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

• US 20060147937A1
• Filed: 09/02/2003
• Published: 07/06/2006
• Est. Priority Date: 11/23/1999
• Status: Abandoned Application

• Current Assignee: Chromocell Corporation
• Original Assignee: Chromocell Corporation
• Inventors: Shekdar, Kambiz, Blobel, Gunter
• Application Number:
  US10/654,241
• Publication Number:
  US 20060147937A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

1-21. -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, wherein said molecular beacon comprises nucleotides that are complementary to the RNA and nucleotides that are mutually complementary;

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, an antisense RNA, an RNA that encodes a peptide, a ribosomal RNA, an hnRNA and an snRNA.

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.

27. The method of claim 22, wherein the level of at least one second RNA transcript expression is quantified in said biological sample using a second molecular beacon which fluoresces upon hybridization to said second RNA transcript.

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

104. The method of claim 27, wherein said steps of said second RNA transcript are performed either simultaneously, sequentially, or both with the corresponding steps of said at least one mRNA transcript.

105. The method of claim 27, wherein the fluorophore of said second molecular beacon is the same or different from the fluorophore of the first molecular beacon.

29-39. -39. (canceled)

40. A method for identifying genetic recombinational events in living cells comprising the steps of:

a) exposing a cell to a molecular beacon that fluoresces upon hybridization with a RNA sequence selected from the group consisting of that transcribed from a recombined sequence and that transcribed from the nonrecombined sequence;

b) detecting said cell expressing said RNA sequence.

106. The method of claim 40, wherein the fluorescence is detected by fluorescence microscopy or fluorescence-activated cell sorter technology.

41-43. -43. (canceled)

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

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

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

c) isolating said cells that fluoresce.

45. The method of claim 44, further comprising the step of generating a plurality of cell lines expressing said at least one RNA by growing said isolated cells.

46. The method of claim 44, wherein the method is performed simultaneously or sequentially with a plurality of different DNAs encoding a plurality of different RNAs to isolate cells that express at least one RNA, and at least a portion of the cells express at least a different RNA from other cells.

47. The method of claim 46, further comprising the step of generating a plurality of cell lines, each cell line expresses at least one RNA, wherein at least one RNA is different from at least one RNA of the other cell lines, by growing said isolated cells.

68. The method of any one of claims 44, 48, 52, 56, 60, 62, 64 and 66 further comprising the steps of

i) exposing said isolated cells to a molecular beacon that fluoresces upon hybridization to the respective RNA, wherein said molecular beacon comprises nucleotides that are complementary to the respective RNA and nucleotides that are mutually complementary;

and

ii) determining whether the isolated cells express the respective RNAs based on said fluorescence;

or quantitating the level of fluorescence to determine the level of expression of the respective RNAs.

84. The method of any one of claims 44, 48, 52, 56, 60, 62, 64 and 66, wherein the step of isolating said cells that fluoresce is carried out using fluorescence activated cell sorter technology.

85. The method of any one of claims 44, 48, 52, 56, 60, 62, 64 and 66, wherein the RNA comprises one or more of a messenger RNA that encodes a protein, an RNA that encodes a peptide, an antisense RNA, a structural RNA, a ribosomal RNA, an hnRNA and an snRNA.

86. The method of claim 85, wherein said RNA is an antisense RNA.

87. The method of claim 86, wherein the isolated cells are functionally null for expression of at least one preselected protein, wherein said antisense RNA binds essentially all mRNA transcripts of said at least one preselected protein.

88. The method of any one of claims 44, 48, 52, 56, 60, 62, 64 and 66, wherein said DNA is operably linked to a conditional promotor.

89. The method of claim 88, wherein the RNA is lethal to the cell.

90. The method of claim 88, wherein the promotor is inducible, and an inducer is applied prior to exposing said cells to the molecular beacon.

91. The method of any one of claims 44, 48, 52, 56, 60, 62, 64 and 66, further comprising the step of selecting the cells after step a) but prior to exposing said cells to said molecular beacons.

92. The method of any one of claims 44, 48, 52, 56, 60, 62, 64 and 66, 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.

