A reverse transcriptase-polymerase chain reaction assay (RT-PCR) was used quantitatively to measure accumulated levels of RNA transcripts in total mouse RNAs derived from male germ cells at various spermatogenic stages. RNA levels for two X-linked enzymes, phosphoglycerate kinase (PGK-1) and hypoxanthine phosphoribosyl transferase (HPRT), both decrease during spermatogenesis, although the transcript levels decrease much more rapidly for PGK-1. RNA for the Y-linked ZFY (zinc finger protein) is elevated in all spermatogenic cell fractions tested, being particularly high in leptotene/zygotene spermatocytes and round spermatids. RNA for adenine phosphoribosyltransferase (APRT) increases 5-fold to a peak during late pachynema. RNA for PGK-2, undetectable in spermatogonial cells, increases at least 50-fold by the round spermatid stage. DNA (cytosine-5-)-methyltransferase (MTase) transcript levels are over an order of magnitude higher throughout spermatogenesis than in non-dividing liver cells.

A HpaII-PCR assay was used to study DNA methylation in individual mouse embryos. It was found thatHpaII site H-7 in the CpG island of the X-chromosome-linked Pgk-1 gene is l10% methylated in oocytes and male embryos but becomes 40% methylated in female embryos at 6.5 days, about the time of X-chromosome inactivation of the inner cell mass.An assay based on the use of HpaII prior to the polymerase chain reaction (PCR) (20,22) allowed us to study DNA methylation in individual mouse embryos at the time of X-chromosome inactivation. We probed methylation in the CpG-rich island at the 5' end of the X-linked gene for phosphoglycerate kinase (Pgk-J), at a HpaII site (H-7) showing female-specific methylation in adult somatic tissues (Fig. 1). We found that site H-7 is not significantly methylated in male embryos but that in female embryos DNA methylation begins near 5.5 days and then rises, in both the embryo proper and the extraembryonic tissues, reaching nearly the adult level by 6.5 days.X-chromosome inactivation occurs in the inner cell mass of female mouse embryos between 5.5 and 6.5 days, at about the time of implantation (3, 10), a stage when the embryo consists of fewer than a thousand cells and the sex is not easily distinguishable. For this reason, only one prior study has been done correlating methylation with X-chromosome inactivation in the early embryo. Lock et al. (9) pooled many nonsexed embryos and found that methylation at sites in the first intron of the Hprt gene takes place between 9.5 and 13.5 days, well after the time of X-chromosome inactivation and the methylation we observe in the Pgk-1 gene.The promoter and first exon of Pgk-J are part of a CpG island that has been well characterized in both mice and humans (Fig. 1) (15) (Fig. 2). To assay for methylation, we made use of the fact that PCR amplification occurs only if the DNA between the two primer sites is intact. We chose PCR primers that flank H-7 and used a quantitative PCR procedure (20) to assay for HpaII-resistant (methylated) molecules. We had previously found, using this assay, that while male-embryo DNA was not amplified by PCR after HpaII digestion, female-embryo DNA was amplified, giving a signal 50% that of undigested DNA. Our interpretation was that site H-7 is methylated only on the inactive X chromosome (22). Figure 3 shows results of the HpaII-PCR assay on DNA from ectoplacental cone, extraembryonic ectoderm, and embryonic ectoderm dissected from two 6.5-day embryos, one male and one female. HpaII-resistant genomic molecules were present (giving band G in lanes marked +) in all three tissues of the female but not the male embryo. The larger fragments seen in the lanes without HpaII treatment came from M13 DNA, which was added as a carrier and also as a control for completeness of HpaII digestion. Band I in Fig. 3 is the PCR product of an internal standard, amplified by the same primers used for genomic DNA. The internal standard was a DNA fragment identical to genomic DNA except for a deletion internal to the primer binding sit...

Methylation-sensitive restriction enzymes and Southern blot analysis are commonly used to assay for DNA methylation, usually requiring DNA from about 105 cells. However sensitivity adequate for only a few hundred cells is needed in many cases, for example, the study of DNA methylation changes in germ cells or pre-implantation embryos. We earlier reported a sensitive PCR assay using primers that bracket a Hpall site; if the DNA is treated with HpaII prior to PCR, an amplified product is seen only from a methylated genomic template (1). We report here two improvements which make the assay quantitatively applicable to 100-300 cells, with as little as 10% DNA methylation detectable.We now include in each reaction an internal standard sharing primer binding sites with the genomic template, but distinguishable by size (2). The inset in Figure 1 shows the PCR products from the internal standard (band # 1), and from the genomic template (female blood DNA) with or without prior treatment with Hpall (band # 2). Also shown in Figure 1 are the densitometric tracings of these ethidium bromide-stained bands. Plotted in Figure 2 is the ratio of the internal standard band (I) to the genomic band (G) in an experiment in which the internal standard was varied from 80 to 240 molecules, while the genomic DNA (500 pg, approximately 100 molecules) was held constant. We find that female mouse spleen and blood DNA are methylated 52.5 2.3% and 50.6 i 3.5%, respectively, in agreement with previous data (1). To prepare DNA suitable for the HpaII-PCR assay, we used a guanidine HCl procedure (3), modified as described in the legend to Figure 1.

Monoallelic expression is an integral component of regulation of a number of essential genes and gene families. To probe for allele-specific expression in cells of CNS origin, we used next-generation sequencing (RNA-seq) to analyze four clonal neural stem cell (NSC) lines derived from Mus musculus C57BL/6 (B6)×Mus musculus molossinus (JF1) adult female mice. We established a JF1 cSNP library, then ascertained transcriptome-wide expression from B6 vs. JF1 alleles in the NSC lines. Validating the assay, we found that 262 of 268 X-linked genes evaluable in at least one cell line showed monoallelic expression (at least 85% expression of the predominant allele, p-value<0.05). For autosomal genes 170 of 7,198 genes (2.4% of the total) showed monoallelic expression in at least 2 evaluable cell lines. The group included eight known imprinted genes with the expected pattern of allele-specific expression. Among the other autosomal genes with monoallelic expression were five members of the glutathione transferase gene superfamily, which processes xenobiotic compounds as well as carcinogens and cancer therapeutic agents. Monoallelic expression within this superfamily thus may play a functional role in the response to diverse and potentially lethal exogenous factors, as is the case for the immunoglobulin and olfactory receptor superfamilies. Other genes and gene families showing monoallelic expression include the annexin gene family and the Thy1 gene, both linked to inflammation and cancer, as well as genes linked to alcohol dependence (Gabrg1) and epilepsy (Kcnma1). The annotated set of genes will provide a resource for investigation of mechanisms underlying certain cases of these and other major disorders.

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