Merck Millipore CpG Oct4 Amplification Kit

Oct-4 (POU5F1, Oct-3) is a member of the POU domain family of homeodomain transcription factors, named for its ability to bind octamer DNA binding sites resembling 5'-ATGCAAAT-3'. In mouse, the expression pattern of Oct-4 mRNA is restricted to cells of the inner cell mass in pre- and post-implantation embryos, and primordial germ cells of adult mice (1). Oct-4 expression is essential for maintenance of pluripotentiality of murine ES cells in culture, and withdrawal of Oct-4 activity results in trans-differentiation of embryonic stem cells into trophoectoderm both in culture and in animals (2, 3). As such, Oct-4 represents a well known candidate for a master regulator of stem cell identity, self renewal and maintenance of the undifferentiated state (4).

One of the mechanisms implicated in regulation of Oct-4 expression in both mouse and human cells is epigenetic silencing via methylation (5, 6). There is evidence for regulation both at the level of DNA methylation of the Oct-4 regulatory region, as well as chromatin remodeling via methylation of key residues on histone proteins. With respect to DNA, methylation of cytosines located 5' to guanosine is known to have a profound effect on the expression of several eukaryotic genes (7). In normal cells, methylation occurs predominantly in CG-poor regions, while CG-rich areas, called CpG islands, remain unmethylated. Aberrant methylation of normally unmethylated CpG islands has been documented as a relatively frequent event in immortalized and transformed cells (8) and has been associated with transcriptional inactivation in various genes (9). Similarly, normal development processes can be controlled by epigenetic inactivation via DNA methylation (imprinting) or promoter methylation of key developmental regulatory genes.
Previously developed methods to determine the methylation status of cytosine include digestion with methylation sensitive restriction enzymes and genomic DNA sequencing. Both techniques have limitations: restriction enzymes can only detect methylation sites within their recognition sequence and sequencing is time consuming. Methylation-specific PCR (MSP) is a new technology for sensitive detection of abnormal gene methylation utilizing small amounts of DNA (9). This process employs an initial bisulfite reaction to modify the DNA, followed by PCR amplification with specific primers designed to distinguish methylated from unmethylated DNA. The CpG WIZ Oct-4 Amplification Kit provides a method for evaluating methylation of the murine Oct-4 promoter in methylation studies (10). Furthermore, this assay provides a sensitive molecular tool to evaluate the differentiation status of murine ES cells in culture.
The CpGenome DNA Modification Kit (Cat. No. S7820 or S7824) contains the reagents necessary to perform the initial bisulfite reactions, while the CpG WIZ Oct-4 Amplification Kit contains the reagents required for the PCR amplification reactions.