Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling

S. M. Mitalipov, Q. Zhou, J. A. Byrne, W. Z. Ji, Robert B Norgren, D. P. Wolf

Research output: Contribution to journalArticle

87 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2232-2242
Number of pages11
JournalHuman Reproduction
Volume22
Issue number8
DOIs
StatePublished - Aug 2007

Fingerprint

Primates
Nuclear Envelope
Lamin Type A
Chromosomes
Embryo Transfer
Blastocyst
Maturation-Promoting Factor
Macaca mulatta
Cell Nucleus
Fluorescent Dyes
Haplorhini
Embryonic Structures
Fibroblasts

Keywords

  • Lamin A/C
  • Nuclear remodeling
  • Premature cytoplast activation
  • Primate
  • Somatic cell nuclear transfer

ASJC Scopus subject areas

  • Reproductive Medicine
  • Obstetrics and Gynecology

Cite this

Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling. / Mitalipov, S. M.; Zhou, Q.; Byrne, J. A.; Ji, W. Z.; Norgren, Robert B; Wolf, D. P.

In: Human Reproduction, Vol. 22, No. 8, 08.2007, p. 2232-2242.

Research output: Contribution to journalArticle

Mitalipov, S. M. ; Zhou, Q. ; Byrne, J. A. ; Ji, W. Z. ; Norgren, Robert B ; Wolf, D. P. / Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling. In: Human Reproduction. 2007 ; Vol. 22, No. 8. pp. 2232-2242.
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abstract = "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.",
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