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Promoter analysis of the rabbit POU5F1 gene and its expression in preimplantation stage embryos

Julianna Kobolak1, Katalin Kiss1,2, Zsuzsanna Polgar3, Solomon Mamo4,1, Claire Rogel-Gaillard5, Zsuzsanna Tancos1,6, Istvan Bock7,6, Arpad G Baji1, Krisztina Tar7, Melinda K Pirity7 and Andras Dinnyes1,6*

Author Affiliations

1 Micromanipulation and Genetic Reprogramming Group, Agricultural Biotechnology Center, Szent-Györgyi A. u. 4. H-2100 Gödöllő, Hungary

2 National Medical Center Cell Biology Department; Daróci u. 24. H-1113 Budapest, Hungary

3 Faculty of Natural Sciences, Constantine the Philosopher University, Slovakia

4 University College Dublin, Lyons Research Farm, Newcastle Co. Dublin, Ireland

5 INRA, UMR1313, Laboratoire de Génétique Animale et Biologie Intégrative , 78350 Jouy en Josas , France

6 Molecular Animal Biotechnology Laboratory, Szent Istvan University, Pater K. u. 1. H-2103 Gödöllő, Hungary

7 BioTalentum Ltd., Aulich Lajos u. 26. H-2100 Gödöllő, Hungary

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BMC Molecular Biology 2009, 10:88 doi:10.1186/1471-2199-10-88

Published: 4 September 2009

Abstract

Background

The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4). It is crucial in the regulation of pluripotency during embryonic development and widely used as molecular marker of embryonic stem cells (ESCs). The objective of this study was to identify and to analyse the promoter region of rabbit POU5F1 gene; furthermore to examine its expression pattern in preimplantation stage rabbit embryos.

Results

The upstream region of rabbit POU5F1 was subcloned sequenced and four highly conserved promoter regions (CR1-4) were identified. The highest degree of similarity on sequence level was found among the conserved domains between rabbit and human. Among the enhancers the proximal enhancer region (PE-1A) exhibited the highest degree of homology (96.4%). Furthermore, the CR4 regulator domain containing the distal enhancer (DE-2A) was responsible for stem cell-specific expression. Also, BAC library screen revealed the existence of a processed pseudogene of rabbit POU5F1. The results of quantitative real-time PCR experiments showed that POU5F1 mRNA was abundantly present in oocytes and zygotes, but it was gradually reduced until the activation of the embryonic genome, thereafter a continuous increase in POU5F1 mRNA level was observed until blastocyst stage. By using the XYClone laser system the inner cell mass (ICM) and trophoblast portions of embryos were microdissected and examined separately and POU5F1 mRNA was detected in both cell types.

Conclusion

In this study we provide a comparative sequence analysis of the regulatory region of rabbit POU5F1 gene. Our data suggest that the POU5F1 gene is strictly regulated during early mammalian development. We proposed that the well conserved CR4 region containing the DE-2A enhancer is responsible for the highly conserved ESC specific gene expression. Notably, we are the first to report that the rabbit POU5F1 is not restricted to ICM cells only, but it is expressed in trophoblast cells as well. This information may be well applicable to investigate further the possible phylogenetic role and the regulation of POU5F1 gene.