96. A method for generating a transgenic animal that expresses the RNA according to any one of claims 44, 48, 52, 56, 60, 62, 64 and 66, comprising carrying out the steps of any one of claims 44, 48, 52, 56, 60, 62, 64 and 66, utilizing embryonic stem cells, determining the viability of said stem cells, and using said viable embryonic stem cells to produce said transgenic animal.

97. The method of claim 96, wherein the RNA is an antisense RNA, and the transgenic animal is functionally null expressing for at least one preselected protein by the binding of the antisense RNA to essentially all mRNA transcripts of said at least one preselected protein.

98. The method of claim 97, wherein said preselected protein is an alternatively spliced form of a gene product.

48. A method for isolating cells that express at least one of two or more RNAs, comprising the steps of:

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

b) introducing into said cells a second DNA encoding at least a second RNA;

c) exposing said cells to a first molecular beacon that fluoresces upon hybridization to said first RNA, wherein said molecular beacon comprises nucleotides that are complementary to the first RNA and nucleotides that are mutually complementary;

d) exposing said cells to at least a second molecular beacon that fluoresces upon hybridization to said at least second RNA, wherein said molecular beacon comprises nucleotides that are complementary to the second RNA and nucleotides that are mutually complementary; and

e) isolating cells that exhibit fluorescence upon hybridization of at least one of said molecular beacons to their respective RNA.

49. The method of claim 48, further comprising the step of generating a cell line or a plurality of cell lines expressing said at least one of said two or more RNAs by growing said isolated cells.

50. The method of claim 48, wherein the method is performed simultaneously or sequentially with a plurality of different DNAs encoding a plurality of different RNAs to isolate cells that express said one of two or more RNAs, wherein a portion of the cells express at least a different RNA than the other cells.

51. The method of claim 50, further comprising the step of generating a plurality of cell lines expressing at least one of said two or more RNAs, wherein at least one RNA is different from at least one RNA of the other cell lines, by growing said isolated cells.

74. The method of any one of claims 48, 56, 60 and 62, wherein said second molecular beacon has the same or a different fluorophore than that of the first molecular beacon.

76. The method of claim 48 or 56, wherein said steps of said first RNA are performed either simultaneously, sequentially or both, with the corresponding steps of said second RNA.

81. The method of any one of claims 48, 56 and 62, wherein said first DNA and said second DNA are on the same construct or different constructs.

93. The method of claim 48 or 56, wherein the two or more RNAs or proteins encoded thereof form a complex.

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

a) introducing into cells DNA encoding said at least one RNA and at least one tag sequence;

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

c) isolating said cells that fluoresce.

53. The method of claim 52, further comprising the step of generating a plurality of cell lines expressing said at least one RNA by growing said isolated cells.

54. The method of claim 52, wherein the method is performed simultaneously or sequentially with a plurality of different DNAs encoding a plurality of different RNAs to isolate cells that express at least one RNA, and at least a portion of the cells express at least a different RNA from other cells.

55. The method of claim 54, further comprising the step of generating a plurality of cell lines, each cell line expresses at least one RNA, wherein at least one RNA is different from at least one RNA of the other cell lines, by growing said isolated cells.

70. The method of claim 54 or 58, wherein the plurality of DNA encode the same or different tag sequence.

71. The method of any one of claims 52, 56, 60, 62, 64 and 66, wherein the DNA encoding the tag sequence encodes one or more identical tag sequences.

72. The method of any one of claims 52, 56, 60, 62, 64 and 66, wherein the DNA encoding said tag sequence is in frame with the DNA encoding said RNA.

73. The method of any one of claims 52, 56, 60, 62, 64 and 66, wherein the DNA encoding said tag sequence is out of frame with the DNA encoding said RNA.

56. A method for isolating cells expressing at least one of two or more RNAs, comprising the steps of:

a) introducing into cells a first DNA encoding a first RNA and at least a first tag sequence;

b) introducing into said cells a second DNA encoding at least a second RNA and at least a second tag sequence;

c) exposing said cells to at least a first molecular beacon that fluoresces upon hybridization with the first tag sequence, wherein said molecular beacon comprises nucleotides that are complementary to the RNA transcript of the first tag sequence and nucleotides that are mutually complementary;

d) exposing said cells to at least a second molecular beacon that fluoresces upon hybridization with the second tag sequence, wherein said molecular beacon comprises nucleotides that are complementary to the RNA transcript of said second tag sequence and nucleotides that are mutually complementary; and

e) isolating cells that exhibit fluorescence upon hybridization of at least one of said molecular beacons to their respective RNA transcripts of said tag sequences.

