JP5753134B2 - Selection and isolation of viable cells using mRNA binding probes - Google Patents

Description

This application claims priority to provisional application 60 / 166,987, filed on November 23, 1999. The entire application is incorporated herein by reference.

BACKGROUND OF THE INVENTION Molecular beacons are nucleic acid probes that recognize and convey the presence of certain nucleic acid sequences. This probe is a hairpin type sequence with a central linear portion of nucleotides complementary to the target sequence and a terminal portion containing short mutually complementary sequences. One end is covalently attached to the fluorophore and the other end is covalently attached to the quencher group. In the natural state having hybridized ends, the proximity of the fluorophore and the quenching molecule is such that no fluorescence is emitted. When this beacon is hybridized to a target nucleic acid, it naturally undergoes a fluorescent three-dimensional structural change. See, for example, US Pat. No. 5,925,517.

  This property has allowed researchers to detect specific nucleic acids primarily in in vitro reactions and in some cases in living cells. In vivo visualization of messenger RNA was achieved using molecular beacons transported to living cells in liposomes (Matsuo, 1998, Biochem. Biophys. Acta 1379: 178-184). So far, research dealing with cells has used beacons generated by the very weak fluorophore EDANS. This is because this molecule was the only one known to be quenched by the quenching molecule EDAC. The result was a body that could be discriminated but barely made, and it was not promising to apply this direction of research to in vivo detection.

