Isolation of living cells and preparation of cell lines based on detection and quantification of preselected cellular ribonucleic acid sequences

• US 6,692,965 B1
• Filed: 11/22/2000
• Issued: 02/17/2004
• Est. Priority Date: 11/23/1999
• Status: Expired due to Term

• Current Assignee: Chromocell Corporation
• Original Assignee: Chromocell Corporation
• Inventors: Shekdar, Kambiz, Blobel, Gunter
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): Sullivan, Daniel M
• Application Number:
  US09/718,448
• Time in Patent Office: 1,182 Days
• Field of Search: 536/23.1, 536/23.2, 536/23.4, 536/23.5, 536/24.1, 536/24.2, 536/24.3, 536/24.31, 536/24.32, 424/93.21, 424/93.7, 435/320.1, 435/455.471
• US Class Current: 435/455
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2521/537 Protease
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Abstract

The invention is directed to reliable and efficient detection of mRNAs as well as other RNAs in living cells and its use to identify and, if desired, separate cells based on their desired characteristics. Such methods greatly simplify and reduce the time necessary to carry out previously-known procedures, and offers new approaches as well, such as selecting cells that generate a particular protein or antisense oligonucleotide, generating cell lines that express multiple proteins, generating cell lines with knock-out of one or more protein, and others.

Claims

1. A method for generating a cell nine expressing at least one RNA of interest, comprising the steps of:

a) introducing into cells DNA encoding said at least one RNA of interest;

b) exposing said cells to at least one molecular beacon that fluoresces upon hybridization to said at least one RNA of interest, wherein said molecular beacon comprises nucleotides complementary to the RNA of interest and nucleotides mutually complementary;

c) isolating said cells that fluoresce; and

d) generating a cell line expressing said at least one RNA of interest by growing said isolated cells.

3. The method of claim 1 or 2, wherein the step of isolating said cells that fluoresce is carried out using fluorescence activated cell sorter technology.

5. The method of claim 1 or 2, wherein the RNA of interest comprises one or more of a messenger RNA that encodes a protein, an antisense RNA molecule, a structural RNA, a ribosomal RNA, an hnRNA and an snRNA.

22. The method of any one of claims 1, 2, 6, 7, 8 and 16, further comprising the step of selecting the cells after step a) but prior to exposing said cells to said molecular beacons.

23. The method of any one of claims 1, 2, 6, 7, 8 and 16, wherein at least one DNA further encodes at least one drug resistance marker, and said method further comprises the step of selecting cells resistant to at least one drug to which said marker confers resistance.

24. The method of any one of claims 1, 2, 6, 7, 8 and 16, wherein said DNA is operably linked to a conditional promotor.

25. The method of claim 24, wherein said DNA encodes one or more of a messenger RNA that encodes a protein, an antisense RNA molecule, a structural RNA, a ribosomal RNA, an hnRNA and an snRNA.

26. The method of claim 24, wherein the RNA of interest is lethal to the cell.

27. The method of claim 24, wherein the promotor is inducible, and an inducer is applied prior to exposing said cells to the molecular beacon.

2. A method for generating a cell line that expresses two or more RNA molecules of interest, comprising the steps of:

a) introducing into cells DNA encoding a first RNA of interest;

b) introducing into said cells DNA encoding at least one additional RNA of interest;

c) exposing said cells to at least one molecular beacon that fluoresces upon hybridization to said first RNA of interest, wherein said molecular beacon comprises nucleotides complementary to the first RNA of interest and nucleotides mutally complementary;

d) exposing said cells to at least one molecular beacon that, fluoresces upon hybridization to said at least one additional RNA of interest, wherein said molecular beacon comprises nucleotides complementary to the additional RNA of interest and nucleotides mutually complementary;

e) isolating cells that exhibit fluorescence upon hybridization of said molecular beacons to their respective RNA of interest; and

f) generating a cell line expressing said two or more RNA molecules of interest by growing said isolated cells.

4. The method of claim 2, wherein said steps of said first RNA of interest are performed either simultaneously or sequentially with the corresponding steps of said at least one additional RNA of interest.

15. The method of any one of claims 2, 7 and 8, wherein said DNA encoding two or more RNA molecules of interest is on the same construct or on different constructs.

6. A method for generating a cell line expressing an RNA of interest, comprising the steps of:

a) introducing into cells DNA encoding said at least one RNA of interest and at least one epitope tag;

b) exposing said cells to at least one molecular beacon that fluoresces upon hybridization with the RNA transcript of an epitope tag, wherein said molecular beacon comprises nucleotides complementary to the RNA transcript of the epitope tag and nucleotides mutually complementary;

c) isolating said cells that fluoresce; and

d) generating a cell line expressing said RNA of interest by growing said isolated cells.

9. The method of any one of claims 6 to 8, wherein the step of isolating said cells that fluoresce is carried out using fluorescence activated cell sorter technology.

12. The method of any one of claims 6 to 8, wherein the RNA of interest comprises one or more of a messenger RNA that encodes a protein, an antisense RNA molecule, a structural RNA, a ribosomal RNA, an hnRNA and an snRNA.

13. The method of any one of claims 6 to 8, wherein the DNA encoding said epitope tag is in frame with the DNA encoding said RNA of interest.

