siRNAライブラリーを用いたマイクロRNAの解析

使用 siRNA 文库进行 MicroRNA 分析

• 批准号: 04F04869
• 负责人: 藁科 知子 (桑原 知子) (2006)
• 金额: $ 1.34万
• 依托单位: National Institute of Advanced Industrial Science and Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04F04869/
• 关键词: siRNA; アルツハイマー病; レンチウイルス; 神経; 海馬; 脳; GFP; 遺伝子治療; RNAi; miRNA

アルツハイマー病の発症メカニズムに深く関わるBeta Secretase遺伝子に対して、効率よく発現のノックダウンを誘導できるsiRNAの様々な設計を行った。設計したsiRNA中、in vitroの幹細胞培養系にて最も抑制効率の高かったsiRNAを、in vivoで効果を検証するためのレンチウイルスコンストラクトに組み込んだ。siRNA発現カセットが導入された感染細胞を識別するために、マーカーとしてGFPをともに発現する様に設計した。分化に影響を与えない緩衝液で懸濁され濃縮したウイルス液をコントロールとし、siRNAを発現するウイルスを、マイクロインジェクションによって神経新生が頻繁に起きる脳海馬領域に導入した。導入して2ヶ月経過後、脳を抽出し海馬領域の切片を作成し、siRNAが与える影響を各分化経路のマーカーとなる神経遺伝子や、グリア系遺伝子の多重染色を行い、評価した。さらに、脳領域での神経新生、前駆細胞の細胞増殖および成熟ニューロンへの分化誘導にBeta Secretase遺伝子がどのような役割を果たしているかも、関連するシグナル伝達系のキナーゼ群の活性化や幾つかの転写因子の発現、およびそれらのノックダウンを指標に、多重染色によって調べた。これらの結果、設計し導入したsiRNAによって、どの標的遺伝子の場合も安定に脳内で起きていることをin vivoにおいても確認できた。また、同時に行ったシグナル伝達系のキナーゼのノックダウン細胞で、Beta Secretase遺伝子の発現抑制が見られた。これらは、アルツハイマー病の発症メカニズムに深く関わるBeta Secretase遺伝子が、健常体の動物の脳内で神経分化に深く関わる転写因子の制御機構によって、厳密に発現がコントロールされていることを示唆する。今後さらにこのメカニズムを深く追求し、遺伝子治療に向けて、より効率の良い標的遺伝子とノックダウンツールを研究開発して行く予定である。

In this case, we need to learn more about the symptoms and signs of the disease. We need to know that there is a problem in the Beta Secretase health care system. The rate is very high. We need to know how to improve the performance of the siRNA system. In the design of siRNA, the in vitro cell culture system has the highest inhibition rate, the highest inhibition rate, the highest inhibition SiRNA detects the number of people who are infected. The number of people who are infected is different. The number of people who are infected is different. The number of people who are infected with the virus is different from that of GFP. The difference between the two is different from each other. The difference between them is that there is no difference between them. The difference between them is that there is a difference between them. The difference between them is that there is a difference between them. The difference between them is that there is a difference between them. The difference between them is that there are many problems in the field of sea, such as the birth of the gods, the birth of the gods and the birth of the gods. After two months of entry, the sea area was cut into slices, siRNA and so on, and the differentiation pathways were analyzed. In the field and in the field, the cells were born, the cells matured, the cells differentiated, and the Beta Secretase cells were differentiated. The results showed that the population was active, the population was active, the factor was detected, and the multiple staining was used. Check the results, design and input the siRNA filter, and the sub-cluster of the switch will start the emergency response in vivo filter to confirm that the message is in the safety zone. At the same time, it is necessary to make sure that the cells are affected, and that the Beta Secretase cells are suppressed in the meantime. The symptoms of the disease, the symptoms, the signs, the signs. In the future, there will be a lot of research in the future, such as the deep pursuit of the future, the improvement of the quality of life, and the improvement of the rate of improvement.