For example, the present invention provides the following.
(Item 1)
A method of isolating cells that express at least one RNA comprising the steps of:
a) introducing a DNA encoding the at least one RNA into a cell;
b) exposing the cell to at least one molecular beacon that fluoresces when hybridized to the at least one RNA, the molecular beacon comprising nucleotides complementary to the RNA and nucleotides complementary to each other; A waste process; and
c) A method comprising isolating said cells that fluoresce.
(Item 2)
A method for isolating a cell that expresses at least one of two or more RNAs, comprising:
a) introducing a first DNA encoding the first RNA into a cell;
b) introducing a second DNA encoding at least one second RNA into the cell;
c) exposing the cell to a first molecular beacon that fluoresces when hybridized to the first RNA, the molecular beacon comprising nucleotides complementary to the first RNA and nucleotides complementary to each other; ;
d) exposing the cell to at least one second molecular beacon that fluoresces when hybridized to the at least second RNA, the molecular beacon comprising nucleotides complementary to the second RNA and nucleotides complementary to each other; A process comprising: and
e) isolating cells that exhibit fluorescence when at least one of the molecular beacons is hybridized to its corresponding RNA;
Including methods.
(Item 3)
A method of isolating cells that express at least one RNA comprising the steps of:
a) introducing DNA encoding the at least one RNA and at least one tag sequence into a cell;
b) exposing the cell to at least one molecular beacon that fluoresces when hybridized to the RNA transcript of the tag sequence, wherein the molecular beacon is mutually complementary to nucleotides complementary to the RNA transcript of the tag sequence; And complementary nucleotides; and
c) A method comprising isolating said cells that fluoresce.
(Item 4)
A method for isolating a cell that expresses at least one of two or more RNAs, comprising:
a) introducing a first DNA encoding a first RNA and at least one first tag sequence into a cell;
b) introducing a second DNA encoding at least one second RNA and at least one second tag sequence into the cell;
c) exposing the cell to at least one first molecular beacon that fluoresces when hybridized to a first tag sequence, the molecular beacon comprising nucleotides complementary to an RNA transcript of the first tag sequence; Comprising nucleotides complementary to each other;
d) exposing the cell to at least one second molecular beacon that fluoresces when hybridized to a second tag sequence, the molecular beacon comprising nucleotides complementary to the RNA transcript of the second tag sequence; And comprising nucleotides complementary to each other; and
e) isolating cells that exhibit fluorescence when at least one of the molecular beacons is hybridized to a corresponding RNA transcript of the tag sequence;
Including methods.
(Item 5)
A method of isolating cells that overexpress at least one RNA comprising:
a) at least one first DNA encoding the at least one RNA and at least one first tag sequence; and at least one second DNA encoding the at least one RNA and at least one second tag sequence. Introducing into a cell, wherein the first tag sequence is different from the second tag sequence;
b) exposing the cell to at least one first molecular beacon that fluoresces when hybridized to the RNA transcript of the at least first tag sequence, wherein the molecular beacon is against the RNA transcript of the first tag sequence; Including complementary nucleotides and complementary nucleotides;
c) exposing the cell to at least one second molecular beacon that fluoresces when hybridized to the RNA transcript of the at least second tag sequence, wherein the molecular beacon is against the RNA transcript of the second tag sequence. And d) isolating cells that fluoresce when at least one of the molecular beacons is hybridized to a corresponding RNA transcript of the tag sequence. Process,
Including methods.
(Item 6)
A method of isolating a cell that overexpresses at least one first protein but has at least one functional zero expression or reduced expression of at least one second protein comprising:
a) at least one RNA encoding the at least one first protein; at least one first DNA encoding at least one first tag sequence; and the at least one RNA and at least one second tag. Introducing at least one second DNA encoding the sequence into the cell, which is different from the first and second tag sequences;
b) introducing into the cell at least one third DNA encoding at least one antisense RNA that binds to the mRNA transcript of said at least second protein;
c) exposing the cell to at least one first molecular beacon that fluoresces when hybridized to the RNA transcript of the at least first tag sequence, wherein the molecular beacon is against the RNA transcript of the first tag sequence; Including complementary nucleotides and complementary nucleotides;
d) exposing the cell to at least one second molecular beacon that fluoresces when hybridized to the RNA transcript of the at least second tag sequence, wherein the molecular beacon is against the RNA transcript of the second tag sequence. Including complementary nucleotides and complementary nucleotides;
e) exposing the cell to at least one third molecular beacon that fluoresces when hybridized to the at least one antisense RNA, wherein the molecular beacon is complementary to nucleotides complementary to the antisense RNA and to each other; And comprising the steps of: and
f) isolating cells that fluoresce when the molecular beacon is hybridized to the respective RNA;
Including methods.
(Item 7)
A method for isolating a cell expressing at least one first RNA comprising:
a) supplying a cell having the ability to express the at least first RNA;
b) exposing the cell to at least one first molecular beacon that fluoresces when hybridized to the at least first RNA, the molecular beacon comprising nucleotides complementary to the first RNA and nucleotides complementary to each other; Including the steps of:
c) isolating the cells that fluoresce;
Including methods.
(Item 8)
The cell further has the ability to express at least one second RNA;
a) exposing the cell to at least one second molecular beacon that fluoresces when hybridized to the at least second RNA, the molecular beacon comprising nucleotides complementary to the second RNA and nucleotides complementary to each other; A process comprising: and
b) isolating cells that exhibit fluorescence when the molecular beacon is hybridized to the second RNA;
The method according to item 7, further comprising:
(Item 9)
Item 8. The method according to Item 7, wherein the protein is selected from the group consisting of a cell surface localized protein and an intracellular protein.
(Item 10)
5. The method according to item 2 or 4, wherein the second tag sequence is the same as or different from the first tag sequence.
(Item 11)
9. The method of any one of items 2, 4, 5, and 8, wherein the second molecular beacon has a fluorescent material that is the same as or different from that of the first molecular beacon.
(Item 12)
7. The method of item 6, wherein the first, second and third molecular beacons have the same or separate fluorescent materials or combinations thereof.
(Item 13)
