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OwnerNathaniel Heintz, Tito A. Serafini +1inventors
InventorsNathaniel Heintz, Tito A. Serafini, Andrew W. Shyjan
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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.
An isolated ribosome-reagent complex comprising:
An isolated mRNA binding protein-mRNA-reagent complex comprising:
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.
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.
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.
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.
Dependencies are inferred from the claim text.