Method for extracting quantitative information relating to an influence on a cellular response
    1.
    发明授权
    Method for extracting quantitative information relating to an influence on a cellular response 有权
    提取与细胞反应有关的定量信息的方法

    公开(公告)号:US06518021B1

    公开(公告)日:2003-02-11

    申请号:US09417197

    申请日:1999-10-07

    IPC分类号: C12Q168

    摘要: Cells are genetically modified to express a luminophore, e.g., a modified (F64L, S65T, Y66H) Green Flourescent Protein (GFP, EGFP) coupled to a component of an intracellular signalling pathway such as a transcription factor, a cGMP- or cAMP-dependent protein kinase, a cyclin-, calmodulin- or phospholipid-dependent or mitogen-activated serine/threonin protein kinase, a tryosine protein kinase, or a protein phosphatase (e.g. PKA, PKC, Erk, Smad, VASP, actin, p38, Jnkl, PKG, IkappaB, CDK2, Grk5, Zap70, p85, protein-tyrosine phosphatase 1C, Stat5, NFAT, NFkappaB, RhoA, PKB). An influence modulates the intracellular signaling pathway in such a way that the luminophore is being redistributed or translocated with the component in living cells in a manner experimentally determined to be correlated to the degree of influence. Measurement of redistribution is performed by recording of light intensity, flourescence lifetime, polarization, wavelength shift, resonance energy transfer, or other properties by an apparatus consisting of e.g. a flourescence microscope and a CCD camera. Data stored as digital images are processed to numbers representing the degree of redistribution. The method can be used as a screening program for identifying a compound that modulates a component and is capable of treating a disease related to the function of the component.

    摘要翻译: 细胞被遗传修饰以表达发光体,例如,与细胞内信号传导途径的组分偶联的经修饰的(F64L,S65T,Y66H)绿色荧光蛋白(GFP,EGFP),例如转录因子,cGMP-或cAMP依赖性 蛋白激酶,细胞周期蛋白,钙调蛋白或磷脂依赖性或丝裂原活化丝氨酸/苏氨酸蛋白激酶,软骨蛋白激酶或蛋白磷酸酶(例如PKA,PKC,Erk,Smad,VASP,肌动蛋白,p38,Jnk1, PKG,IkappaB,CDK2,Grk5,Zap70,p85,蛋白质 - 酪氨酸磷酸酶1C,Stat5,NFAT,NFkappaB,RhoA,PKB)。 影响调节细胞内信号传导途径,使发光体以活体细胞中的成分重新分配或易位,其方式通过实验确定与影响程度相关。 重新分布的测量是通过记录光强度,荧光寿命,极化,波长偏移,共振能量转移或其它性质通过由例如包含的装置组成的装置进行的。 荧光显微镜和CCD相机。 作为数字图像存储的数据被处理成表示再分配程度的数字。 该方法可以用作鉴定调节成分并能够治疗与该成分的功能有关的疾病的化合物的筛选程序。

    Method for extracting quantitative information relating to an influence on a cellular response
    2.
    发明授权
    Method for extracting quantitative information relating to an influence on a cellular response 有权
    提取与细胞反应有关的定量信息的方法

