Method for Isolating Cell-Type Specific mRNAs

Abstract

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 quan tification 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.

What is claimed is: 
     
         1 . An isolated ribosome-reagent complex comprising:
 (a) an intact ribosome comprising a ribosomal fusion protein, wherein said ribosomal fusion protein comprises a ribosomal protein, or fragment thereof, fused to a peptide tag;   (b) an mRNA bound to said intact ribosome; and   (c) a reagent bound to said peptide tag.   
     
     
         2 . The isolated complex of  claim 1 , wherein said reagent is bound to a solid support. 
     
     
         3 . The isolated complex of  claim 1 , wherein said peptide tag is not a ribosomal protein. 
     
     
         4 . The isolated complex of  claim 1 , wherein said ribosomal protein is S6, S15, S18, L10a, L32, or L37. 
     
     
         5 . The isolated complex of  claim 1 , wherein said peptide tag is placed at the N- or C-terminus of said ribosomal protein. 
     
     
         6 . The isolated complex of  claim 1 , wherein said peptide tag does not inhibit or interfere with a function of said ribosomal protein. 
     
     
         7 . An isolated mRNA binding protein-mRNA-reagent complex comprising:
 (a) a mRNA binding fusion protein comprises a mRNA binding protein, or fragment thereof, fused to a peptide tag;   (b) a mRNA bound to said mRNA binding fusion protein; and   (c) a reagent bound to said peptide tag.   
     
     
         8 . The isolated complex of  claim 7 , wherein said reagent is bound to a solid support. 
     
     
         9 . The isolated complex of  claim 7 , wherein said peptide tag is not a mRNA binding protein. 
     
     
         10 . The isolated complex of  claim 7 , wherein said mRNA binding fusion protein is not a polyA binding protein. 
     
     
         11 . The isolated complex of  claim 7 , wherein said peptide tag is placed at the N- or C-terminus of said mRNA binding protein. 
     
     
         12 . The isolated complex of  claim 7 , wherein said peptide tag does not inhibit or interfere with a function of said mRNA binding protein. 
     
     
         13 . A transgenic plant comprising a transgene comprising a nucleotide sequence encoding a ribosomal fusion protein, wherein said ribosomal fusion protein comprises a ribosomal protein, or fragment thereof, fused to a peptide tag, wherein said nucleotide sequence is operably linked to a plant endogenous promoter, wherein said plant endogenous promoter causes expression of said ribosomal fusion protein in a chosen cell type, and wherein said ribosomal fusion protein is incorporated in an intact ribosome that translates and/or binds mRNA. 
     
     
         14 . The transgenic plant of  claim 13 , wherein said peptide tag is not a ribosomal protein. 
     
     
         15 . The transgenic plant of  claim 13 , wherein said ribosomal protein is S6, S15, S18, L10a, L32, or L37. 
     
     
         16 . The transgenic plant of  claim 13 , wherein said peptide tag is placed at the N- or C-terminus of said ribosomal protein. 
     
     
         17 . The transgenic plant of  claim 13 , wherein said peptide tag does not inhibit or interfere with a function of said ribosomal protein. 
     
     
         18 . A transgenic plant comprising a transgene comprising a nucleotide sequence encoding a mRNA binding fusion protein, wherein said mRNA binding fusion protein comprises a mRNA binding protein, or fragment thereof, fused to a peptide tag, wherein said nucleotide sequence is operably linked to a plant endogenous promoter, wherein said plant endogenous promoter causes expression of said mRNA binding fusion protein in a chosen cell type. 
     
     
         19 . The transgenic plant of  claim 18 , wherein said peptide tag is not a mRNA binding protein. 
     
     
         20 . The transgenic plant of  claim 18 , wherein said mRNA binding fusion protein is not a polyA binding protein. 
     
     
         21 . The transgenic plant of  claim 18 , wherein said peptide tag is placed at the N- or C-terminus of said mRNA binding protein. 
     
     
         22 . The transgenic plant of  claim 18 , wherein said peptide tag does not inhibit or interfere with a function of said mRNA binding protein. 
     
     
         23 . The transgenic plant of  claim 13 , wherein said ribosomal fusion protein is not translated in a cell type that is not chosen. 
     
     
         24 . The transgenic plant of  claim 13 , wherein said chosen cell type comprises neural cells. 
     
     
         25 . The transgenic plant of  claim 24 , wherein said neural cells comprise neuronal cells. 
     
     
         26 . The transgenic plant of  claim 13 , wherein said peptide tag is streptavidin. 
     
     
         27 . The transgenic plant of  claim 13 , wherein said peptide tag comprises 200 or more amino acids. 
     
     
         28 . The transgenic plant of  claim 13 , wherein said plant endogenous promoter is from a characterizing gene. 
     
     
         29 . The transgenic plant of  claim 28 , wherein said expression of said nucleotide sequence is substantially similar to expression of said characterizing gene. 
     
     
         30 . The transgenic plant of  claim 28 , wherein said expression of said nucleotide sequence is in at least 80% of cells shown to express said characterizing gene in said mammal. 
     
     
         31 . The transgenic plant of  claim 13 , wherein said plant endogenous promoter is part of an endogenous regulatory sequence. 
     
     
         32 . The transgenic plant of  claim 31 , wherein said endogenous regulatory sequence is at least or about 100 kilobases in length. 
     
     
         33 . The transgenic plant of  claim 31 , wherein said endogenous regulatory sequence is at least or about 200 kilobases in length. 
     
     
         34 . The transgenic plant of  claim 31 , wherein said endogenous regulatory sequence comprises a transcription regulatory element. 
     
