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Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the oosight imaging system.

ABSTRACT: In somatic cell nuclear transfer (SCNT) procedures, exquisite enucleation of the recipient oocyte is critical to cloning efficiency. The purpose of this study was to compare the effects of two enucleation systems, Hoechst staining and UV irradiation (hereafter, irradiation group) and Oosight imaging (hereafter, Oosight group), on the in vitro production of bovine SCNT embryos. In the Oosight group, the apoptotic index (2.8 ± 0.5 vs. 7.3 ± 1.2) was lower, and the fusion rate (75.6% vs. 62.9%), cleavage rate (78.0% vs. 63.7%), blastocyst rate (40.2% vs. 29.2%), and total cell number (128.3±4.8 vs. 112.2 ± 7.6) were higher than those in the irradiation group (all p<0.05). The overall efficiency after SCNT was twice as high in the Oosight group as that in the irradiation group (p<0.05). The relative mRNA expression levels of Oct4, Nanog, Interferon-tau, and Dnmt3A were higher and those of Caspase-3 and Hsp70 were lower in the Oosight group compared with the irradiation group (p<0.05). This is the first report to show the positive effect of the Oosight imaging system on molecular gene expression in the SCNT embryo. The Oosight imaging system may become the preferred choice for enucleation because it is less detrimental to the developmental potential of bovine SCNT embryos.

SUBMITTER: Kim EY

PROVIDER: S-EPMC3411338 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the oosight imaging system.

Kim Eun Young EY Park Min Jee MJ Park Hyo Young HY Noh Eun Ji EJ Noh Eun Hyung EH Park Kyoung Sik KS Lee Jun Beom JB Jeong Chang Jin CJ Riu Key Zung KZ Park Se Pill SP

Cellular reprogramming 20120720 4

In somatic cell nuclear transfer (SCNT) procedures, exquisite enucleation of the recipient oocyte is critical to cloning efficiency. The purpose of this study was to compare the effects of two enucleation systems, Hoechst staining and UV irradiation (hereafter, irradiation group) and Oosight imaging (hereafter, Oosight group), on the in vitro production of bovine SCNT embryos. In the Oosight group, the apoptotic index (2.8 ± 0.5 vs. 7.3 ± 1.2) was lower, and the fusion rate (75.6% vs. 62.9%), cl ...[more]

PMID: 22816525

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Project description:Since the first cloned animal Dolly Sheep was successfully created by using somatic cell nuclear transfer(SCNT) technique. It has become an irreplaceable tool to understand nuclear reprogramming and totipotency and holds huge potentials for regenerative medicine. However, extremely poor development rate of SCNT embryos indicates it is still questionable. The nature of reprogramming oocyte factors and their mechanism of action remain largely unknown.It is evident that the major barrier that hinders the developing iSCNT embryo mainly appears at the time of embryonic genome activation (EGA), which primarily occurs at the eight-cell stage in mammalian. The interspecies somatic cell nuclear transfer (iSCNT) is desired model for nuclear reprogramming research and a powerful tool for discovering the master genome activation genes. In this study, a valuable transcriptome recourse of iSCNT embryos was established, which derived from more than 2000 clone embryos of four different inter-family donor cells. Based on weighted gene co-expression network (WGCNA) approach, we provide an extensive transcriptome analysis of differentially expressed genes(DEG) for iSCNT embryos. The total gene expression patterns of different iSCNT embryos were discussed. 26 cell-specific modules with were identified, and those module significance and GO enriched categories were analyzed. The regulatory pathways of reprogramming barriers were further enriched. As master genome trigger genes, the transcripts related to TFIID subunit, RNA polymerase and Mediator were incomplete activated in iSCNT embryos. This indicated that pioneer factors, present in the cytoplasm of the oocyte, were failed to bind the sequence target on the heterology nuclear genome. This genomic incompatibility between the nuclear donor cell and the cytoplast may be as a major contributing factor causes the developmental failure of iSCNT cloned embryos. This study demonstrates that the iSCNT embryos undergoes only partial or incomplete reprogramming at eight-cell stage in mammalian. Our results offered convincing evidence that the abnormal expression of key master pathways may be caused the embryo developmental block of cloning embryo. This work will contribute to a better understanding of the molecular interaction between nuclear–cytoplasmic interaction and provides insight into the molecular determinants of nuclear reprogramming, human embryonic stem cell (hESC)-based therapies and rescuing highly endangered species.

Dataset Information

Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the oosight imaging system.

Publications

Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the oosight imaging system.

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