27 - 30 May 2005

EPIGENETICS IN CLONING Eckhard Wolf Institute of Molecular Animal Breeding and Biotechnology Gene Center Ludwig-Maximilian University of Munich Feodor-Lynen-Str. 25 D-81377 Munich Germany ewolf@lmb.uni-muenchen.de

With the exception of lymphocytes, the various cell types in a higher multicellular organism have basically an identical genotype but are functionally and morphologically different. This is due to tissue-specific, temporal, and spatial gene expression patterns which are controlled by genetic and epigenetic mechanisms.

Successful cloning of mammals by transfer of nuclei from differentiated tissues into enucleated oocytes demonstrates that these genetic and epigenetic programs can be largely reversed and that cellular totipotency can be restored. Although these experiments indicate an enormous plasticity of nuclei from differentiated tissues, somatic cloning is a rather inefficient and unpredictable process, and a plethora of anomalies have been described in cloned embryos, fetuses, and offspring.

Accumulating evidence indicates that incomplete or inappropriate epigenetic reprogramming of donor nuclei is likely to be the primary cause of failures in nuclear transfer. Various epigenetic mechanisms, including DNA methylation, imprinting, X chromosome inactivation, chromatin remodeling, telomere maintenance, and epigenetic inheritance are essential for normal embryonic development and have been shown to be abnormal in clones from different species.

Nuclear transfer represents an invaluable tool to experimentally address fundamental questions related to epigenetic reprogramming. Understanding the dynamics and mechanisms underlying epigenetic control will help to solve problems inherent in nuclear transfer technology and enable many applications, including the modulation of cellular plasticity for human cell therapies.

References:

Dean W, Santos F, Stojkovic M, Zakhartchenko V, Walter J, Wolf E, Reik W (2001) Conservation of methylation reprogramming in mammalian embryos: aberrant reprogramming in cloned embryos. PNAS 98, 13734-13738

Hiendleder S, Mund C, Reichenbach HD, Wenigerkind H, Brem G, Zakhartchenko V, Lyko F, Wolf E (2004) Tissue-specific elevated genomic cytosine methylation levels are associated with an overgrowth phenotype of bovine fetuses derived by in vitro techniques. Biol Reprod 71, 217-223

Hiendleder S, Prelle K, Brüggerhoff K, Reichenbach H-D, Wenigerkind H, Bebbere D, Stojkovic M, Müller S, Brem G, Zakhartchenko V, Wolf E (2004) Nuclear-cytoplasmic interactions affect in utero developmental capacity, phenotype and cellular metabolism of bovine nuclear transfer fetuses. Biol Reprod 70, 1196-1205

Hiendleder S, Zakhartchenko V, Wenigerkind H, Reichenbach H-D, Brüggerhoff K, Prelle K, Brem G, Stojkovic M, Wolf E (2003) Heteroplasmy in bovine fetuses produced by intra- and intersubspecific somatic cell nuclear transfer: neutral segregation of nuclear donor mitochondrial DNA in various tissues and evidence for recipient cow mitochondria in fetal blood. Biol Reprod 68, 159-166

Santos F, Zakhartchenko V, Stojkovic M, Peters A, Jenuwein T, Wolf E, Reik W, Dean W (2003) Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos. Curr Biol 13, 1116-1121

Shi W, Dirim F, Wolf E, Zakhartchenko V, Haaf T (2004) Methylation reprogramming and chromosomal aneuploidy in in vivo fertilized and cloned rabbit preimplantation embryos. Biol Reprod 71, 340-247

Shi W, Hoeflich A, Flaswinkel H, Stojkovic M, Wolf E, Zakhartchenko V (2003) Induction of a senescent-like phenotype does not confer the ability of bovine immortal cells to support the development of nuclear transfer embryos. Biol Reprod 69, 301-309

Shi, W, Zakhartchenko V, Wolf E (2003) Mammalian nuclear transfer and epigenetic reprogramming. Differentiation 71, 91-113

List of abstracts from the 3rd International Conference on the Female Reproductive Tract