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BOR - Papers in Press, published online ahead of print February 21, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.058776
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biolreprod.106.058776v1
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Submitted November 10, 2006
Returned for revision December 22, 2006
Accepted February 14, 2007

Embryo


Quantitative Monitoring of Pluripotency Gene Activation after Somatic Cloning in Cattle

Annegret Wuensch *, Felix A. Habermann , Satoshi Kurosaka , Regina Klose , Valeri Zakhartchenko , Horst-Dieter Reichenbach , Fred Sinowatz , K. John McLaughlin , and Eckhard Wolf

* To whom correspondence should be addressed. E-mail: wuensch{at}lmb.uni-muenchen.de.

Abstract
The development of somatic cell nuclear transfer (SCNT) embryos critically depends on appropriate reprogramming and expression of pluripotency genes, such as Pou5f1/POU5F1 (previously known as Oct4/OCT4). To study POU5F1 transcription activation in living bovine SCNT embryos without interference by maternal POU5F1 mRNA we generated chromosomally normal fetal fibroblast donor cells stably carrying a mouse Pou5f1 promoter-driven EGFP reporter gene at a single integration site without detectable EGFP expression. Morphological and quantitative analysis of whole mount SCNT embryos by confocal microscopy revealed robust initial activation of the Pou5f1 reporter gene during the fourth cell cycle. In Day 6 SCNT embryos EGFP expression levels were markedly higher than in Day 4 embryos but varied substantially between individual embryos even at comparable cell numbers. Embryos with low EGFP levels had far more morphologically abnormal cell nuclei than those with high EGFP levels. Our data strongly suggest that bovine SCNT embryos consistently start activation of the POU5F1 promoter during the fourth cell cycle, while later in development the expression level substantially differs between individual embryos, which may be associated with developmental potential. In fibroblasts from phenotypically normal SCNT fetuses recovered on Day 34, the Pou5f1 reporter promoter was silent, but was activated by second round SCNT. The restoration of pluripotency can be directly observed in living cells or SCNT embryos from such Pou5f1-EGFP transgenic fetuses providing an attractive model for systematic investigation of epigenetic reprogramming in higher mammals.

Key words: Embryo • Early development


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Reproduction, February 1, 2008; 135(2): 165 - 179.
[Abstract] [Full Text] [PDF]


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