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    Method for Isolating Cell-Type Specific mRNAs
    1.
    发明申请
    Method for Isolating Cell-Type Specific mRNAs 审中-公开
    分离细胞型特异性mRNA的方法

    公开(公告)号：US20140196176A1

    公开(公告)日：2014-07-10

    申请号：US13930864

    申请日：2013-06-28

    申请人： Nathaniel Heintz ,  Tito A. Serafini ,  Andrew W. Shyjan

    发明人： Nathaniel Heintz ,  Tito A. Serafini ,  Andrew W. Shyjan

    IPC分类号： C12N15/10 ,  C12N15/82 ,  C12Q1/68

    CPC分类号： C12N15/1041 ,  C12N15/1006 ,  C12N15/82 ,  C12N15/8222 ,  C12Q1/6827

    摘要： The invention provides methods for isolating cell-type specific mRNAs by selectively isolating ribosomes or proteins that bind mRNA in a cell type specific manner, and, thereby, the mRNA hound to the ribosomes or proteins that bind mRNA. Ribosomes, which are riboprotein complexes, bind mRNA that is being actively translated in cells. According to the methods of the invention, cells are engineered to express a molecularly tagged ribosomal protein or protein that binds mRNA by introducing into the cell a nucleic acid comprising a nucleotide sequence encoding a ribosomal protein or protein that binds mRNA fused to a nucleotide sequence encoding a peptide tag. The tagged ribosome or mRNA binding protein can then be isolated, along with the mRNA bound to the tagged ribosome or mRNA binding protein, and the mRNA isolated and further used for gene expression analysis. The methods of the invention facilitate the analysis and quantification of gene expression in the selected cell type present within a heterogeneous cell mixture, without the need to isolate the cells of that cell type as a preliminary step.

    摘要翻译： 本发明提供了通过选择性分离以细胞类型特异性方式结合mRNA的核糖体或蛋白质分离细胞型特异性mRNA的方法，从而将mRNA捕获到结合mRNA的核糖体或蛋白质上。 作为核糖体复合物的核糖体结合正在细胞中积极翻译的mRNA。 根据本发明的方法，将细胞工程化以表达分子标记的核糖体蛋白质或蛋白质，其通过向细胞中引入核酸，所述核酸包含编码核糖体蛋白质或蛋白质的核苷酸序列，所述核糖体蛋白质或蛋白质与编码 肽标签。 然后可以分离标记的核糖体或mRNA结合蛋白以及与标记的核糖体或mRNA结合蛋白结合的mRNA，并分离mRNA并进一步用于基因表达分析。 本发明的方法促进了在异质细胞混合物中存在的所选细胞类型中的基因表达的分析和定量，而不需要将该细胞类型的细胞作为初步步骤分离。

    Method for Isolating Cell-Type Specific mRNAS
    2.
    发明申请
    Method for Isolating Cell-Type Specific mRNAS 有权
    隔离细胞型特异性mRNA的方法

    公开(公告)号：US20110314565A1

    公开(公告)日：2011-12-22

    申请号：US13104316

    申请日：2011-05-10

    申请人： Nathaniel Heintz ,  Tito A. Serafini ,  Andrew W. Shyjan

    发明人： Nathaniel Heintz ,  Tito A. Serafini ,  Andrew W. Shyjan

    IPC分类号： A01K67/027 ,  C12N1/19 ,  C07K14/00 ,  A01K67/033 ,  A01H5/00 ,  C07K19/00 ,  C07H1/08 ,  C12N5/10

    CPC分类号： C12N15/1041 ,  C12N15/1006 ,  C12N15/82 ,  C12N15/8222 ,  C12Q1/6827

    摘要： The invention provides methods for isolating cell-type specific mRNAs by selectively isolating ribosomes or proteins that bind mRNA in a cell type specific manner, and, thereby, the mRNA hound to the ribosomes or proteins that bind mRNA. Ribosomes, which are riboprotein complexes, bind mRNA that is being actively translated in cells. According to the methods of the invention, cells are engineered to express a molecularly tagged ribosomal protein or protein that binds mRNA by introducing into the cell a nucleic acid comprising a nucleotide sequence encoding a ribosomal protein or protein that binds mRNA fused to a nucleotide sequence encoding a peptide tag. The tagged ribosome or mRNA binding protein can then be isolated, along with the mRNA bound to the tagged ribosome or mRNA binding protein, and the mRNA isolated and further used for gene expression analysis. The methods of the invention facilitate the analysis and quantification of gene expression in the selected cell type present within a heterogeneous cell mixture, without the need to isolate the cells of that cell type as a preliminary step.

