76 SCREENING Oct-4 PROMOTER ACTIVITY IN BOVINE FIRST AND SECOND ROUND SCNT EMBRYOS USING AN EGFP REPORTER CONSTRUCT

Abstract

Oct-4, also known as Oct-3 or POU5F1, belongs to the POU (Pit-Oct-Unc) transcription factor family. So far it has been identified in mice, cattle, pigs, and humans. In mice, pluripotent cells of the early pre-implantation embryo express Oct-4. Therefore, Oct-4 expression is considered to be a marker for pluripotency in mice. In bovine blastocysts, Oct-4 protein is not restricted to the pluripotent cells of the inner cell mass (ICM) but is also present in the trophectoderm (TE). It has been reported, however, that at this stage the transcript level of Oct-4 was already downregulated in the TE suggesting a similar regulation of Oct-4 transcription in bovine and mice. To obtain insight into the regulation of the Oct-4 promoter after somatic cell nuclear transfer (SCNT), we transfected bovine fetal fibroblasts (BFF) with GOF18-¿PE-EGFP, a reporter gene construct for the Oct-4 promoter (kindly provided by Dr. Hans R. Schoeler, Director, Max Planck Institute for Molecular Biomedicine, Münster, Germany). Six stably transfected colonies (BFFGOF), none of which exhibited green fluorescence, were used for SCNT, with subsequent examination of the resulting embryos on Days 5-7 by fluorescence microscopy. SCNT embryos originating from BFF colonies 3, 12, and 82 showed fluorescence both in ICM and TE. SCNT with BFF colony 8 resulted in embryos with no detectable fluorescence. This might have been due to a positional effect of the reporter gene construct or an incomplete reprogramming of the used fibroblast colonies during SCNT. To study the consequences of another round of SCNT on Oct-4 promoter activity, cloned embryos from BFF colonies 12 (positive) and 8 (negative) were transferred to recipients. At Day 33, three BFFGOF12 and four BFFGOF8 fetuses were recovered and BFF cultures were established from all cloned fetuses. The presence of GOF18-¿PE-EGFP was verified by PCR and Southern blot analysis. BFF cultures from two cloned BFFGOF12 and BFFGOF8 fetuses each were used for SCNT, and fluorescence was examined in Day 6 embryos. SCNT embryos derived from BFFGOF12 fetuses exhibited weaker fluorescence than embryos directly derived from the original transfected colony. SCNT embryos derived from BFFGOF8 fetuses showed no fluorescence. GFP-positive embryos will be examined by immunocytochemistry using an antibody against Oct-4 to evaluate the correlation between endogenous Oct-4 and Oct-4 reporter gene expression. Further fluorescence in situ hybridization (FISH) analyses are underway to localize the reporter gene integration sites in BFFGOF8 and BFFGOF12. In summary, stable GOF18-¿PE-EGFP transfected cells are an interesting tool for monitoring Oct-4 promoter activation after using different protocols of SCNT or in consecutive rounds of SCNT. Furthermore, it will be possible to correlate Oct-4 promoter activity with epigenetic mechanisms, such as DNA methylation and histone modifications, in SCNT embryos.