Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos

Abstract

The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (P<1.0×10–26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1–5% per embryos transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.

Aberrant imprinting in mouse trophoblast stem cells established from somatic cell nuclear transfer-derived embryos

“…Furthermore, extraembryonic tissues of SCNT-derived conceptuses also showed an abnormal phenotype shortly after implantation. They had a proliferative deficiency caused by precocious differentiation, leading to poor development of the ectoplacental cone and cytotrophoblast areas at implantation sites [7,8]. Based on these cumulative findings, we speculate that some unidentified epigenetic errors are inherent to extraembryonic lineages derived by SCNT throughout development.…”

“…The IG-DMR regulates many imprinted genes within its region for their parental originspecific expressions in the developing fetus and placenta. Indeed, upregulation of Dlk1, one of the IG-DMR-regulated genes, is often associated with death in early cloned fetuses, probably because of hypermethylation of the IG-DMR [7,9]. It is probable that the abnormal development of placentas in cloned mice is also affected by the hypermethylated IG-DMR.…”

Generation of Cloned Mice from Adult Neurons by Direct Nuclear Transfer1

“…Thus, somatic-type LOCKs might affect the epigenetic status and gene expression patterns of SCNT-derived embryos, and prevent proper genome reprogramming and term development. Indeed, microarray analysis of single SCNT-derived embryos revealed that they had a unique gene expression profile that was strictly dependent upon the type of donor cells [34,38,42]. In other words, it is expected that SCNT-derived embryos from differentiated somatic cells with low levels of H3K9me2 might have few LOCKs and enhance the efficiency of mouse cloning.…”

Understanding the X chromosome inactivation cycle in mice

“…Intriguingly, SCNT embryos also showed Xist-independent correction of gene expression profiles, indicating the presence of extensive reprogramming of the genomic state during implantation. 23 This NT study has demonstrated that, in mouse embryos, the maternal Xist-repressing imprint is also erased in placental tissues, specifically before day 4.5. Therefore, in a strict sense, the preferential inactivation of the paternal X chromosome in the mouse placentas is not imprinted but may be "biased" or "skewed" (Fig.…”