a clear picture of when methylation of Rasgrf1 is established.

Filed under: 2004,Davis, Dr. Tamara,Rhan-Lee, Charlotte by Ann Dixon

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