Methods and compositions for elucidating relative protein expression levels in cells

• US 20010034028A1
• Filed: 03/19/2001
• Published: 10/25/2001
• Est. Priority Date: 03/20/2000
• Status: Active Grant

• Current Assignee: Newlink Genetics - Iowa State University
• Original Assignee: Newlink Genetics - Iowa State University
• Inventors: Shulka, Sachet A., Seregina, Tatiana, Link, Charles J., Vahanian, Nicholas N., Higginbotham, James N., Powers, Bradley J., Young, Won Bin, Ramsey, W. Jay
• Granted Patent: US 6,897,020 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 comprising:

introducing to the genome of said test cell a polynucleotide construct, said polynucleotide construct encoding an assay marker peptide, the expression of which is obtained only upon integration of said polynucleotide construct to an actively transcribing genome region of said test cell;

sorting said cell based upon the expression of said assay marker peptide;

correlating said expression with a genetic locus and

comparing said expression with a reference cell to identify a protein expression profile for the test cell indicative of the test cell.

2. The method of

claim 1

wherein said polynucleotide construct is a vector.

3. The method of

claim 1

wherein said vector is a viral vector.

4. The method of

claim 3

wherein said viral vector is based upon a virus selected from the group consisting of:

retrovirus, lenti-virus, adenovirus or adeno-associated virus.

5. The vector of

claim 4

wherein said vector is a retroviral vector.

6. The method of

claim 1

wherein said assay marker peptide is a fluorescent protein.

7. The marker of

claim 6

wherein said peptide is green fluorescent protein.

8. The marker of

claim 7

wherein said marker is a hrGFP.

9. The method of

claim 1

wherein said polynucleotide construct further comprises a polyadenylation signal.

10. The method of

claim 1

wherein said polynucleotide construct further comprises an internal ribosome entry site.

11. The method of

claim 1

wherein said polynucleotide construct further comprises a splice acceptor site.

12. The method of

claim 1

wherein said polynucleotide construct further comprises a selectable marker gene.

13. The method of

claim 1

wherein said sorting is accomplished by separating said cells on the basis of chemiluminescence, fluorescence, or mechanical means.

14. The method of

claim 13

wherein said sorting is by fluorescence activated flow cytometry.

15. The method of

claim 1

wherein said test cell is a cancer cell and said reference cell is a normal cell.

16. The method of

claim 15

wherein said test cell is a colon cell and said reference cell is a colon cancer cell.

17. The method of

claim 1

wherein said correlating step comprises the following steps:

isolating nucleic acid material from said separated cell;

cleaving said nucleic acid material so that the region of integration of said polynucleotide is separated;

amplifying said cleaved region to form an amplicon comprising known and unknown sequence;

sequencing said amplicon; and

correlating said unknown amplicon sequence with a sequence database to identify a genetic locus associated with said amplicon.

18. The method of

claim 17

wherein said step of cleaving said nucleic acid includes cleaving said nucleic acid so that the inserted polynucleotide construct is cleaved once and flanking nucleic acid sequence is cleaved in a region contiguous to the inserted polynucleotide.

19. The method of

claim 18

wherein said step of cleaving said nucleic acid includes cleaving said nucleic acid at a site that is a fixed distance from said integrated polynucleotide.

20. The method of

claim 19

further comprising the step of:

ligating the ends of said cleaved nucleic acid so that a concatamer is formed.

22. The method of

claim 1

wherein said polynucleotide construct is a vector.

23. The method of

claim 22

wherein said vector is a viral vector.

24. The method of

claim 23

wherein said viral vector is based upon a virus selected from the group consisting of:

retrovirus, lenti-virus, adenovirus or adeno-associated virus.

25. The vector of

claim 24

wherein said vector is a retroviral vector.

41. The method of

claim 1

wherein said correlating step comprises the following steps:

isolating nucleic acid material from said separated cell;

cleaving said nucleic acid material so that the region of integration of said polynucleotide is separated;

amplifying said cleaved region to form an amplicon comprising known and unknown sequence;

sequencing said amplicon; and

correlating said unknown amplicon sequence with a sequence database to identify a genetic locus associated with said amplicon.

42. The method of

claim 41

wherein said step of cleaving said nucleic acid includes cleaving said nucleic acid so that the inserted polynucleotide construct is cleaved once and flanking nucleic acid sequence is cleaved in a region contiguous to the inserted polynucleotide.