14. The method of any one of claims 6 to 8, wherein the DNA encoding said epitope tag is out of frame with the DNA encoding said RNA of interest.

7. A method for generating a cell line expressing two or more RNA molecules of interest, comprising the steps of:

a) introducing into cells a first DNA encoding a first RNA of interest and at least one epitope tag;

b) introducing into said cells DNA encoding at least one additional RNA of interest and at least one epitope tag;

c) exposing said cells to at least one molecular beacon that fluoresces upon hybridization with the RNA transcript of an epitope tag for said first RNA of interest, wherein said molecular beacon comprises nucleotides complementary to the RNA transcript of the epitope tag and nucleotides mutually complementary;

d) exposing said cells to at least one molecular beacon that fluoresces upon hybridization with the RNA transcript of an epitope tag for said at least one additional RNA of interest, wherein said molecular beacon comprises nucleotides complementary to the RNA transcript of said epitope tag and nucleotides mutually complementary;

e) isolating cells that exhibit fluorescence upon hybridization of said molecular beacons with their respective RNA transcripts; and

f) generating a cell line expressing said two or more RNA molecules of interest by growing said isolated cells.

10. The method of claim 7, wherein said steps of said first RNA of interest are performed either simultaneously or sequentially with the corresponding steps of said at least one additional RNA of interest.

8. A method for generating a cell line expressing two or more RNA molecules of interest, comprising the steps of:

a) introducing into cells DNA encoding two or more RNA molecules of interest, wherein the DNA encoding at least one of said RNA molecules of interest further encodes at least one epitope tag;

b) exposing said cells to at least one molecular beacon that fluoresces upon hybridization with an RNA of interest, wherein said molecular beacon comprises nucleotides complementary to the RNA of interest and nucleotides mutually complementary, and at least one molecular beacon that fluoresces upon hybridization with the RNA transcript of an epitope tag, wherein said molecular beacon comprises nucleotides complementary to the RNA transcript of an epitope tag and nucleotides mutually complementary;

c) isolating said cells that fluoresce; and

d) generating a cell line expressing said two or more RNA molecules of interest by growing said isolated cells.

11. The method of claim 8, wherein the steps relating to each of said two or more RNA molecules of interest are performed simultaneously or sequentially.

16. A method for generating a cell line that overexpresses an RNA of interest comprising the steps of:

a) introducing into cells a first DNA encoding said RNA of interest and a first epitope tag; and

at least one additional DNA encoding said RNA of interest and a second epitope tag, wherein the first and second epitope tags are different;

b) exposing said cells to at least one molecular beacon that fluoresces upon hybridization with said RNA transcript of said first epitope tag, wherein said molecular beacon comprises nucleotides complementary to the RNA transcript of said first epitope tag and nucleotides mutually complementary, and to at least one molecular beacon that fluoresces upon hybridization with the RNA transcript of said second epitope tag, wherein said molecular beacon comprises nucleotides complementary to the RNA transcript of said second epitope tag and nucleotides mutually complementary;

c) isolating cells that exhibit fluorescence of at least one of said molecular beacons; and

d) generating a cell line overexpressing said RNA of interest by growing said isolated cells.

17. The method of claim 16, wherein the step of isolating said cells that fluoresce is carried out using fluorescence-activated cell sorter technology.

18. The method of claim 16, wherein the steps relating to the first DNA and said additional DNA are performed simultaneously or sequentially.

19. The method of claim 16, wherein said first DNA and said additional DNA are on the same construct or different constructs.

20. The method of claim 16, wherein said RNA of interest comprises one or more of a messenger RNA that encodes a protein, an antisense RNA molecule, a structural RNA, a ribosomal RNA, an hnRNA and an snRNA.

21. The method of claim 16, wherein said DNA encoding said first epitope tag and said DNA encoding said second epitope tag are each independently in frame or out of frame with said DNA encoding said RNA of interest.

28. A method for isolating cells expressing at least one RNA of interest comprising the steps of:

a) providing cells potentially expressing said at least one RNA of interest;

b) exposing said cells to at least one molecular beacon that fluoresces upon hybridization with said at least one RNA of interest, wherein said molecular beacon comprises nucleotides complementary to the RNA of interest and nucleotides mutually complementary;

c) isolating said cells that fluoresce.

29. The method of claim 28, wherein said cells further potentially express one or more additional RNA molecules of interest, further comprising the steps of:

d) exposing said cells to at least one molecular beacon that fluoresces upon hybridization with said at least one additional RNA of interest, wherein said molecular beacon comprises nucleotides complementary to the additional RNA of interest and nucleotides mutually complementary; and

e) isolating cells exhibiting fluorescence upon hybridization of said molecular beacon with said additional RNA of interest.

30. The method of claim 28 or 29, wherein the step of isolating said cells that fluoresce is carried out using fluorescence-activated cell sorter technology.

31. The method of claim 29, wherein said steps are performed either simultaneously or sequentially with the corresponding steps in claim 28.

32. The method of claim 28 or 29, wherein the RNA of interest comprises one or more of a messenger RNA that encodes a protein, an antisense RNA molecule, a structural RNA, a ribosomal RNA, an hnRNA and an snRNA.

33. The method of claim 32, wherein said protein is selected from the group consisting of a cell surface-localized protein and an intracellular protein.