ISOLATION OF LIVING CELLS AND PREPARATION OF CELL LINES BASED ON DETECTION AND QUANTIFICATION OF PRESELECTED CELLULAR RIBONUCLEIC ACID SEQUENCES

• US 20100212040A1
• Filed: 04/30/2010
• Published: 08/19/2010
• Est. Priority Date: 11/23/1999
• Status: Abandoned Application

• Current Assignee: Chromocell Corporation
• Original Assignee: Chromocell Corporation
• Inventors: Blobel, Gunter, Shekdar, Kambiz
• Application Number:
  US12/771,223
• Publication Number:
  US 20100212040A1
• Time in Patent Office: Days
• US Class Current: 800/21
• 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 line expressing at least one preselected protein comprising the steps of:

a) transfecting cell lines with at least one DNA construct encoding said at least one preselected protein and at least one drug resistance marker;

b) selecting for cells resistant to a drug to which said marker confers resistance, said cells transcribing the at least one DNA construct and the at least one drug resistance marker;

c) exposing said selected cells to a first molecular beacon that fluoresces upon hybridization to an RNA transcript of said at least one preselected protein;

d) isolating said cells that fluoresces;

e) generating a cell line expressing said at least one preselected protein by growing said isolated cells.

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

3. The method of claim 1, wherein said cell line expresses a second preselected protein, said steps further including transfecting said cell line with a second DNA construct encoding a second preselected protein and a drug resistance marker;

selecting for cells expressing said second marker;

exposing said cells to a second molecular beacon which fluoresces upon hybridization to an RNA transcript of said second preselected protein and isolating cells that exhibit fluorescence of each of said at least one and said second mRNA transcripts.

4. The method of claim 3 wherein said step is performed either simultaneously or sequentially.

5-11. -11. (canceled)

12. A method for generating a cell line expressing at least one preselected antisense RNA molecule comprising the steps of:

a) transfecting a cell line with a DNA construct encoding said at least one preselected antisense RNA molecule and at least one drug resistance gene;

b) selecting for cells resistant to a drug to which said marker confers resistance, said cells transcribing the at least one DNA construct and the at least one drug resistance marker;

c) exposing said cells to a molecular beacon that fluoresces upon hybridization to said antisense RNA molecule;

d) isolating said cells that fluoresce; and

e) generating a cell line expressing said preselected antisense RNA sequence by growing said isolated cells.

13. The method of claim 12 wherein isolating said cells that fluoresce is carried out using fluorescence activated cell sorter technology.

14. The method of claim 12, wherein said cell line expresses at least one second preselected antisense RNA molecule, said steps further including transfecting said cell line with a second DNA construct encoding a second preselected antisense RNA molecule and a second drug resistence marker;

selecting for cellular expressing said second marker;

exposing said cells to a second molecular beacon which fluoresces upon hybridization to a said second antisense RNA molecule; and

isolating cells that exhibit fluorescence of each of said at least one and said second mRNA transcripts.

35. A method for generating cells functionally null for expression of at least one preselected protein comprising the steps of providing in said cells a plurality of antisense RNAs to said preselected protein, each provided in accordance with claim 12, wherein said plurality of antisense RNA to said at least one preselected protein binds essentially all mRNA transcripts of said at least one preselected protein.

36. The method of claim 35, wherein said preselected protein is an alternatively spliced form of a gene product.

37. A method for generating a transgenic animal that is a functionally null-expressing mutant of at least one preselected protein comprising carrying out the steps of claim 35 utilizing embryonic stem cells, determining the viability of said stem cells functionally null expressing said preselected protein, and using said viable embryonic stem cells to produce said transgenic animal.

39. A method for generating a cell line expressing a lethal antisense RNA under control of an inducible promoter by carrying out the method of claim 12 wherein step (a) is performed in the presence of a minimal amount of an inducer.

15-21. -21. (canceled)

22. A method for quantifying the level of at least one RNA transcript expression in a biological sample comprising the steps of

a) exposing said biological sample to a first molecular beacon which fluoresces upon hybridization with said RNA transcript;

b) quantitating the level of fluorescence in said biological sample; and

c) correlating said level of fluorescence with said level of said at least one mRNA transcript.

23. The method of claim 22, wherein said biological sample is selected from the group consisting of cellular sample and a tissue sample.

24. The method of claim 22 wherein said RNA transcript is selected from the group consisting of RNA that encodes a protein, a structural RNA, and an antisense RNA.

25. The method of claim 22, wherein said biological sample is fixed.

26. The method of claim 22, wherein said fluorescence is quantitated by fluorescence microscopy or fluorescence-activated cell sorter technology.

28. The method of claim 22 wherein said cells expressing said RNA are isolated using fluorescence cell sorter technology.

27. (canceled)

29-34. -34. (canceled)

38. (canceled)

40-43. -43. (canceled)