57. The method of claim 56, further comprising the step of generating a cell line or a plurality of cell lines expressing said at least one of said two or more RNAs by growing said isolated cells.

58. The method of claim 56, wherein the method is performed simultaneously or sequentially with a plurality of different DNAs encoding a plurality of different RNAs to isolate cells that express said one of two or more RNA, wherein a portion of the cells express at least a different RNA than the other cells.

59. The method of claim 58, further comprising the step of generating a plurality of cell lines expressing at least one of said two or more RNAs, wherein at least one RNA is different from at least one RNA of the other cell lines, by growing said isolated cells.

69. The method of claim 56 or 60, wherein the second tag sequence is the same or different from the first tag sequence.

60. A method of isolating cells expressing at least one of two or more sets of plurality of RNA, comprising the steps of:

a) introducing into cells a first set of plurality of DNAs encoding at least a first set of plurality of RNAs and at least a first tag sequence;

b) introducing into cells a second set of plurality of DNAs encoding at least a second set of plurality of RNAs and at least a second tag sequence;

c) exposing said cells to at least a first molecular beacon that fluoresces upon hybridization to the RNA transcript of said at least first tag sequence, wherein said molecular beacon comprises nucleotides that are complementary to the RNA transcript of the first tag sequence and nucleotides that are mutually complementary;

d) exposing said cells to at least a second molecular beacon that fluoresces upon hybridization to the RNA transcript of said at least second tag sequence, wherein said molecular beacon comprises nucleotides that are complementary to the RNA transcript of the second tag sequence and nucleotides that are mutually complementary; and

e) isolating said cells that exhibit fluorescence upon hybridization of at least one of said molecular beacons to their respective RNA transcripts of said tag sequences.

61. The method of claim 60, further comprising the step of generating a cell line or a plurality of cell lines expressing at least one of said two or more sets of plurality of RNA by growing said isolated cells.

77. The method of claim 60, wherein said steps of said first set of plurality of RNA are performed either simultaneously, sequentially or both, with the corresponding steps of said second set of plurality of RNA.

82. The method of claim 60, wherein said first set of plurality of DNA and said second set of plurality of DNA are on the same construct or different constructs.

94. The method of claim 60, wherein the first set of plurality of RNA or proteins encoded thereof form a complex, the second set of plurality of RNA or proteins encoded thereof form a complex, the first and second set of plurality of RNA or proteins encoded thereof form a complex.

62. A method of isolating cells that overexpress at least one RNA comprising the steps of:

a) introducing into cells at least a first DNA encoding said at least one RNA and at least a first tag sequence; and

at least a second DNA encoding said at least one RNA and at least a second tag sequence;

wherein said first tag sequence is different from said second tag sequence;

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

c) exposing said cells to at least a second molecular beacon that fluoresces upon hybridization with the RNA transcript of said at least second tag sequence, wherein said molecular beacon comprises nucleotides that are complementary to the RNA transcript of the second tag sequence and nucleotides that are mutually complementary; and

d) isolating cells that fluoresce upon hybridization of at least one of said molecular beacons to their respective RNA transcripts of said tag sequences.

63. The method of claim 62 further comprising the step of generating a plurality of cell lines that overexpress said RNA by growing said isolated cells.

78. The method of claim 62, wherein said steps of said first tag sequence are performed either simultaneously, sequentially or both, with the corresponding steps of said second tag sequence.

64. A method for isolating cells that overexpress at least a first protein and which are functionally null expressing for at least a second protein, comprising the steps of:

a) introducing into cells at least a first DNA encoding at least one RNA that encodes said at least first protein, and at least a first tag sequence; and

at least a second DNA encoding said at least one RNA and at least a second tag sequence, wherein said first and second tag sequences are different;

b) introducing into cells at least a third DNA encoding at least one antisense RNA that binds to the mRNA transcript of said at least second protein;

c) exposing said cells to at least a first molecular beacon that fluoresces upon hybridization with the RNA transcript of said at least first tag sequence, wherein said molecular beacon comprises nucleotides that are complementary to the RNA transcript of the first tag sequence and nucleotides that are mutually complementary;