     
         35 . The transgenic plant of  claim 34 , wherein said transcription regulatory element comprises a transcriptional enhancer sequence, insulator sequence, or a combination thereof. 
     
     
         36 . The transgenic plant of  claim 13 , wherein said nucleotide sequence is operably linked to a regulatory sequence associated with or part of a bacterial artificial chromosome (BAC). 
     
     
         37 . A cell of said transgenic plant of  claim 13  comprising a nucleotide sequence encoding a ribosomal fusion protein, wherein said ribosomal fusion protein comprises a ribosomal protein, or fragment thereof, fused to a peptide tag, wherein said nucleotide sequence is operably linked to a plant endogenous promoter, wherein said plant endogenous promoter causes expression of said ribosomal fusion protein said cell, and wherein said ribosomal fusion protein is incorporated in an intact ribosome that translates and/or binds mRNA. 
     
     
         38 . A method of isolating an actively translated mRNA from said transgenic plant of  claim 13 , comprising:
 (a) contacting a cell isolated from said transgenic plant or a lysate of said cell that comprises said ribosomal fusion protein with a reagent that binds to said peptide tag;   (b) isolating said ribosomal fusion protein containing said peptide tag; and   (c) isolating said actively translated mRNA from said ribosomes.   
     
     
         39 . The method of  claim 38 , wherein said reagent is bound to a solid support. 
     
     
         40 . The method of  claim 38 , wherein said peptide tag is streptavidin and said reagent specifically binds streptavidin. 
     
     
         41 . The method of  claim 38 , wherein said chosen cell type is a neural cell. 
     
     
         42 . The method of  claim 41 , wherein said neural cells comprise neuronal cells. 
     
     
         43 . The method of  claim 38 , wherein said peptide tag comprises 200 or more amino acids. 
     
     
         44 . The method of  claim 38 , wherein said plant endogenous promoter is from a characterizing gene. 
     
     
         45 . The method of  claim 44 , wherein said expression of said nucleotide sequence is substantially similar to expression of said characterizing gene. 
     
     
         46 . The method of  claim 44 , wherein said expression of said nucleotide sequence is in at least 80% of cells shown to express said characterizing gene in said mammal. 
     
     
         47 . The method of  claim 38 , wherein said plant endogenous promoter is part of an endogenous regulatory sequence. 
     
     
         48 . The method of  claim 47 , wherein said endogenous regulatory sequence is at least or about 100 kilobases in length. 
     
     
         49 . The method of  claim 47 , wherein said endogenous regulatory sequence is at least or about 200 kilobases in length. 
     
     
         50 . The method of  claim 47 , wherein said endogenous regulatory sequence comprises a transcription regulatory element. 
     
     
         51 . The method of  claim 50 , wherein said transcription regulatory element comprises a transcriptional enhancer sequence, insulator sequence, or a combination thereof. 
     
     
         52 . The method of  claim 38 , wherein said nucleotide sequence is operably linked to a regulatory sequence associated with or part of a bacterial artificial chromosome (BAC). 
     
     
         53 . The method of  claim 38 , further comprising contacting said cell with an agent capable of arresting translation. 
     
     
         54 . The method of  claim 38 , further comprising determining a gene expression profile for said cell. 
     
     
         55 . The method of  claim 38 , further comprising identifying said actively translated mRNA. 
     
     
         56 . The method of  claim 38 , further comprising quantifying said actively translated mRNA of said cell. 
     
     
         57 . A transgene comprising a nucleotide sequence encoding a ribosomal fusion protein, wherein said ribosomal fusion protein comprises a ribosomal protein, or fragment thereof, fused to a peptide tag, wherein said nucleotide sequence is operably linked to plant endogenous promoter, wherein said plant endogenous promoter causes expression of said ribosomal fusion protein in a chosen cell type, and wherein said ribosomal fusion protein is incorporated in an intact ribosome that translates and/or binds mRNA. 
     
     
         58 . The transgene of  claim 57 , wherein said ribosomal protein is S6, S15, S18, L10a, L32, or L37. 
     
     
         59 . The transgene of  claim 57 , wherein said peptide tag is placed at the N- or C-terminus of said ribosomal protein. 
     
     
         60 . A transgene comprising a nucleotide sequence encoding a mRNA binding fusion protein, wherein said mRNA binding fusion protein comprises a mRNA binding protein, or fragment thereof, fused to a peptide tag, wherein said nucleotide sequence is operably linked to a plant endogenous promoter, wherein said plant endogenous promoter causes expression of said mRNA binding fusion protein in a chosen cell type. 
     
     
         61 . The transgene of  claim 60 , wherein said peptide tag is placed at the N- or C-terminus of said mRNA binding protein. 
     
     
         62 . The transgene of  claim 57 , wherein said plant endogenous promoter is part of an endogenous regulatory sequence that is at least or about 100 kilobases in length. 
     
     
         63 . The transgene of  claim 57 , wherein said plant endogenous promoter is part of an endogenous regulatory sequence that is at least or about 200 kilobases in length. 
     
     
         64 . The transgene of  claim 57 , wherein said plant endogenous promoter is part of an endogenous regulatory sequence that comprises a transcription regulatory element. 
     
     
         65 . The transgene of  claim 64 , wherein said transcription regulatory element comprises a transcriptional enhancer sequence, insulator sequence, or a combination thereof. 
     
     
         66 . The transgene of  claim 57 , wherein said nucleotide sequence is operably linked to a regulatory sequence associated with or part of a bacterial artificial chromosome (BAC). 
     
     
         67 . A method of making said transgenic plant of  claim 13 , comprising introducing into a founder plant or embryo a transgene comprising a nucleotide sequence encoding a ribosomal fusion protein.