113 CHARACTERIZATION OF THE BOVINE EMBRYO-DERIVED CT-1 CELL LINE FOR STUDYING REGULATION OF TRANSCRIPTION FACTORS EXPRESSED WITHIN THE BOVINE TROPHECTODERM

Abstract

Cloned bovine embryos have been shown to display deviations in gene expression of trophectodermal (TE) lineage-specific transcription factors, including Cdx2, Errβ, Eomes, Mash2, and Hand1. Misregulation of these genes in the early TE has been implicated to play a causal role in abnormal placental formation and fetal loss. Because suboptimal in vitro culture conditions fail to support development beyond hatching, very little is known about regulation of peri-attachment bovine embryo development. Trophectodermal-derived bovine cell lines, such as the CT-1 cell line produced by Talbot and colleagues (Talbot et al. 2000 Biol. Reprod. 62, 235–247) could prove beneficial for understanding how regulatory factors govern early bovine TE development. The CT-1 cell line has been cultured continuously for over 2 years and appears morphologically similar to primary bovine TE outgrowths. Furthermore, these cells produce interferon τ, a cytokine unique to the early bovine TE. The objective of this study was to develop a gene expression profile of selected developmental genes that play a potential role in commitment to the TE fate, maintenance of multipotency, or differentiation into a binucleate phenotype. The CT-1 colonies were initially expanded on gamma-irradiated STO cells at 37°C and 5% CO₂ and passaged weekly. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1 mm sodium pyruvate and were passaged whenever colony edges touched. Cultures designated for RNA extraction and RT-PCR were then passaged into collagen-coated flasks or Cellbind^® flasks (Corning, Lowell, MA, USA) at an average dilution of 1:4 for at least 6 feeder-free passages. Feeder-free culture at 37°C and 8.5% CO₂ was optimal for adherence and growth. There were no observable differences in morphology, adherence, or growth rate between CT-1 cells grown on collagen film or a Cellbind^® surface. The RT-PCR results indicate that the transcription factor Cdx2, which is homologous to the mouse master TE regulator, is abundantly expressed, whereas pluripotency specific Oct4 is weakly expressed. However, pluripotency specific transcription factors Nanog and Sox2 were undetectable. Transcription factors Eomes, Ets2, Errβ, and Id2, which may be required for proper bovine trophoblast elongation and developmental potential, were also expressed. Hand1 mRNA, a basic helix-loop-helix transcription factor that directs mouse trophoblast cells to differentiate into a giant cell phenotype, was also present and may play a role in binucleate cell formation. Together the data indicate that the CT-1 cell line can be utilized as a permissive cell line for studying transcriptional control of key TE regulatory factors that define the bovine TE lineage and allow better interpretation of gene expression data collected from assisted reproductive technology-derived embryos.