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 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.

1 - 67 . (canceled) 
     
     
         68 . A non-human mammal comprising:
 (1) a first nucleic acid comprising a transactivator-responsive conditional regulatory element and a first coding sequence, wherein the first coding sequence encodes a ribosomal fusion protein comprising a ribosomal protein, or fragment thereof, fused to a peptide tag, and wherein the first coding sequence is operably linked to the transactivator-responsive conditional regulatory element; and   (2) a second nucleic acid comprising a mammalian endogenous promoter that directs gene expression in a chosen cell type and a second coding sequence, wherein the second coding sequence encodes the transactivator, and wherein the second coding sequence is operably linked to the mammalian endogenous promoter;   wherein the mammalian endogenous promoter controls expression of the transactivator in the chosen cell type, the transactivator controls expression of the ribosomal fusion protein, and the ribosomal fusion protein is incorporated in an intact ribosome that translates and/or binds mRNA.   
     
     
         69 . The non-human mammal of  claim 68 , wherein the transactivator is tetracycline, doxycycline, interferon, estrogen, ecdysone, progesterone antagonist RU486, or rapamycin. 
     
     
         70 . The non-human mammal of  claim 69 , wherein the transactivator is tetracycline. 
     
     
         71 . The non-human mammal of  claim 68 , wherein the transactivator-responsive conditional regulatory element induces expression of the ribosomal fusion protein in the presence of the transactivator. 
     
     
         72 . The non-human mammal of  claim 68 , wherein the transactivator-responsive conditional regulatory element represses expression of the ribosomal fusion protein in the presence of the transactivator. 
     
     
         73 . The non-human mammal of  claim 68 , wherein the peptide tag is a myc epitope. 
     
     
         74 . The non-human mammal of  claim 68 , wherein the peptide tag is positioned at the N-terminus of the ribosomal fusion protein or at the C-terminus of the ribosomal fusion protein. 
     
     
         75 . The non-human mammal of  claim 68 , wherein the chosen cell type is a neuronal cell type. 
     
     
         76 . The non-human mammal of  claim 68 , wherein the mammal is a mouse or rat. 
     
     
         77 . The non-human mammal of  claim 68 , wherein the mammalian endogenous promoter is from a characterizing gene and expression of the nucleic acid encoding the transactivator is substantially similar to expression of the characterizing gene. 
     
     
         78 . The non-human mammal of  claim 77 , wherein expression of the nucleic acid encoding the transactivator is in at least 80% of cells shown to express the characterizing gene in the mammal. 
     
     
         79 . A non-human mammalian cell comprising:
 (1) a first nucleic acid comprising a transactivator-responsive conditional regulatory element and a first coding sequence, wherein the first coding sequence encodes a ribosomal fusion protein comprising a ribosomal protein, or fragment thereof, fused to a peptide tag, and wherein the first coding sequence is operably linked to the transactivator-responsive conditional regulatory element; and   (2) a second nucleic acid comprising a mammalian endogenous promoter that directs gene expression in the non-human mammalian cell and a second coding sequence, wherein the second coding sequence encodes the transactivator, and wherein the second coding sequence is operably linked to the mammalian endogenous promoter;   wherein the mammalian endogenous promoter controls expression of the transactivator in the non-human mammalian cell, the transactivator controls expression of the ribosomal fusion protein, and the ribosomal fusion protein is incorporated in an intact ribosome that translates and/or binds mRNA.   
     
     
         80 . A nucleic acid vector comprising:
 (1) a first nucleic acid comprising a transactivator-responsive conditional regulatory element and a first coding sequence, wherein the first coding sequence encodes a ribosomal fusion protein comprising a ribosomal protein, or fragment thereof, fused to a peptide tag, and wherein the first coding sequence is operably linked to the transactivator-responsive conditional regulatory element; and   (2) a second nucleic acid comprising a mammalian endogenous promoter and a second coding sequence, wherein the second coding sequence encodes the transactivator, and wherein the second coding sequence is operably linked to the mammalian endogenous promoter.   
     
