Single cell restriction enzyme-based analysis of methylation at genomic imprinted regions in preimplantation mouse embryos

Abstract

The methylation of cytosines in DNA is a fundamental epigenetic regulatory mechanism. During preimplantation development, mammalian embryos undergo extensive epigenetic reprogramming, including the global erasure of germ cell-specific DNA methylation marks, to allow for the establishment of the pluripotent state of the epiblast. However, DNA methylation marks at specific regions, such as imprinted gene regions, escape this reprogramming process, as their inheritance from germline to soma is paramount for proper development. To study the dynamics of DNA methylation marks in single blastomeres of mouse preimplantation embryos, we devised a new approach—single cell restriction enzyme analysis of methylation (SCRAM). SCRAM allows for reliable, fast, and high-throughput analysis of DNA methylation states of multiple regions of interest from single cells. In the method described below, SCRAM is specifically used to address loss of DNA methylation at genomic imprints or other highly methylated regions of interest.