    摘要翻译： 本发明提供了通过选择性分离以细胞类型特异性方式结合mRNA的核糖体或蛋白质分离细胞型特异性mRNA的方法，从而将mRNA捕获到结合mRNA的核糖体或蛋白质上。 作为核糖体复合物的核糖体结合正在细胞中积极翻译的mRNA。 根据本发明的方法，将细胞工程化以表达分子标记的核糖体蛋白质或蛋白质，其通过向细胞中引入核酸，所述核酸包含编码核糖体蛋白质或蛋白质的核苷酸序列，所述核糖体蛋白质或蛋白质与编码 肽标签。 然后可以分离标记的核糖体或mRNA结合蛋白以及与标记的核糖体或mRNA结合蛋白结合的mRNA，并分离mRNA并进一步用于基因表达分析。 本发明的方法促进了在异质细胞混合物中存在的所选细胞类型中的基因表达的分析和定量，而不需要将该细胞类型的细胞作为初步步骤分离。

    Non human transgenic mammal comprising a transgene comprising a nucleotide sequence encoding a ribosomal protein fused to a peptide tag
    3.
    发明授权
    Non human transgenic mammal comprising a transgene comprising a nucleotide sequence encoding a ribosomal protein fused to a peptide tag 有权
    包含转基因的非人转基因哺乳动物，其包含编码与肽标签融合的核糖体蛋白的核苷酸序列

    公开(公告)号：US08513485B2

    公开(公告)日：2013-08-20

    申请号：US13104316

    申请日：2011-05-10

    申请人： Nathaniel Heintz ,  Tito A. Serafini ,  Andrew W. Shyjan

    发明人： Nathaniel Heintz ,  Tito A. Serafini ,  Andrew W. Shyjan

    IPC分类号： A01K67/027 ,  G01N33/00 ,  C12N15/00

    CPC分类号： C12N15/1041 ,  C12N15/1006 ,  C12N15/82 ,  C12N15/8222 ,  C12Q1/6827

    摘要： The invention provides methods for isolating cell-type specific mRNAs by selectively isolating ribosomes or proteins that bind mRNA in a cell type specific manner, and, thereby, the mRNA hound to the ribosomes or proteins that bind mRNA. Ribosomes, which are riboprotein complexes, bind mRNA that is being actively translated in cells. According to the methods of the invention, cells are engineered to express a molecularly tagged ribosomal protein or protein that binds mRNA by introducing into the cell a nucleic acid comprising a nucleotide sequence encoding a ribosomal protein or protein that binds mRNA fused to a nucleotide sequence encoding a peptide tag. The tagged ribosome or mRNA binding protein can then be isolated, along with the mRNA bound to the tagged ribosome or mRNA binding protein, and the mRNA isolated and further used for gene expression analysis. The methods of the invention facilitate the analysis and quantification of gene expression in the selected cell type present within a heterogeneous cell mixture, without the need to isolate the cells of that cell type as a preliminary step.

    摘要翻译： 本发明提供了通过选择性分离以细胞类型特异性方式结合mRNA的核糖体或蛋白质分离细胞型特异性mRNA的方法，从而将mRNA捕获到结合mRNA的核糖体或蛋白质上。 作为核糖体复合物的核糖体结合正在细胞中积极翻译的mRNA。 根据本发明的方法，将细胞工程化以表达分子标记的核糖体蛋白质或蛋白质，其通过向细胞中引入核酸，所述核酸包含编码核糖体蛋白质或蛋白质的核苷酸序列，所述核糖体蛋白质或蛋白质与编码 肽标签。 然后可以分离标记的核糖体或mRNA结合蛋白以及与标记的核糖体或mRNA结合蛋白结合的mRNA，并分离mRNA并进一步用于基因表达分析。 本发明的方法促进了在异质细胞混合物中存在的所选细胞类型中的基因表达的分析和定量，而不需要将该细胞类型的细胞作为初步步骤分离。