9. The method of any one of items 2, 4, and 8, wherein the step of the first RNA is performed either simultaneously with the corresponding step of the second RNA, sequentially, or both.
(Item 14)
6. The method of item 5, wherein the steps of the first tag sequence and the second tag sequence are performed either simultaneously, sequentially, or both.
(Item 15)
7. The method of item 6, wherein said steps of said first tag sequence, second tag sequence and antisense RNA are performed either simultaneously, sequentially or both.
(Item 16)
6. The method according to any one of items 2, 4 and 5, wherein the first DNA and the second DNA are present on the same construct or on different constructs.
(Item 17)
7. The method of item 6, wherein the first DNA, second DNA, and third DNA are present on the same construct, different constructs, or a combination thereof.
(Item 18)
5. The method of item 2 or 4, wherein the two or more RNAs are the same or different.
(Item 19)
Item 9. The method according to any one of Items 1 to 8, wherein the step of isolating the cells that emit fluorescence is performed by fluorescence activated cell sorter technology or fluorescence microscopy.
(Item 20)
9. The method according to any one of items 1 to 8, wherein the RNA is antisense RNA.
(Item 21)
The isolated cell is functionally zero or reduced in expression of at least one preselected protein, and the antisense RNA comprises the at least one preselected protein 21. The method of item 20, wherein the method binds to substantially all mRNA, or a substantial portion of its transcript.
(Item 22)
Item 7. The method according to any one of Items 1 to 6, wherein the DNA is operably linked to a conditional promoter.
(Item 23)
24. The method of item 22, wherein the promoter is inducible and an inducer is applied prior to exposing the cell to a molecular beacon.
(Item 24)
7. The method of any one of items 1 to 6, further comprising the step of selecting a cell after step a) but before exposing the cell to the molecular beacon.
(Item 25)
The at least one DNA further encodes at least one drug resistance marker, and the method comprises the step of selecting cells that have been rendered resistant by the marker and become resistant to at least one drug. The method according to any one of items 1 to 6, further comprising:
(Item 26)
A method for producing a transgenic animal that expresses RNA according to any one of items 1 to 6, comprising performing the step according to any one of items 1 to 6, utilizing embryonic stem cells, the stem cells And determining the survival rate of the cell and producing the transgenic animal using the living embryonic stem cells.
(Item 27)
The RNA is an antisense RNA, and the transgenic animal binds to substantially all of, or a substantial portion of, the mRNA transcript of the at least one preselected protein, 27. A method according to item 26, wherein the expression of at least one preselected protein is functionally zero or expression is reduced.
(Item 28)
A method for quantifying the level of at least one first RNA expression in a biological sample comprising:
a) exposing the biological sample to a first molecular beacon that fluoresces when hybridized with the first RNA, the molecular beacon comprising nucleotides complementary to the first RNA and sequences complementary to each other; Steps to include;
b) quantifying the fluorescence level in the biological sample; and
c) correlating the fluorescence level with the level of the at least first RNA;
Including methods.
(Item 29)
30. The method of item 28, wherein the biological sample is selected from the group consisting of a cell sample and a tissue sample.
(Item 30)
29. A method according to item 28, wherein the biological sample is fixed.
(Item 31)
29. The method of item 28, wherein the fluorescence is quantified by fluorescence microscopy or fluorescence activated cell sorter technology.
(Item 32)
The level of expression of at least one second RNA is quantified in the biological sample by a second molecular beacon that fluoresces when hybridized to the second RNA, the second molecular beacon being complementary to the second RNA 29. A method according to item 28, comprising a nucleotide and nucleotides complementary to each other.
(Item 33)
34. The method of item 32, wherein the step of the second RNA is performed either simultaneously with the corresponding step of the at least first RNA, sequentially, or both.
(Item 34)
33. The method of item 32, wherein the second molecular beacon has a fluorescent material that is the same as or different from that of the first molecular beacon.
(Item 35)
33. A method according to item 28 or 32, wherein the cell sample is a cell.
(Item 36)
36. The method of item 35, further comprising isolating the cell based on the expression level of the at least first RNA or second RNA or both.
(Item 37)
38. The method of item 36, wherein the cells are isolated using fluorescence activated cell sorter technology or fluorescence microscopy.
(Item 38)
The method according to any one of items 1 to 8 and 36, further comprising the step of growing the cells.
(Item 39)
40. The method of item 38, wherein one cell line or a plurality of cell lines are generated.
(Item 40)
33. Any one of items 1 to 8 and 28 to 32, wherein the RNA is one or more of RNA encoding a protein, structural RNA, antisense RNA, RNA encoding a peptide, ribosomal RNA, hnRNA, and snRNA. The method described in 1.
(Item 41)
A single hybridization probe that produces proteolytic activity comprising:
A single-stranded target complementary sequence complementary to the target sequence;
A pair of oligonucleotide arms sandwiching a target complementary sequence on both sides, comprising a 5 ′ arm sequence covalently linked to the 5 ′ end and a 3 ′ arm sequence covalently linked to the 3 ′ end, A pair of oligonucleotide arms forming a double strand;
An interactive pair comprising a proteolytic enzyme and an inhibitor of said proteolytic enzyme, wherein one member of each pair is conjugated to a 5 ′ arm sequence and the other member is conjugated to a 3 ′ arm sequence; When the inhibitor interacts with the proteolytic enzyme, an interaction pair capable of at least partially inactivating the proteolytic activity of the proteolytic enzyme,
A hybridization probe comprising:
(Item 42)
Item 44. The probe according to Item 41, wherein the proteolytic activity level is a function representing the degree of interaction between the proteolytic enzyme and the inhibitor under assay conditions in the absence of the target sequence. A probe having proteolytic activity, wherein under conditions where the target sequence is present in excess, hybridization of a target complementary sequence to the target sequence increases the level of the characteristic proteolytic activity.
(Item 43)
43. A probe according to item 42, wherein the assay conditions have a detection temperature for detecting a target sequence.
(Item 44)
42. A probe according to item 41, wherein the protease inhibitor is a peptide.
(Item 45)
The proteolytic enzyme and the inhibitor of the proteolytic enzyme are aminopeptidase and amasstatin, trypsin-like cysteine protease and antipain, aminopeptidase and bestatin, chymotrypsin-like cysteine protease and chymostatin, aminopeptidase and diprotin A or B, carboxy 42. The probe according to item 41, which is selected from the group consisting of peptidase A and EDTA, elastase-like serine protease and elastinal, and thermolysin or aminopeptidase M and 1,10-phenanthroline.

