Protocol

DNA Methylation Analysis of Human Imprinted Loci by Bisulfite Genomic Sequencing

¹Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA
²Institute for Stem Cell Biology and Regenerative Medicine, Department of Obstetrics and Gynecology, Stanford University, Palo Alto, CA 94304-5542, USA

³Corresponding authors (reneer{at}stanford.edu; vangeles{at}stanford.edu)

INTRODUCTION

The genomic imprinting mechanism regulates differential expression of paternally and maternally derived genes. During development, primordial germ cells (PGCs) have a unique methylation pattern which undergoes demethylation, both globally and at imprinted loci. Later, the methylation status of imprinted genes is re-established during gametogenesis by de novo methylation. Errors in the methylation patterns of imprinted genes have been associated with syndromes such as Beckwith-Wiedemann, Angelman, and Prader-Willi in humans. Human embryonic stem cells (hESCs) have unique methylation patterns compared to somatic stem cells and cancer cells. The status of allelic expression or methylation of human imprinted genes can be studied in detail in human embryos/blastocysts and hESCs to understand early development and differentiation. This protocol describes the bisulfite genomic sequencing method for analysis of methylation patterns of imprinted loci in human embryonic stem cells. Multiple imprinted loci can be analyzed by performing the following protocol with gene-specific primers.