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

Shoukhrat Mitalipov, Q. Zhou, J. A. Byrne, W. Z. Ji, R. B. Norgren, D. P. Wolf

    Research output: Contribution to journalArticle

    85 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

    • Physiology
    • Developmental Biology
    • Obstetrics and Gynecology
    • Reproductive Medicine

    Cite this

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

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

    Research output: Contribution to journalArticle

    Mitalipov, Shoukhrat ; Zhou, Q. ; Byrne, J. A. ; Ji, W. Z. ; Norgren, R. 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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