43. The method of

claim 42

wherein said step of cleaving said nucleic acid includes cleaving said nucleic acid at a site that is a fixed distance from said integrated polynucleotide.

44. The method of

claim 43

further comprising the step of:

ligating the ends of said cleaved nucleic acid so that a concatamer is formed.

21. A method of elucidating a relative quantitative protein expression profile of a population of test cells comprising:

introducing to the genome of a plurality of said test cells a polynucleotide construct, said polynucleotide construct encoding an assay marker peptide, the expression of which is obtained only upon integration of said polynucleotide construct to an actively transcribing genome region of said test cell population;

sorting said cells based upon the expression level of said assay marker peptide;

correlating said expression with a genetic locus and

comparing said expression with a reference cell population to identify a protein expression profile for the test cell population indicative of the test cell population.

26. The method of

claim 21

wherein said assay marker peptide is a fluorescent protein.

27. The marker of

claim 26

wherein said peptide is green fluorescent protein.

28. The marker of

claim 27

wherein said marker is a hrGFP.

29. The method of

claim 21

wherein said polynucleotide construct further comprises a polyadenylation signal.

30. The method of

claim 21

wherein said polynucleotide construct further comprises an internal ribosome entry site.

31. The method of

claim 21

wherein said polynucleotide construct further comprises a splice acceptor site.

32. The method of

claim 21

wherein said polynucleotide construct further comprises a selectable marker gene.

33. The method of

claim 21

wherein said sorting is accomplished by separating said cells on the basis of chemiluminescence, fluorescence, or mechanical means.

34. The method of

claim 33

wherein said sorting is by fluorescence activated flow cytometry.

35. The method of

claim 21

wherein said test cell population is a cancer cell population and said reference cell population is a normal cell population.

36. The method of

claim 35

wherein said test cell population is a colon cell and said reference cell population is a colon cancer cell.

37. The method of

claim 21

wherein said correlating step comprises the following steps:

isolating nucleic acid material from said separated cells;

cleaving said nucleic acid material so that the region of integration of said polynucleotide is separated;

amplifying said cleaved region to form an amplicon comprising known and unknown sequence;

sequencing said amplicon; and

correlating said unknown amplicon sequence with a sequence database to identify a genetic locus associated with said amplicon.

38. The method of

claim 37

wherein said step of cleaving said nucleic acid includes cleaving said nucleic acid so that the inserted polynucleotide construct is cleaved once and flanking nucleic acid sequence is cleaved in a region contiguous to the inserted polynucleotide.

39. The method of

claim 38

wherein said step of cleaving said nucleic acid includes cleaving said nucleic acid at a site that is a fixed distance from said integrated polynucleotide.

40. The method of

claim 39

further comprising the step of:

ligating the ends of said cleaved nucleic acid so that a concatamer is formed.

45. A polynucleotide construct pGT5A.

46. A polynucleotide construct pGT5AH.

47. A polynucleotide construct pGT5Z.

48. A polynucleotide construct pGT7A.

49. A polynucleotide construct pGT7AH.

50. A polynucleotide construct pGT7Z.

51. A method of elucidating a protein expression profile of a test cell comprising:

introducing to the genome of said test cell a polynucleotide construct, said polynucleotide construct encoding an assay marker peptide, the expression of which is obtained only upon integration of said polynucleotide construct to an actively transcribing genome region of said test cell;

sorting said cell based upon the expression of said assay marker peptide;

sequencing the region marked by said expression marker using sequence tag acquisition and reporting method;

correlating said expression with a genetic locus and

comparing said sequence with a reference cell to identify a protein expression profile for the test cell indicative of the test cell.

52. A method of elucidating a protein expression profile of a test cell comprising:

introducing to the genome of said test cell a polynucleotide construct, said polynucleotide construct encoding an assay marker peptide, the expression of which is obtained only upon integration of said polynucleotide construct to an actively transcribing genome region of said test cell;

sorting said cell based upon the expression of said assay marker peptide;

sequencing the region marked by said expression marker using serial analysis of viral integration method;

correlating said sequence with a genetic locus and

comparing said expression with a reference cell to identify a protein expression profile for the test cell indicative of the test cell.