    公开(公告)号:US08058008B2

    公开(公告)日:2011-11-15

    申请号:US10072036

    申请日:2002-02-05

    IPC分类号: G01N33/53 G01N21/76 C12P21/02

    摘要: Cells are genetically modified to express a luminophore, e.g., a modified (F64L, S65T, Y66H) Green Fluorescent Protein (GFP, EGFP) coupled to a component of an intracellular signalling pathway such as a transcription factor, a cGMP- or cAMP-dependent protein kinase, a cyclin-, calmodulin- or phospholipid-dependent or mitogen-activated serine/threonin protein kinase, a tyrosine protein kinase, or a protein phosphatase (e.g. PKA, PKC, Erk, Smad, VASP, actin, p38, Jnk1, PKG, IkappaB, CDK2, Grk5, Zap70, p85, protein-tyrosine phosphatase 1C, Stat5, NFAT, NFkappaB, RhoA, PKB). An influence modulates the intracellular signalling pathway in such a way that the luminophore is being redistributed or translocated with the component in living cells in a manner experimentally determined to be correlated to the degree of the influence. Measurement of redistribution is performed by recording of light intensity, fluorescence lifetime, polarization, wavelength shift, resonance energy transfer, or other properties by an apparatus consisting of e.g. a fluorescence microscope and a CCD camera. Data stored as digital images are processed to numbers representing the degree of redistribution. The method can be used as a screening program for identifying a compound that modulates a component and is capable of treating a disease related to the function of the component.

    摘要翻译: 细胞被遗传修饰以表达发光体,例如,与细胞内信号传导途径的组分偶联的经修饰的(F64L,S65T,Y66H)绿色荧光蛋白(GFP,EGFP),例如转录因子,cGMP-或cAMP依赖性 蛋白激酶,细胞周期蛋白,钙调蛋白或磷脂依赖性或丝裂原活化的丝氨酸/苏氨酸蛋白激酶,酪氨酸蛋白激酶或蛋白磷酸酶(例如PKA,PKC,Erk,Smad,VASP,肌动蛋白,p38,Jnk1, PKG,IkappaB,CDK2,Grk5,Zap70,p85,蛋白质 - 酪氨酸磷酸酶1C,Stat5,NFAT,NFkappaB,RhoA,PKB)。 影响调节细胞内信号传导途径,使得发光体在活体细胞中以组分重新分配或移位,其方式通过实验确定与影响程度相关。 重新分布的测量是通过记录光强度,荧光寿命,极化,波长偏移,共振能量转移或其它性质通过由例如, 荧光显微镜和CCD相机。 作为数字图像存储的数据被处理成表示再分配程度的数字。 该方法可以用作鉴定调节成分并能够治疗与该成分的功能有关的疾病的化合物的筛选程序。

    METHOD FOR EXTRACTING QUANTITATIVE INFORMATION RELATING TO AN INFLUENCE ON A CELLULAR RESPONSE
    3.
    发明申请
    METHOD FOR EXTRACTING QUANTITATIVE INFORMATION RELATING TO AN INFLUENCE ON A CELLULAR RESPONSE 审中-公开
    提取与细胞响应有关的量化信息的方法

    公开(公告)号:US20090023598A1

    公开(公告)日:2009-01-22

    申请号:US11927246

    申请日:2007-10-29

    IPC分类号: C40B30/06

    摘要: A method for screening a library of compounds to detect a biologically active compound that modulates intracellular translocation of a subunit of a component of an intracellular pathway affecting intracellular processes includes: culturing one or more cells containing a nucleotide sequence coding for a hybrid polypeptide comprising a luminophore linked to the subunit of the component; introducing a compound of the library of compounds into the cell culture; screening the compound to determine whether the compound modulates the intracellular translocation of the subunit of the component; measuring light emitted from the luminophore to determine a first distribution; measuring light emitted from the luminophore to determine a second distribution; computing a variation between the first distribution and the second distribution by processing the measured light, any variation is indicative that the compound is biologically active. The method is also performed with a library of compounds.

    摘要翻译: 筛选化合物文库以检测调节影响细胞内过程的细胞内途径的组分的亚单位的细胞内易位的生物活性化合物的方法包括:培养含有编码包含发光体的杂合多肽的核苷酸序列的一个或多个细胞 连接到组件的子单元; 将化合物的化合物引入细胞培养物中; 筛选化合物以确定化合物是否调节组分亚单位的细胞内易位; 测量从发光体发射的光以确定第一分布; 测量从发光体发射的光以确定第二分布; 通过处理测量的光来计算第一分布和第二分布之间的变化,任何变化表明化合物具有生物活性。 该方法也用化合物文库进行。