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

Ryutaro Hirasawa,Kimiko Inoue,Shogo Matoba,Atsuo Ogura

doi:10.1371/journal.pone.0076422

Ryutaro Hirasawa, Kimiko Inoue + Show 2 more

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https://doi.org/10.1371/journal.pone.0076422

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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.

Highlights

Somatic cell nuclear transfer (SCNT) is a unique technology that produces a cloned animal from a single donor somatic cell nucleus
They were collected from the uteri at E6.5 (Figure 1A left) and were dissected into four parts: epiblast (EPI), extraembryonic (EXE), visceral endoderm (VE) and ectoplacental cone (EPC) (Figure 1A right)
The EPI part was treated as an embryonic sample and a mixture of EXE and EPC was treated as a pooled extraembryonic sample

Summary

Introduction

Somatic cell nuclear transfer (SCNT) is a unique technology that produces a cloned animal from a single donor somatic cell nucleus This technology is potentially promising for many practical applications in the fields of medicine and drug manufacturing, the animal industries and in conservation of genetic resources [1]. To achieve these goals, the somatic cell genomes should be precisely reprogrammed into the state seen in normally fertilized embryos: so-called totipotency. It has been reported that preimplantation cloned SCNT-derived embryos are prone to several epigenetic abnormalities such as global DNA hypermethylation [6], satellite repeat DNA hypermethylation [7,8] and histone methylation [9], which are thought to be legacies from the somatic cell genome

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