Protocol development optimal for cell-cycle stage of somatic donor cells in production of cloned rats
| Project/Area Number |
16300139
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Laboratory animal science
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| Research Institution | National Institutes of Natural Sciences |
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Principal Investigator |
HIRABAYASHI M. National Institutes of Natural Sciences, National Institute for Physiological Sciences, Center for Brain Experiment, Associate Professor, 行動・代調分子解析センター, 助教授 (20353435)
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| Co-Investigator(Kenkyū-buntansha) |
HOCHI S. Shinshu University, Faculty of Textile Science and Technology, Associate Professor, 繊維学部, 助教授 (10283243)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 2005: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | Cloned rat / MPF / Activation / Calcium / Kinase / Meiosis / PCC / Proteasome / 核移植 / 核移殖 / 自発的活性化 |
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| Research Abstract |
1. Qi Zhou et al. (2003) have first reported that ooplasmic injection of M-phase donor cell nucleus, followed by removal of maternal karyoplate in a one-step manner and the subsequent activation treatment with buthylactone-I, resulted in the birth of somatic cell-derived cloned rats. In the present study, the reproducibility of the Qi Zhou's protocol for production of cloned rats was investigated. None of 166 embryos reconstructed from fetal fibroblasts arrested at the metaphase stage (DA origin) and ovulated oocytes (SD origin) implanted and developed to full-term after transfer into 3 recipient females.
2. For the successful production of cloned mice using GO/G1-phase cumulus cells, nuclear injection into enucleated oocytes, followed by incidence of premature chromosome condensation (PCC) and parthenogenetic activation with strontium (Wakayama et al., 1998), was proved to be a reproducible and convenient technique in our laboratory. In the present study, the Wakayama's protocol was ap
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plied to produce cloned rats with minor modifications. Only 17% of the reconstructed oocytes (67/394) cleaved 24 h after a combined treatment for activation with ionomycin + 6-DMAP + cycloheximide. Transfer of 188 presumptive cloned zygotes into 9 recipients failed to produce any offspring.
3. Rat oocytes activate spontaneously once they are exposed to an in vitro condition. The incidence of PCC in the reconstructed rat oocytes was interfered with a rapid loss of maturation-promoting factor (MPF ; p34^<cdc2> kinase) activity due to the spontaneous oocyte activation. In addition, enucleation per se caused a complete loss of oocyte potential to support PCC of the donor nuclear materials. Therefore in the present study, regimens to inhibit spontaneous activation of rat oocytes were explored using chemicals associated with regulation of cell cycle via phosphorylation or cascades for ubichitination. Treatment with MG-132, a proteasome inhibitor, contributed to an increase of PCC incidence in enucleated oocytes receiving cumulus cell nuclei. Less
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Report
(4 results)
Research Products
(20 results)
