| Project/Area Number |
20590866
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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 |
Circulatory organs internal medicine
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| Research Institution | Tottori University |
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Principal Investigator |
HISATOME Ichiro Tottori University, 大学院・医学系研究科, 教授 (60211504)
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| Co-Investigator(Kenkyū-buntansha) |
SHIRAYOSHI Yasuaki 鳥取大学, 大学院・医学系研究科, 准教授 (90249946)
YAMAMOTO Yasutaka 鳥取大学, 大学院・医学系研究科, 助教 (20362882)
MIAKE Junichiro 鳥取大学, 医学部附属病院, 助教 (40372677)
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| Project Period (FY) |
2008 – 2010
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| Project Status |
Completed (Fiscal Year 2010)
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| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2010: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2009: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2008: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
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| Keywords | ペースメーカ / ES細胞 / イオンチャネル / 生物学的ペースメーカ / HCN4 / 自動能 / 房室ブロックモデル / 細胞移植 |
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| Research Abstract |
We are attempting to establish the cardiac pacemaker cells derived from embryonic stem (ES) cells. To visualize cardiac pacemaker cells in differentiating embryoid bodies (EBs), EGFP gene was knocked in at HCN4 locus, which encodes ion channels responsible for pacemaker activity. In the present study, we conducted to establish EGFP-knock in ES cell line at HCN4 locus. PCR screening revealed two positive clones as possible knock in ES cell line. Southern blot analysis showed that one of them, H7 clone, is correctly targeted at HCN4 locus. After induction to cardiac differentiation through the formation of EBs in H7 clone, the expression of GFP was specifically localized at their contracting region in the outgrowth of EBs. Analysis of cell sorting revealed that 0.1～0.3% of differentiated H7 cells in EBs were GFP positive and that 70～90% of the sorted GFP positive cells were morphologically homogenous and beat spontaneously. Patch clamp analysis showed that spontaneously beating cells revealed the comparable electrophysiological properties with cardiac pacemaker cells, such as automaticity and If current. These results assured that H7 cell line was suitable to investigate and purify cardiac pacemaker cells.
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