# US 2014/0196176 A1 · Method for Isolating Cell-Type Specific mRNAs

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- Number: US 2014/0196176 A1
- Document ID: US20140196176A1
- Status: Published application
- Kind: A1
- Application number: 13/930,864
- Filed: 2013-06-28
- Published: 2014-07-10
- Earliest claimed: 2004-08-16
- Inventors: Nathaniel Heintz; Tito A. Serafini; Andrew W. Shyjan
- Web page: https://patentgrep.com/patent/US20140196176A1

## CPC

- A01K 67/0275: Genetically modified vertebrates, e.g. transgenic
- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers}
- C12N 15/1041: {Ribosome/Polysome display, e.g. SPERT, ARM}
- C12N 15/1072: {Differential gene expression library synthesis, e.g. subtracted libraries, differential screening}
- C12N 15/82: for plant cells {, e.g. plant artificial chromosomes (PACs)}
- C12N 15/8222: {Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation}
- C12Q 1/6827: for detection of mutation or polymorphism
- A01K 2217/05: Animals comprising random inserted nucleic acids (transgenic)
- A01K 2217/206: Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
- A01K 2227/10: Mammal
- A01K 2227/105: Murine
- A01K 2267/0393: Animal model comprising a reporter system for screening tests
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

The isolated complex of claim 1, wherein said reagent is bound to a solid support.

### Claim 3 (depends on 1)

The isolated complex of claim 1, wherein said peptide tag is not a ribosomal protein.

### Claim 4 (depends on 1)

The isolated complex of claim 1, wherein said ribosomal protein is S6, S15, S18, L10a, L32, or L37.

### Claim 5 (depends on 1)

The isolated complex of claim 1, wherein said peptide tag is placed at the N- or C-terminus of said ribosomal protein.

### Claim 6 (depends on 1)

The isolated complex of claim 1, wherein said peptide tag does not inhibit or interfere with a function of said ribosomal protein.

### Claim 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.

### Claim 8 (depends on 7)

The isolated complex of claim 7, wherein said reagent is bound to a solid support.

### Claim 9 (depends on 7)

The isolated complex of claim 7, wherein said peptide tag is not a mRNA binding protein.

### Claim 10 (depends on 7)

The isolated complex of claim 7, wherein said mRNA binding fusion protein is not a polyA binding protein.

### Claim 11 (depends on 7)

The isolated complex of claim 7, wherein said peptide tag is placed at the N- or C-terminus of said mRNA binding protein.

### Claim 12 (depends on 7)

The isolated complex of claim 7, wherein said peptide tag does not inhibit or interfere with a function of said mRNA binding protein.

### Claim 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.

### Claim 14 (depends on 13)

The transgenic plant of claim 13, wherein said peptide tag is not a ribosomal protein.

### Claim 15 (depends on 13)

The transgenic plant of claim 13, wherein said ribosomal protein is S6, S15, S18, L10a, L32, or L37.

### Claim 16 (depends on 13)

The transgenic plant of claim 13, wherein said peptide tag is placed at the N- or C-terminus of said ribosomal protein.

### Claim 17 (depends on 13)

The transgenic plant of claim 13, wherein said peptide tag does not inhibit or interfere with a function of said ribosomal protein.

### Claim 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.

### Claim 19 (depends on 18)

The transgenic plant of claim 18, wherein said peptide tag is not a mRNA binding protein.

### Claim 20 (depends on 18)

The transgenic plant of claim 18, wherein said mRNA binding fusion protein is not a polyA binding protein.

### Claim 21 (depends on 18)

The transgenic plant of claim 18, wherein said peptide tag is placed at the N- or C-terminus of said mRNA binding protein.

### Claim 22 (depends on 18)

The transgenic plant of claim 18, wherein said peptide tag does not inhibit or interfere with a function of said mRNA binding protein.

### Claim 23 (depends on 13)

The transgenic plant of claim 13, wherein said ribosomal fusion protein is not translated in a cell type that is not chosen.

### Claim 24 (depends on 13)

The transgenic plant of claim 13, wherein said chosen cell type comprises neural cells.

### Claim 25 (depends on 24)

The transgenic plant of claim 24, wherein said neural cells comprise neuronal cells.

### Claim 26 (depends on 13)

The transgenic plant of claim 13, wherein said peptide tag is streptavidin.

