New Approach for the generation of transgenic frogs
| Project/Area Number |
12680812
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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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 | Shimane University |
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Principal Investigator |
NISHIKAWA Akio Shimane Univ., Fac. of Life and Environmental Science, Assoc. Professor, 生物資源科学部, 助教授 (30192686)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | Primary culture / Xenopus laevis / subculture / heart endothelial cells / clone culture / cell-chimera / myoblasts / cell proliferation / 初代培養 / 心臓細胞 / クローン増殖 |
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| Research Abstract |
The methods for the primary culture of various tissues from adult frogs were developed in order to establish the host cells for gene introduction and homologous recombination. The main result was as follows.
1. We at first examine the cell types that grew rapidly by cultivation of various types of cells isolated from heart, liver, testis, skin (dermis), and skeletal muscles of adult frog (Xenopus laevis). We found that heart endothelial cells grew most rapidly. The cell number increased 10-fold within 1 week. Since serial subculture could be succeeded until the PD (population doubling) 50, these cells can be used for the experiment for gene introduction. Other than endothelial cells, the myoblasts isolated from skeletal muscles also proliferated rapidly in vitro while some of them underwent differentiation.
2. The maximum (10-times/week) proliferation rate of heart endothelial cells was observed when they were inoculated at 3x10^4/22-mm well. However, the growth rate gradually decreased as the PD number increase, suggested that these cells were not transformed. The clone culture from single cell was not succeeded.
3. In order to find out another possibility of host cells for the gene transfer experiment, we tried to keep the isolated skeletal myoblasts in undifferentiated condition by high serum-concentration in medium. The result showed that myoblast differentiation was completely suppressed, suggesting the possible use of myoblasts as the host cells for gene introduction.
4. Since there is a necessity for the cell-chimera analysis before the transgenesis experiment by nuclear transfer, we established the culture of the cells with nuclear marker (Xenopus borealis cells) in order to trace the cell fate. If we make hybrid myotube with isolated X. borealis and X. laevis myoblasts, each nuclear fate could be easily traced by quinacrine staining.
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Report
(3 results)
Research Products
(7 results)
