Regulation and expression of Nanog, Oct4, and Sox2 in the bovine blastocyst following somatic cell nuclear transfer.

摘要

A live birth from a somatic cell nuclear transfer (SCNT) embryo represents a small percentage of donor cells that survived the reprogramming gauntlet. The inability to reprogram histone modifications in the donor cell line could add to the reprogramming deficiencies associated with SCNT. The effects of two histone modifications associated with transcriptional activation (H3K4m3 and H4K16ac) and two histone modifications associated with repressing transcription (H3K9m2 and H3K27me3) were evaluated in the context of their association to three genes known to contribute to maintaining totipotency: Nanog, Oct4, and Sox2. A µChIP assay was utilized using antibodies specific for each histone modification followed by real time PCR (qPCR) analysis to quantify the percentage of each gene associated with each particular histone modification. Gene expression analysis was followed by immunofluorescence and protein analysis. Results of these analyses suggest that gene association to certain histone modifications did not accurately predict gene expression in bovine blastocyst embryos. Of the three genes studied, only Oct4 expression differed significantly between in vitro fertilized (IVF; control) and SCNT blastocysts. Protein levels detected through immunofluorescence correlated directly with the gene expression analysis. Nanog and Sox2 expression profiles of IVF and SCNT bovine blastocysts are similar, yet the histone modification profiles associated with all three genes differ significantly. Altered expression levels in developmentally important genes will likely result in abnormal activity of the associated cellular pathway. Aberrant histone modifications, along with abnormal Oct4 expression, may contribute to the low percentage of SCNT embryos that result in live offspring.