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Quantitative Monitoring of Pluripotency Gene Activation after Somatic Cloning in Cattle¹

Center for Animal Transgenesis and Germ Cell Research,⁶ The School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania 19348 Institute of Molecular Animal Breeding and Biotechnology,⁴ Ludwig-Maximilians University, D-81377 Munich, Germany Biotechnology Unit,⁷ Institute of Animal Breeding, Bavarian State Institute for Agriculture, D-85586 Poing, Germany Institute of Veterinary Anatomy, Histology and Embryology,⁵ Ludwig-Maximilians University, D-80539 Munich, Germany Laboratory for Functional Genome Analysis (LAFUGA),⁸ Gene Center, Ludwig-Maximilians University, D-81377 Munich, Germany

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 enhanced green fluorescent protein (EGFP) reporter gene at a single integration site without detectable EGFP expression. Morphologic and quantitative analyses 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, whereas 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 a second round of 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 large mammals.

early development,, embryo

¹Supported in part by the Deutsche Forschungsgemeinschaft (FOR 478, ZA 425/1-1, GRK 1029), the Marion Dilley and David George Jones Funds, the Commonwealth and General Assembly of Pennsylvania, and the National Institute of Child Health & Human Development.

Correspondence: ²Eckhard Wolf, Institute of Molecular Animal Breeding and Biotechnology, Feodor-Lynen-Str. 22, D-81377 Munich, Germany. FAX: 49 89 2180 76849; e-mail: ewolf{at}lmb.uni-muenchen.de

Correspondence: ³These authors contributed equally to this work.

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