title: Effects of donor fibroblasts expressing OCT4 on bovine embryos generated by somatic cell nuclear transfer.
abstract: The production of healthy, live, cloned animals by somatic cell nuclear transfer (SCNT) has been hampered by low efficiencies. Significant epigenetic changes must take place to ensure proper chromatin remodeling in SCNT. We hypothesized that exogenous expression of OCT4 in donor fibroblasts prior to its fusion with enucleated oocytes would facilitate SCNT reprogramming. We infected bovine adult fibroblasts with retroviral vectors containing yellow fluorescent protein (YFP) only, or the OCT4 gene fused to YFP (YO). We found that development to the blastocyst stage was not different between NT-YFP and NT-YO groups. NT-YFP embryos had the fewest trophoblast cells, measured by numbers of CDX2-positive cells. Fibroblasts expressing OCT4 had reduced levels of histone 3 lysine 9 or 27 trimethylation (H3K9me3 and H3K27me3, respectively). NT-YO blastocysts displayed higher H3K9me3 levels than IVF and NT-YFP embryos; however, they did not have different H3K27me3 levels. Levels of XIST mRNA expression in NT-YO and NT-YF were higher when compared to in vitro-fertilized blastocysts. We observed no differences in the expression of SOX2, NANOG, and CDX2. Although overexpression of OCT4 in donor cells increased H3K9me3 and did not reduce XIST gene expression, we show that a single transcription factor can affect the number of trophectoderm cells in bovine SCNT embryos.
authors.affiliation: Department of Animal Science, Michigan State University, East Lansing, USA.
authors.forename: Marcelo D
authors.initials: MD
authors.lastname: Goissis
authors.forename: Steven T
authors.initials: ST
authors.lastname: Suhr
authors.forename: Jose B
authors.initials: JB
authors.lastname: Cibelli
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology* / Q000235:genetics; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism*; D002417:Cattle; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D019976:Cloning, Organism / Q000379:methods; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008745:Methylation; D053652:Nuclear Transfer Techniques; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis* / Q000235:genetics; D062085:RNA, Long Noncoding / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
chemical_list: D006657:Histones; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D062085:RNA, Long Noncoding; D011993:Recombinant Fusion Proteins