     
         81 . A method of isolating an actively translated mRNA from a non-human mammal comprising:
 (1) providing a cell isolated from a non-human mammal, or a lysate thereof, the non-human mammal comprising:
 (a) a first nucleic acid comprising a transactivator-responsive conditional regulatory element and a first coding sequence, wherein the first coding sequence encodes a ribosomal fusion protein comprising a ribosomal protein, or fragment thereof, fused to a peptide tag, and wherein-the first coding sequence is operably linked to the transactivator-responsive conditional regulatory element; and 
 (b) a second nucleic acid comprising a mammalian endogenous promoter that directs gene expression in the non-human mammalian cell and a second coding sequence, wherein the second coding sequence encodes the transactivator, and wherein the second coding sequence is operably linked to the mammalian endogenous promoter; 
 wherein the mammalian endogenous promoter controls expression of the transactivator in the non-human mammalian cell, the transactivator controls expression of the ribosomal fusion protein, and the ribosomal fusion protein is incorporated in an intact ribosome that translates and/or binds mRNA; 
   (2) isolating ribosomal protein-mRNA complexes comprising the ribosomal fusion protein comprising the peptide tag; and   (3) isolating the actively translated mRNA molecules of the ribosomal protein-mRNA complexes.   
     
     
         82 . The method of  claim 81 , wherein the step (2) of isolating the ribosomal protein-mRNA complexes comprises contacting the cell or lysate with a reagent that binds the peptide tag. 
     
     
         83 . The method of  claim 81 , further comprising the step of identifying the actively translated mRNA. 
     
     
         84 . The method of  claim 81 , further comprising the step of quantifying actively translated mRNA. 
     
     
         85 . A non-human mammal comprising:
 (1) a first nucleic acid comprising a transcription-regulatory sequence, a recombinase-responsive conditional regulatory element, and a first coding sequence, wherein:
 (a) the first coding sequence encodes a ribosomal fusion protein comprising a ribosomal protein, or fragment thereof, fused to a peptide tag; and 
 (b) the recombinase-responsive conditional regulatory element comprises first and second recombinase sites, which first and second recombinase sites are positioned such that exposure to the recombinase causes the first coding sequence to become operably linked to the transcription-regulatory sequence; and 
   (2) a second nucleic acid comprising a mammalian endogenous promoter that directs gene expression in a chosen cell type and a second coding sequence, wherein the second coding sequence encodes the recombinase, and wherein the second coding sequence is operably linked to the mammalian endogenous promoter;   wherein the mammalian endogenous promoter controls expression of the recombinase in the chosen cell type, the recombinase causes the first coding sequence to become operably linked to the transcription-regulatory sequence resulting in expression of the ribosomal fusion protein, and the ribosomal fusion protein is incorporated in an intact ribosome that translates and/or binds mRNA.   
     
     
         86 . The non-human mammal of  claim 85 , wherein the first and second recombinase sites are FRT sites and the recombinase is an FLP recombinase. 
     
     
         87 . The non-human mammal of  claim 85 , wherein the first and second recombinase sites are lox sites and the recombinase is a Cre recombinase. 
     
     
         88 . The non-human mammal of  claim 85 , wherein the peptide tag is a myc epitope. 
     
     
         89 . The non-human mammal of  claim 85 , wherein the peptide tag is positioned at the N-terminus of the ribosomal fusion protein or at the C-terminus of the ribosomal fusion protein. 
     
     
         90 . The non-human mammal of  claim 85 , wherein the chosen cell type is a neuronal cell type. 
     
     
         91 . The non-human mammal of  claim 85 , wherein the mammal is a mouse or rat. 
     
     
         92 . The non-human mammal of  claim 85 , wherein the mammalian endogenous promoter is from a characterizing gene and expression of the nucleic acid encoding the recombinase is substantially similar to expression of the characterizing gene. 
     
     
         93 . The non-human mammal of  claim 92 , wherein expression of the nucleic acid encoding the recombinase is in at least 80% of cells shown to express the characterizing gene in the mammal. 
     
