Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling.
BACKGROUND
Somatic cell nuclear transfer (SCNT) requires cytoplast-mediated reprogramming of the donor nucleus. Cytoplast factors such as maturation promoting factor are implicated based on their involvement in nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC). Given prior difficulties in SCNT in primates using conventional protocols, we hypothesized that the ability of cytoplasts to induce nuclear remodeling was instrumental in efficient reprogramming.


METHODS
NEBD and PCC in monkey (Macaca mulatta) SCNT embryos were monitored by lamin A/C immunolabeling.


RESULTS
Initially, a persistent lamin A/C signal from donor cell nuclei after fusion with cytoplasts was observed indicative of incomplete NEBD following SCNT and predictive of developmental arrest. We then identified fluorochrome-assisted enucleation and donor cell electrofusion as likely candidates for inducing premature cytoplast activation and a consequent lack of nuclear remodeling. Modified protocols designed to prevent premature cytoplast activation during SCNT showed robust NEBD and PCC. Coincidently, over 20% of SCNT embryos reconstructed with fetal fibroblasts progressed to blastocysts. Similar results were obtained with other somatic cells. Reconstructed blastocysts displayed patterns of Oct-4 expression similar to fertilized embryos reflecting successful reprogramming.


CONCLUSIONS
Our results represent a significant breakthrough in elucidating the role of nuclear remodeling events in reprogramming following SCNT.
Mitalipov
S M
SM
Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health and Science University, 505 NW 185th Avenue, Beaverton, OR 97006, USA. mitalipo@ohsu.edu
Zhou
Q
Q
Byrne
J A
JA
Ji
W Z
WZ
Norgren
R B
RB
Wolf
D P
DP
D000818:Animals; D002467:Cell Nucleus / Q000235:genetics*; D042002:Chromatin Assembly and Disassembly / Q000502:physiology*; D005260:Female; D034904:Lamin Type A / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008253:Macaca mulatta / Q000196:embryology; D008297:Male; D016200:Maturation-Promoting Factor / Q000502:physiology; D053652:Nuclear Transfer Techniques
D034904:Lamin Type A; D007976:Leupeptins; C440308:lamin C; D016200:Maturation-Promoting Factor; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde