Investigating the roles of OCT4 and CDX2 in directing bovine trophectoderm lineage development
Schiffmacher, Andrew Thomas
Keefer, Carol L
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Domestic animal embryo technologies would benefit from a better understanding of the molecular mechanisms that direct early embryonic development. Failure to establish a normal transcriptional regulatory program in the early trophectoderm of nuclear transfer or in vitro derived bovine embryos has been implicated as an underlying cause of placental abnormalities and fetal death. Misexpression of trophoblast–specific genes in these embryos has been identified, but the functions and roles of these genes remain poorly understood. The main focus of this study was to study genes involved in bovine trophectoderm lineage development using the bovine trophectoderm derived CT–1 cell line as a genetic model. Specifically, we investigated the roles of the regulatory transcription factors OCT4 and CDX2 in directing the developmental program of the early bovine trophectoderm via gene regulation of other trophectoderm–specific transcription factors. First, we overcame certain technical limitations of CT–1 cells by improving nucleic acid transfection, CT–1 cell dispersal, and culture protocols, demonstrating for the first time that overexpression assays using Lipitoid are feasible in the hard–to–transfect CT–1 cell line. We expanded the list of trophoblast genes known to be expressed in CT–1 cells and determined that the expression profile was similar to that of the ovoid stage of bovine pre–attachment embryogenesis. We measured relative levels of these genes in response to OCT4 and CDX2 overexpression and knockdown. Our results indicated that CDX2 may be a regulator of transcription of many bovine trophoblast genes and should be the focus of further study. We identified a novel OCT4 retrocopy transcribed into both sense and natural antisense transcripts, which may have a role in post–transcriptionally regulating OCT4 expression within the early bovine trophoblast. Together, these studies validate the CT–1 cell line as an appropriate genetic model for studying gene regulation in the bovine trophectoderm.