Methods and compositions for elucidating relative protein expression levels in cells

• US 6,897,020 B2
• Filed: 03/19/2001
• Issued: 05/24/2005
• Est. Priority Date: 03/20/2000
• Status: Expired due to Fees

• Current Assignee: Newlink Genetics - Iowa State University
• Original Assignee: Newlink Genetics Corporation (Lumos Pharma, Inc.)
• Inventors: Seregina, Tatiana, Vahanian, Nicholas N., Young, Won Bin, Ramsey, W. Jay, Link, Charles J., Shukla, Sachet A., Higginbotham, James N., Powers, Bradley J.
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): Lambertson, David A.
• Application Number:
  US09/811,842
• Publication Number:
  US 20010034028A1
• Time in Patent Office: 1,527 Days
• Field of Search: 435/6, 435/69.1, 435/463, 435/320.1
• US Class Current: 435/6.14
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries

Abstract

The present invention relates generally to methods and compositions for the identification of differential protein expression patterns and concomitantly the active genetic regions that are directly or indirectly involved in different tissue types, disease states, or other cellular differences desirable for diagnosis or for targets for drug therapy.

Claims

1. A method of elucidating a protein expression profile of a test cell line or group of cells, the method comprising:

(A) randomly introducing into the genome of a cell or group of cells a promoterless polynucleotide construct, the construct comprising in a 5′

to 3′

orientation;

i) a splice acceptor consensus sequence;

ii) the complementary sequence of a type IIS restriction enzyme recognition sequence;

iii) an oligonucleotide sequence encoding assayable marker peptide; and

iv) a polyadenylation sequence;

(B) wherein said promoterless polynucleotide construct when introduced into an actively expressed gene results in the generation of a truncated cellular protein fused at its C-terminal truncated end to the marker peptide; and

i) identifying those cells expressing said marker peptide fused to said truncated cellular protein;

ii) determining the identity of the truncated proteins to which the marker peptide is fused in each group of cells,

 

thereby elucidating a protein expression profile of a test cell line of group of cells.

2. The method of claim 1 further comprising sorting cells identified in step (B)(i) into subpopulations based on their different levels of expression of said marker peptide.

3. The method of claim 1 wherein the identity of the protein to which the marker peptide is fused is determined using Serial Analysis of Viral Integration (SAVI).

4. The method of claim 3 wherein Serial Analysis of Viral Integration (SAVI) is performed by:

i) isolating mRNA from each subgroup of cells;

ii) reverse transcribing the mRNA into double stranded cDNA;

iii) subjecting the cDNA to a restriction enzyme that recognizes the type IIS restriction enzyme recognition sequence, and cleaves the cDNA upstream of the recognition sequence, thereby generating one or more cDNA fragments, wherein each of these fragments comprise the oligonucleotide sequence corresponding to an upstream exon directly fused to the marker peptide, the type IIS restriction enzyme recognition sequence and a portion of a native sequence corresponding to the peptide marker;

iv) adding an adaptor sequence to the end of the unknown oligonucleotide sequence;

v) amplifying by the polymerase chain reaction, the fragments containing the oligonucleotide sequences of the exons fused to the marker peptide with oligonucleotide primers complementary to the adaptor and peptide marker encoding sequences;

vi) cloning and sequencing said amplified fragments; and

vii) comparing the sequence of each oligonuclcotide against oligonucleotide sequences in a one or more nucleotide sequence database thereby identifying one or more fusion proteins present in each subgroup of cells.

7. The method of claim 4 or 6 wherein addition of the adaptor sequence is performed by ligation of a double stranded adaptor.

8. The method of claim 4 or 6, wherein addition of the adaptor sequence is performed by poly-deoxyribonucleotide tailing extension.

9. The methods of claim 3, 4 or 6 wherein following amplification of the one or more extended cDNA fragments, and prior to cloning and sequencing the one or more cDNA fragments, the fragments are ligated together to form a concatenated molecule.

10. The methods of claims 1, 5, or 6 where the peptide marker encoding sequence lacks a translation initiation codon and possesses a translation STOP codon.

11. The methods of claims 1, 5 or 6 where the peptide marker encoding sequence lacks a translation initiation and STOP codons.

12. The methods of claim 1, 5 or 6 wherein said separation of cells into subpopulations of cells based on the levels of expression of the peptide marker is performed by fluorescent activated cell sorting.

13. The methods of claims 1, 5 or 6 wherein marker peptide is an epitope recognized by fluorescently or enzymatically labeled antibodies.

14. The methods of claims 1, 5 or 6 wherein the marker peptide requires interaction with another protein in order to generate fluorescent signal.

15. The methods of claims 1, 5 or 6 wherein the polynucleotide construct further comprised, downstream of the oligonucleotide encoding a marker peptide and before the polyadenylation signal, an internal ribosome entry site followed by another protein expression marker.

16. The methods of claims 1, 5 or 6 wherein the polynucleotide construct further comprises, downstream of the oligonucleotide having a specified sequence, a sequence encoding, upon expression, a selectable marker.

17. The methods of claims 1, 5 or 6 wherein the oligonucleotide sequence is a fluorescent protein coding oligonucleotide sequence.

18. The methods of claim 17, wherein the fluorescent protein encoding oligonucleotide is a green fluorescent protein (GFP) coding sequence.

19. The method of claim 18, wherein the GFP oligonucleotide coding sequence is a humanized renilla GFP (hrGFP) coding sequence.

20. The methods of claims 1, 5 or 6 wherein the polynucleotide construct is introduced into the genome of the cell via a vector.

21. The methods of claim 20, wherein the vector is a viral vector.

22. The methods of claim 21, wherein the viral vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector and an adeno-associated viral vector.

5. A method of identify differentially expressed proteins in two different populations of cells, the method comprising:

(A) randomly introducing into the genomes of a reference group of cells and into the genomes of a test group of cells a promoterless polynucleotide construct, wherein the construct comprises in a 5′

to 3′

orientation;

i) a splice acceptor consensus sequence;

ii) the complementary sequence of a type IIS restriction enzyme recognition sequence;

ii) an oligonucleotide sequence encoding assayable marker peptide; and

iv) a polyadenylation sequence;

(B) thereby generating a population of randomly truncated cellular proteins fused at their C-terminals truncated end to the marker peptide; and

i) sorting both groups of cells into subpopulations of cells based on their differential expression levels of the marker peptide;

ii) determining the identity of the fusion proteins generated in each subgroup of sorted cells; and

iii) comparing by statistical methods the protein expression profiles obtained for the test group of cells against the protein expression profiles obtained for the reference group of cells,

 

thereby identifying differences in the expression levels of fusion proteins among the two groups of cells.

6. The method of claim 5 wherein the identity of the protein to which the marker peptide is fused is determined by SAVI.