| Project/Area Number |
23791757
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Multi-year Fund |
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| Research Field |
Urology
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| Research Institution | Kobe University |
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Principal Investigator |
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| Research Collaborator |
LI Fuping 神戸大学, 大学院・医学研分野, 大学院生
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| Project Period (FY) |
2011 – 2012
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| Project Status |
Completed (Fiscal Year 2012)
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| Budget Amount *help |
¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
Fiscal Year 2012: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2011: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
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| Keywords | セルトリ細胞 / エレクトロポレーション / トランスフェクション / EGFP / hTERT |
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| Research Abstract |
In this study, we described an optimized protocol for electroporation-based transfection of Sertoli cells and compared its efficiency with conventional lipofection. Sertoli cells were transfected with pCMV-GFP plasmid by square-wave electroporation in different conditions. According for both cell survival and the percentage expressing EGFP, 250 V was determined to produce the greatest number of transiently transfected cells. Keeping voltage consistent (250 V), the pulse length of 20μm was observed relatively higher cell survival (76.5 ± 3.4%) and transfection efficiency (30.6 ± 5.6%). The yield of positive cells increased with increasing concentrations of plasmid DNA (range 10-50μg/ml) from 14.0 ± 2.8% to 35.0 ± 6.3% respectively, but cells viability steadily decreased following 20μg/ml plasmid DNA from 73.1 ± 4.9% to 57.0 ± 6.6%. We described the process of optimizing electroporation conditions, and the successful electroporation of plasmid DNA into primarily cultured Sertoli cells. Our results indicated that the method of electroporation is more suitable for transfection of Sertoli cells.
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