d) exposing said cells to at least a second molecular beacon that fluoresces upon hybridization with the RNA transcript of said at least second tag sequence, wherein said molecular beacon comprises nucleotides that are complementary to the RNA transcript of the second tag sequence and nucleotides that are mutually complementary;

e) exposing said cells to at least a third molecular beacon that fluoresces upon hybridization to said at least one antisense RNA, wherein said molecular beacon comprises nucleotides that are complementary to the antisense RNA and nucleotides that are mutually complementary; and

e) isolating cells that fluoresce upon hybridization of said molecular beacons to their respective RNAs.

65. The method of claim 64, further comprising the step of generating a cell line or a plurality of cell lines overexpressing at least a first protein and which are functionally null expressing for at least a second protein.

75. The method of claim 64, wherein said first, second and third molecular beacons have the same or different fluorophores, or a combination thereof.

79. The method of claim 64, wherein said steps of said first tag sequence, second tag sequence and antisense RNA are performed either simultaneously, sequentially or both.

83. The method of claim 64, wherein said first DNA, second DNA and third DNA are on the same construct, different constructs or a combination thereof.

66. A method for isolating cells expressing at least one exogenous RNA and one endogenous RNA, comprising the steps of:

a) introducing into cells DNA encoding said at least one exogenous RNA and optionally, a tag sequence, wherein said cells potentially express at least one endogenous RNA;

b) exposing said cells to at least a first molecular beacon that fluoresces upon hybridization to said at least one exogenous RNA or RNA transcript of said tag sequence, wherein said molecular beacon comprises nucleotides that are complementary to the exogenous RNA or RNA transcript of said tag sequence and nucleotides that are mutually complementary;

c) exposing said cells to at least a second molecular beacon that fluoresces upon hybridization to said at least one endogenous RNA, wherein said molecular beacon comprises nucleotides that are complementary to the endogenous RNA and nucleotides that are mutually complementary; and

c) isolating said cells that fluoresce upon hybridization of said molecular beacons to their respective RNAs.

67. The method of claim 66, further comprising the step of generating a cell line or a plurality of cell lines expressing said at least one exogenous RNA and at least one endogenous RNA by growing said isolated cells.

80. The method of claim 66, wherein said steps of said exogenous RNA are performed either simultaneously, sequentially or both with the corresponding steps of said endogenous RNA.

95. The method of claim 66, wherein the endogenous RNA and the exogenous RNA form a complex or the proteins encoded thereof form a complex.

99. A proteolytic activity generating unitary hybridization probe comprising:

a single-stranded target complement sequence being complementary to the target sequence;

flanking the target complement sequence, a pair of oligonucleotide arms consisting of a 5′

arm sequence covalently linked to said 5′

terminus and a 3′

arm sequence covalently linked to said 3′

terminus, said pair of oligonucleotide arms forming a stem duplex,

an interacting pair comprising a proteolytic enzyme and an inhibitor of said proteolytic enzyme, one member of each pair conjugated to the 5′

arm sequence and the other member conjugated to the 3′

arm sequence, wherein said inhibitor is capable of inactivating the proteolytic activity of said proteolytic enzyme when interacting therewith.

100. The probe of claim 99 having, under assay conditions in the absence of said target sequence, a characteristic proteolytic activity whose level is a function of the degree of interaction of said proteolytic enzyme and said inhibitor, wherein under conditions in the presence of an excess of said target sequence, hybridization of the target complement sequence to the target sequence increases the level of said characteristic proteolytic activity.

101. The probe of claim 100, wherein the assay conditions have a detection temperature for detecting the target sequence.

102. The probe of claim 100, wherein said proteolytic enzyme inhibitor is a peptide.

103. The probe of claim 100, wherein said proteolytic enzyme and said inhibitor of said proteolytic enzyme is selected from the group consisting of aminopeptidase and amastatin, trypsin-like cysteine proteases and antipain, aminopeptidase and bestatin, chymotrypsin like cysteine proteases and chymostatin, aminopeptidase and diprotin A or B, carboxypeptidase A and EDTA, elastase-like serine proteases and elastinal, and thermolysin or aminopeptidase M and 1,10-phenanthroline.