### Claim 27 (depends on 13)

The transgenic plant of claim 13, wherein said peptide tag comprises 200 or more amino acids.

### Claim 28 (depends on 13)

The transgenic plant of claim 13, wherein said plant endogenous promoter is from a characterizing gene.

### Claim 29 (depends on 28)

The transgenic plant of claim 28, wherein said expression of said nucleotide sequence is substantially similar to expression of said characterizing gene.

### Claim 30 (depends on 28)

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.

### Claim 31 (depends on 13)

The transgenic plant of claim 13, wherein said plant endogenous promoter is part of an endogenous regulatory sequence.

### Claim 32 (depends on 31)

The transgenic plant of claim 31, wherein said endogenous regulatory sequence is at least or about 100 kilobases in length.

### Claim 33 (depends on 31)

The transgenic plant of claim 31, wherein said endogenous regulatory sequence is at least or about 200 kilobases in length.

### Claim 34 (depends on 31)

The transgenic plant of claim 31, wherein said endogenous regulatory sequence comprises a transcription regulatory element.

### Claim 35 (depends on 34)

The transgenic plant of claim 34, wherein said transcription regulatory element comprises a transcriptional enhancer sequence, insulator sequence, or a combination thereof.

### Claim 36 (depends on 13)

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).

### Claim 37 (depends on 13)

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.

### Claim 38 (depends on 13)

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.

### Claim 39 (depends on 38)

The method of claim 38, wherein said reagent is bound to a solid support.

### Claim 40 (depends on 38)

The method of claim 38, wherein said peptide tag is streptavidin and said reagent specifically binds streptavidin.

### Claim 41 (depends on 38)

The method of claim 38, wherein said chosen cell type is a neural cell.

### Claim 42 (depends on 41)

The method of claim 41, wherein said neural cells comprise neuronal cells.

### Claim 43 (depends on 38)

The method of claim 38, wherein said peptide tag comprises 200 or more amino acids.

### Claim 44 (depends on 38)

The method of claim 38, wherein said plant endogenous promoter is from a characterizing gene.

### Claim 45 (depends on 44)

The method of claim 44, wherein said expression of said nucleotide sequence is substantially similar to expression of said characterizing gene.

### Claim 46 (depends on 44)

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.

### Claim 47 (depends on 38)

The method of claim 38, wherein said plant endogenous promoter is part of an endogenous regulatory sequence.

### Claim 48 (depends on 47)

The method of claim 47, wherein said endogenous regulatory sequence is at least or about 100 kilobases in length.

### Claim 49 (depends on 47)

The method of claim 47, wherein said endogenous regulatory sequence is at least or about 200 kilobases in length.

### Claim 50 (depends on 47)

The method of claim 47, wherein said endogenous regulatory sequence comprises a transcription regulatory element.

### Claim 51 (depends on 50)

The method of claim 50, wherein said transcription regulatory element comprises a transcriptional enhancer sequence, insulator sequence, or a combination thereof.

### Claim 52 (depends on 38)

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).

### Claim 53 (depends on 38)

The method of claim 38, further comprising contacting said cell with an agent capable of arresting translation.

### Claim 54 (depends on 38)

The method of claim 38, further comprising determining a gene expression profile for said cell.

### Claim 55 (depends on 38)

The method of claim 38, further comprising identifying said actively translated mRNA.

### Claim 56 (depends on 38)

The method of claim 38, further comprising quantifying said actively translated mRNA of said cell.

### Claim 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.

### Claim 58 (depends on 57)

The transgene of claim 57, wherein said ribosomal protein is S6, S15, S18, L10a, L32, or L37.

### Claim 59 (depends on 57)

The transgene of claim 57, wherein said peptide tag is placed at the N- or C-terminus of said ribosomal protein.

### Claim 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.

### Claim 61 (depends on 60)

The transgene of claim 60, wherein said peptide tag is placed at the N- or C-terminus of said mRNA binding protein.

### Claim 62 (depends on 57)

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.

### Claim 63 (depends on 57)

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.

### Claim 64 (depends on 57)

The transgene of claim 57, wherein said plant endogenous promoter is part of an endogenous regulatory sequence that comprises a transcription regulatory element.

### Claim 65 (depends on 64)

The transgene of claim 64, wherein said transcription regulatory element comprises a transcriptional enhancer sequence, insulator sequence, or a combination thereof.

### Claim 66 (depends on 57)

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).

### Claim 67 (depends on 13)

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.