Submission Number: KEV-4-427-203

Abstract Number: 3

NUCLEAR TRANSFER IN THE LABORATORY MOUSE: PARAMETERS AFFECTING LONG TERM SURVIVAL OF CLONES.

Kevin Eggan ¹, Hidenori Akutsu ², William III Rideout ¹, Teruhiko Wakayama ^2,3, Anton Wutz ¹, Laurie Jackson-Grusby ¹, Ryuzo Yanagimachi ² and Rudolf Jaenisch ¹

Whitehead Institute for Biomedical Research, Cambridge, MA ¹
Department of Anatomy and Reproductive Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI ²
Rockefeller University, New York, NY ³

Abstract:
The full term development of sheep, cows, goats, pigs and mice has been achieved through the transfer of somatic cell nuclei into enucleated oocytes. Despite these successes, mammalian cloning remains an inefficient process, with a preponderance of reconstructed embryos failing at early to midgestation stages of development. The small percentage of conceptuses that survive to term are characterized by a high mortality rate. These neonates frequently display grossly increased placental and birth weights. Because of the inherent inefficiency of the process, it has been difficult to assess the importance of various parameters thought to be critical for cloning success. These parameters include cell cycle state, and genetic constituency of the donor cell as well as susceptibility of the donor genome to reprogramming by the oocyte. In order to more effectively investigate these parameters, we have developed a method for cloning mice from embryonic stem (ES) cells. In these experiments 10 to 40% of oocytes reconstructed with ES cell nuclei developed to the blastocyst stage. This rate is lower than that seen for cumulus cell clones, which complete preimplantation development with 70 to 90% efficiency. The disparity in preimplantation development between these cell types may reflect differences in the cell cycle states of the rapidly dividing ES cells and quiescent cumulus cells. After transfer into pseudo pregnant females, we noted that ES cell clones developed to term with an efficiency of 20 to 40%, which is much higher than for all other previously reported somatic cell types. Interestingly, we observed that genetic background plays a critical role in postnatal survival of ES cell clones. All clones derived from three inbred ES cell lines died shortly after birth. In contrast, the majority of ES cell clones, derived from four different F1 combinations of inbred genotypes, thrived after birth. Recently, we utilized this procedure to clone ES cells with a M. musculus / M. castaneus F1 genotype. Currently, we are taking advantage of polymorphic alleles within these animals to investigate the parental specific expression of imprinted genes. These experiments will test the hypothesis that improper expression of imprinted genes leads to neonatal overgrowth and death of cloned animals. .

Keywords: clones, somatic cell nuclei, oocytes

This abstract is being presented at: 11:30 AM in session:
Minisymposium III: MECHANISMS OF REPROGRAMMING IN NUCLEAR TRANSPLANTATION