| Project/Area Number |
26670582
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Multi-year Fund |
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| Research Field |
General surgery
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| Research Institution | Osaka University |
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Principal Investigator |
Miyagawa Shuji 大阪大学, 医学(系)研究科(研究院), 准教授 (90273648)
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| Project Period (FY) |
2014-04-01 – 2016-03-31
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| Project Status |
Completed (Fiscal Year 2015)
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| Budget Amount *help |
¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Fiscal Year 2015: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2014: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
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| Keywords | ブタ内在性レトロウィルス(PERVｓ) / CRISPR/CRシステム / OFF Targetting効果 / Gag-pol / ブタ内在性レトロウィルス(PERVs) / CRISPR/ＣＲシステム / OFF Ｔａｒｇｅｔｉｎｇ効果 / ブタ内在性レトロウイルス（PERVs） / CRISPR/CS システム / Off targeting 効果 |
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| Outline of Final Research Achievements |
We attempted to knockout the PERV by CRISPR system. The plasmids expressing hCas9 and sgRNA for PERV were prepared by ligating oligos into pX330. pCAG-EGxxFP-target was mixed with pX330 with/without sgRNA sequences and then introduced into HEK293T cells. The EGFP fluorescence was them observed after transfection. Four oligos and primers are nominated in each gag and pol region from 38 and 20 candidates, respectively. Among them, the best one of each region was selected.
Next, the expression of the PERV can be knockouted by CRISPR system. PK15 was used to check the changes in the expression of PERV by the pX330-gag with neomycine. After the transfection of the pX330, 17 clones of PK15 cells with the pX330 were established by neomycine selection. Among them, 15 clones were ascertained the integration of pX330-gag into the genome. The sequence of the 15 clones was then checked. A clone showed certain delation of the targeting site of PERV by the CRISPR system.
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