     
         94 . A non-human mammalian cell comprising:
 (1) a first nucleic acid comprising a transcription-regulatory sequence, a recombinase-responsive conditional regulatory element, and a first coding sequence, wherein:
 (a) the first coding sequence encodes a ribosomal fusion protein comprising a ribosomal protein, or fragment thereof, fused to a peptide tag; and 
 (b) the recombinase-responsive conditional regulatory element comprises first and second recombinase sites, which first and second recombinase sites are positioned such that exposure to the recombinase causes the first coding sequence to become operably linked to the transcription-regulatory sequence; and 
   (2) a second nucleic acid comprising a mammalian endogenous promoter that directs gene expression in the non-human mammalian cell and a second coding sequence, wherein the second coding sequence encodes the recombinase, and wherein the second coding sequence is operably linked to the mammalian endogenous promoter;   wherein the mammalian endogenous promoter controls expression of the recombinase in the non-human mammalian cell, the recombinase causes the first coding sequence to become operably linked to the transcription-regulatory sequence resulting in expression of the ribosomal fusion protein, and the ribosomal fusion protein is incorporated in an intact ribosome that translates and/or binds mRNA.   
     
     
         95 . A nucleic acid vector comprising:
 (1) a first nucleic acid comprising a transcription-regulatory sequence, a recombinase-responsive conditional regulatory element, and a first coding sequence, wherein:
 (a) the first coding sequence encodes a ribosomal fusion protein comprising a ribosomal protein, or fragment thereof, fused to a peptide tag; and 
 (b) the recombinase-responsive conditional regulatory element comprises first and second recombinase sites, which first and second recombinase sites are positioned such that exposure to the recombinase causes the first coding sequence to become operably linked to the transcription-regulatory sequence; and 
   (2) a second nucleic acid comprising a mammalian endogenous promoter and a second coding sequence, wherein the second coding sequence encodes the recombinase, and wherein the second coding sequence is operably linked to the mammalian endogenous promoter.   
     
     
         96 . A method of isolating an actively translated mRNA from a non-human mammal comprising:
 (1) providing a cell isolated from a non-human mammal, or a lysate thereof, the non-human mammal comprising:
 (a) a first nucleic acid comprising a transcription-regulatory sequence, a recombinase-responsive conditional regulatory element, and a first coding sequence, wherein:
 the first coding sequence encodes a ribosomal fusion protein comprising a ribosomal protein, or fragment thereof, fused to a peptide tag; and 
 (ii) the recombinase-responsive conditional regulatory element comprises first and second recombinase sites, which first and second recombinase sites are positioned such that exposure to the recombinase causes the first coding sequence to become operably linked to the transcription-regulatory sequence; and 
 
 (b) a second nucleic acid comprising a mammalian endogenous promoter that directs gene expression in the non-human mammalian cell and a second coding sequence, wherein the second coding sequence encodes the recombinase, and wherein the second coding sequence is operably linked to the mammalian endogenous promoter; 
 wherein the mammalian endogenous promoter controls expression of the recombinase in the non-human mammalian cell, the recombinase causes the first coding sequence to become operably linked to the transcription-regulatory sequence resulting in expression of the ribosomal fusion protein, and the ribosomal fusion protein is incorporated in an intact ribosome that translates and/or binds mRNA; 
   (2) isolating ribosomal protein-mRNA complexes comprising the ribosomal fusion protein comprising the peptide tag; and   (3) isolating the actively translated mRNA molecules of the ribosomal protein-mRNA complexes.   
     
     
         97 . The method of  claim 96 , wherein the step (2) of isolating the ribosomal protein-mRNA complexes comprises contacting the cell or lysate with a reagent that binds the peptide tag. 
     
     
         98 . The method of  claim 96 , further comprising the step of identifying the actively translated mRNA. 
     
     
         99 . The method of  claim 96 , further comprising the step of quantifying actively translated mRNA.