title: Rat Blastocysts from Nuclear Injection and Time-Lagged Enucleation and Their Commitment to Embryonic Stem Cells.
abstract: Pronucleus-like vesicle formation following premature chromosome condensation (PCC) of the donor cell nucleus is the key event for successful generation of cloned rodents by nuclear transplantation (NT). However in rat cloning, this change is difficult to induce in enucleated recipient oocytes because of their inability to maintain maturation-promoting factor levels. In this study, intact oocytes retrieved from nuclear-visualized H2B-tdTomato knock-in rats were injected with Venus-labeled cell nuclei. Because the incidence of PCC under MG-132 treatment significantly increased with the culture period (0%, 10.8%, 36.8%, and 87.5% at 0, 0.5, 1, and 2 h postinjection, respectively), the metaphase plate of the oocyte was removed 1-2 h after the nuclear injection. The NT-derived rat zygotes (n = 748) were activated with ionomycin/cycloheximide and transferred into temporal host mothers, resulting in the harvest of three blastocysts (0.4%) with Venus fluorescence. Two blastocysts were examined for their potential to commit to NT-derived embryonic stem cells (ntESCs). One ntESC line was established successfully and found to be competent in terms of karyotype, stem cell marker expression, and pluripotency. In conclusion, time-lagged enucleation of visualized oocyte nuclei allows the PCC incidence of donor nuclei and generation of NT blastocysts, and the blastocysts can commit to germline-competent ntESCs.
authors.affiliation: 1 Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences , Okazaki, Aichi, 444-8787, Japan .
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Hara
authors.affiliation: 1 Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences , Okazaki, Aichi, 444-8787, Japan .
authors.forename: Teppei
authors.initials: T
authors.lastname: Goto
authors.affiliation: 2 Department of Physiology, Human and Molecular Genetics Center , Medical College of Wisconsin, Milwaukee, Wisconsin, 53226.
authors.forename: Akiko
authors.initials: A
authors.lastname: Takizawa
authors.affiliation: 1 Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences , Okazaki, Aichi, 444-8787, Japan .
authors.forename: Makoto
authors.initials: M
authors.lastname: Sanbo
authors.affiliation: 3 Department of Physiology and Department of Pediatrics, Human and Molecular Genetics Center , Medical College of Wisconsin, Milwaukee, Wisconsin, 53226.|7 Present address: HudsonAlpha Institute for Biotechnology , Huntsville, Alabama, 35806.
authors.forename: Howard J
authors.initials: HJ
authors.lastname: Jacob
authors.affiliation: 4 Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo , Minato-ku, Tokyo, 108-8639, Japan .|5 Japan Science Technology Agency , ERATO, Nakauchi Stem Cell and Organ Regeneration Project, Minato-ku, Tokyo, 108-8639, Japan .|8 Present address: Gurdon Institute, University of Cambridge , Cambridge, CB2 1QN, United Kingdom .
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: 4 Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo , Minato-ku, Tokyo, 108-8639, Japan .|5 Japan Science Technology Agency , ERATO, Nakauchi Stem Cell and Organ Regeneration Project, Minato-ku, Tokyo, 108-8639, Japan .
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Nakauchi
authors.affiliation: 6 Faculty of Textile Science and Technology, Shinshu University , Ueda, Nagano, 386-8567, Japan .
authors.forename: Shinichi
authors.initials: S
authors.lastname: Hochi
authors.affiliation: 1 Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences , Okazaki, Aichi, 444-8787, Japan .
authors.forename: Masumi
authors.initials: M
authors.lastname: Hirabayashi
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D055879:Gene Knock-In Techniques / Q000379:methods; D008297:Male; D053652:Nuclear Transfer Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley