Davelopment of Method to Analyse the Function of Regulatory Proteins in Single by Cells by Gene Handling Technique.

开发利用基因处理技术分析单细胞调节蛋白功能的方法。

• 批准号: 11559016
• 负责人: ISHIWATA Sinichi
• 金额: $ 2.69万
• 依托单位: Waseda University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11559016/
• 关键词: Caged DNA; Teaperature-pulse unicroscopy; DNA tip; POR method; Myoblast cell; GFP-actin; Actin filament; Stress sensor; GFP-アクチン; ケージドDNA; 温度パルス顕微鏡法; 遺伝子ハンドリング; DNAの熱変性

Recovery of DNA fragment from a DNA probe array : We have developed a DNA preparation method using a DNA probe array that utilizes photo-thermal denaturation to recover specific DNA. The protocol for preparing DNA probe array was investigated to realize the stable immobilization of DNA probes on the surface of solid support. We used a glass plate coated with Cr (5-10 nm thick). The Cr surface was modified with 3-glysidoxypropyltrimethoxysilane to introduce active residues that can couple with amino residues at the 5' terminal of DNA probes. The Cr surface acts as a photo-thermal transducer. The preparation method of DNA uses infrared (1053-nm) laser irradiation to thermally denature and release DNA immobilized in a specific area of a DNA probe array. Different DNA fragments fixed in place on the DNA probe array could be separately recovered. There were enough quantities of recovered DNA that can be amplified by using PCR. Trial to synthesize caged DNA : Aiming at synthesizing caged DNA, we tried to chemically modify an amino-residue on DNA with 4, 5-diraethoxy-2-nitrobenzyl bromide (DMNBB). Although the synthesis of chemically modified DNA once appeared to be confirmed by using PCR method, we finally reached a conclusion that the amino-residue on DNA is not reactive to the chemical modification. Expression of GFP-actin in cultured embryonic heart cells : We could confirm that GFP-actin was expressed in cultured embryonic heart cells under fluorescence microscopy. Although we may have not be able to achieve the original goal, the results obtained in this project must be, we believe, very useful for planning a future project.

从DNA探针阵列中回收DNA片段：我们开发了一种使用DNA探针阵列的DNA制备方法，该方法利用光热变性来回收特定的DNA。研究了DNA探针阵列的制备方案，以实现DNA探针在固相支持物表面的稳定固定化。我们使用镀有 Cr 的玻璃板（5-10 nm 厚）。 Cr 表面用 3-缩水甘油醚氧基丙基三甲氧基硅烷进行修饰，引入可与 DNA 探针 5' 末端的氨基残基偶联的活性残基。 Cr 表面充当光热转换器。 DNA的制备方法利用红外(1053nm)激光照射使固定在DNA探针阵列的特定区域中的DNA热变性并释放。固定在 DNA 探针阵列上的不同 DNA 片段可以单独回收。回收的 DNA 数量足够多，可以通过 PCR 进行扩增。笼状DNA的合成试验：为了合成笼状DNA，我们尝试用4,5-二乙氧基-2-硝基苄基溴(DMNBB)对DNA上的氨基残基进行化学修饰。尽管化学修饰DNA的合成一度似乎通过PCR方法得到证实，但我们最终得出的结论是DNA上的氨基残基对化学修饰不产生反应。培养的胚胎心脏细胞中GFP-肌动蛋白的表达：我们可以在荧光显微镜下证实培养的胚胎心脏细胞中表达GFP-肌动蛋白。虽然我们可能无法实现最初的目标，但我们相信，这个项目所取得的成果对于规划未来的项目一定非常有用。

项目成果

Ishiwata, S., et al.: "Molecular Interactions of Actin"Springer Verlag Berlin Heidelberg. 16 (2001)

Ishiwata，S.等人：“肌动蛋白的分子相互作用”Springer Verlag Berlin Heidelberg。

Ishiwata,S. et al.: "Molecular Interactions of Actin (eds.C.G.dos Remedios & D.D.Thomas)"Springer Verlag, Heidelbery. 269 (2000)

石渡，S.

Kawaguchi,K.& Ishiwata,S.: "Temperature dependence of force, velocity and processivity of single kinesin melecules."Biochem.Biophys.Res.Comm.. 272. 895-899 (2000)

川口，K.

Fukuda, N. & Ishiwata, S.: "Effects of pH on spontaneons tension oscillation in skinned bovine cardiac muscle."Pflugers. Aroh-Eur.J.Physiol.. 438. 125-132 (1999)

福田，N.

Kawaguchi, K. and Ishiwata, S.: "Temperature dependence of force, velocity and processivity of single kinesin molecules"Biochem. Biophys. Res. Comm.. 272(3). 895-899 (2000)

Kawaguchi, K. 和 Ishiwata, S.：“单个驱动蛋白分子的力、速度和持续合成能力的温度依赖性”Biochem。