| Project/Area Number |
12358014
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Laboratory animal science
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| Research Institution | Kinki University |
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Principal Investigator |
TSUNODA Yukio Kinki University, College of Agriculture, Professor, 農学部, 教授 (80217364)
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| Co-Investigator(Kenkyū-buntansha) |
YONEMURA Isao Tottori swine and Poultry Experiment Station, Chief, 繁殖科, 科長
TANI Tetsuya Kinki University, College of Agriculture, Research associate, 農学部, 助手 (70319763)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥43,200,000 (Direct Cost: ¥36,000,000、Indirect Cost: ¥7,200,000)
Fiscal Year 2002: ¥15,600,000 (Direct Cost: ¥12,000,000、Indirect Cost: ¥3,600,000)
Fiscal Year 2001: ¥15,600,000 (Direct Cost: ¥12,000,000、Indirect Cost: ¥3,600,000)
Fiscal Year 2000: ¥12,000,000 (Direct Cost: ¥12,000,000)
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| Keywords | Nuclear transfer / Cloned pig / Somatic cells |
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| Research Abstract |
The production of cloned mammals by nuclear transfer of somatic cells into eggs in which the chromosomes were removed at the second metaphase stage (MII) is now feasible. The technology has been extended to apply to the genetic improvement of farm animals, rescue of endangered species, and production of transgenic animals for medical use and organ transplantation. The nuclear transfer technique is currently unreliable, however, because the production efficiency of normal offspring is low. The removal of chromosomes from recipient eggs is one of the key factors affecting cloning efficiency. The chromosomes of rabbit, sheep, goat, bovine , and pig eggs are difficult to observe without DNA staining. Thus, the chromosomes in these species are removed mainly by aspirating or pushing out a large volume of cytoplasm underlying the first polar body with or without Hoechst staining. However, such procedures might decrease the viability of enucleated oocytes.
The present study demonstrated that brief treatment of in vitro-matured porcine oocytes with demecolcine results in a membrane protrusion that contains a condensed chromosome mass, which can be easily removed by aspiration. This simple chemically-assisted method for removing maternal chromosomes enabled the production of a large number of nuclear-transferred porcine eggs. The development of eggs whose chromosomes were removed by this procedure following transfer of somatic cell nuclei to the blastocyst stage was not significantly different among groups activated using different procedures and was also not different among donor cells of different origins, except for cumulus cells. After transfer of 180 to 341 nuclear-transferred eggs that received somatic cells to 6 recipients, 2 of the recipients produced 8 healthy cloned piglets from the heart cells of a female pig. The chemically-assisted method for removing maternal chromosomes was also effective for bovine and rabbit eggs.
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