| Project/Area Number |
16310140
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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 |
Applied genomics
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| Research Institution | Yamaguchi University |
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Principal Investigator |
OTOI Takeshige Yamaguchi University, The United Graduate School of Veterinazy Science, Professor (30311814)
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| Co-Investigator(Kenkyū-buntansha) |
YAMANOTO Yoshimi Yamaguchi University, Faculty of Agriculture, Professor (40115514)
SUTOU Shizuyo Shujitu University, School of Pharmacy, Professor (80368696)
多比良 和誠 東京大学, 大学院・工学系研究科, 教授 (10261778)
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| Project Period (FY) |
2004 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥15,850,000 (Direct Cost: ¥15,700,000、Indirect Cost: ¥150,000)
Fiscal Year 2007: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2005: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 2004: ¥8,900,000 (Direct Cost: ¥8,900,000)
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| Keywords | bovine spongiform encephalopathy / prion gene / RNA interference / prion protein / siRNA / cloned animal / クローン動勅 |
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| Research Abstract |
Prion diseases such as bovine spongiform encephalopathy (BSE) in cattle are caused by propagation of misfolded forms (PrP^<se>) of the normal cellular prion protein (PrP^c). By combining RNA interference (RNAi) technology with the somatic cell nuclear transfer (SCNT) method, we attempted to produce transgenic calves with knocked down prion gene (bPRNP). To achieve this, small interfering RNA (siRNA) expression plasmid vectors harboring a Pol III promoter, human U6 (hU6) or tRNA promoter, were constructed. Plasmid DNA was stably introduced into the genome of primary cultured bovine cells. SCNT embryos were produced by inserting the transgenic cell to an enucleated bovine egg, electro-fusing the two, and in vitro culture to the blastocyst stage. The ability for SCNT embryos to develop to blastocysts was higher in hU6 based vector groups (44-53%) than that of a tRNA group (32%). However, only SCNT embryos with the tRNA promoter could successfully impregnate recipients (6 out of 11 transfers), resulting in four aborted fetuses, one stillbirth, and one live-born calf. The expression of EGFP was detected in all six. The transgene of the tRNA vector was detected in two aborted fetuses and two calves. The bPRNP transcript level in the brain from the calf, subjected to euthanasia 20 days after birth, was 38% of that of the control calf as determined by quantitative RT-PCR. The PrP^c level in the brain determined by Western blot, however, was approximately 90% of the control, suggesting that a PrP^c level close to the normal level could be maintained by reduced bPRNP transcripts.
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