In one aspect, the invention provides a method for generating a cell line that expresses at least one preselected protein comprising:
a) transfecting a cell line with at least one DNA construct encoding at least one preselected protein and at least one drug resistance marker;
b) selecting a cell that has been rendered resistant by a marker and that has become resistant to a drug, wherein said cell has transcribed at least one drug resistance marker;
c) exposing selected cells to a first molecular beacon, wherein the first molecular beacon fluoresces when hybridized to an RNA transcript of at least one preselected protein;
d) isolating cells that fluoresce;
e) generating a cell line expressing at least one preselected protein by culturing the isolated cells;
Directed to a method comprising:

  The method described above can be performed using fluorescence activated cell sorter technology. In yet another aspect, the cell line can be directed to express a second preselected protein. Transfecting said cell line with a second DNA construct encoding a second preselected protein and a second drug resistance marker; selecting cells expressing the second marker; Exposing the cell to a second molecular beacon that fluoresces when hybridized to an RNA transcript of a second preselected protein; and cells that fluoresce in at least one mRNA transcript and each of the second mRNA transcripts The step of isolating is further performed. It is also possible to supply cell lines that express more than two proteins by repeating the above steps. The method of introducing the second protein may be performed simultaneously or sequentially. If the first and second drug resistance markers are the same, simultaneous selection can be achieved by increasing the drug concentration.

In another aspect of the invention, a method for generating a cell line expressing at least one preselected protein comprising:
a) transfecting a cell line with at least one preselected protein, at least one drug resistance marker, and at least one DNA construct encoding at least one first epitope tag;
b) selecting a cell that has been rendered resistant by the marker and that has become resistant to the drug, and wherein the cell has transcribed at least one drug resistance marker;
c) exposing the selected cells to a first molecular beacon that fluoresces when hybridized to the RNA transcript of the first epitope tag;
d) isolating fluorescent cells; and e) generating a cell line expressing at least one preselected protein by culturing the isolated cells;
Is provided.

  The method described above can be performed using fluorescence activated cell sorter technology. The DNA portion of the construct encoding the epitope tag may be within or outside the frame containing the DNA construct portion encoding the at least one protein. In yet another embodiment, the cell line can be directed to express at least a second preselected protein. That is, the step comprises transfecting said cell line with a second preselected protein, a second drug resistance marker, and a second DNA construct encoding a second epitope tag; Selecting a cell that transcribes the marker; exposing the cell to a second molecular beacon that fluoresces when hybridized to the RNA transcript of the second epitope tag; and the fluorescence of each of the first and second epitope tags Further comprising isolating cells exhibiting In the case of two proteins, the portion of the DNA sequence that encodes the second epitope tag may be within or outside the frame of the portion of the DNA sequence that encodes the second protein. The second preselected protein may be transfected simultaneously or sequentially with the first. If the method is performed simultaneously and the same drug resistance marker is used for both constructs, it is possible to select cells that express both constructs using the higher concentration of drug. Furthermore, it is possible to supply more than two proteins into a cell line by repeating the above steps.

In yet another aspect of the invention, a method for generating a cell line expressing at least one preselected antisense RNA molecule comprising:
a) transfecting a cell line with a DNA construct encoding at least one preselected antisense RNA molecule and at least one drug resistance gene;
b) selecting a cell that has been rendered resistant by the marker and that has become resistant to the drug, and wherein the cell has transcribed at least one drug resistance marker;
c) exposing the cell to a molecular beacon that fluoresces when hybridized to an antisense RNA molecule;
d) isolating cells that fluoresce; and
e) generating a cell line expressing a preselected antisense RNA sequence by culturing the isolated cells;
Is provided.

  Isolation of fluorescent cells can be performed using fluorescence activated cell sorter technology. This cell line can also be directed to express at least one second preselected antisense RNA. That is, the process comprises simultaneously or sequentially transfecting a cell line with a second DNA construct encoding a second preselected antisense RNA molecule and a second drug resistance marker; Selecting a cell that expresses the two markers; exposing the cell to a second molecular beacon that fluoresces when hybridized to a second antisense RNA molecule; and at least one mRNA transcript and a second mRNA transcript It further comprises isolating cells that exhibit their respective fluorescence. If the method is performed simultaneously and the same drug resistance marker is used for both constructs, selection can be achieved by using the higher drug concentration. It is possible to supply more than two antisense RNA molecules by repeating the above steps with another construct.

In yet another aspect of the invention, a method for generating a cell line expressing at least one preselected antisense RNA molecule comprising:
a) transfecting a cell line with at least one preselected antisense RNA molecule, at least one drug resistance marker, and a DNA construct encoding a first epitope tag nucleotide sequence;
b) selecting a cell that has been rendered resistant by the marker and that has become resistant to the drug, and wherein the cell has transcribed at least one drug resistance marker;
c) exposing the selected cells to a first molecular beacon that fluoresces when hybridized to the mRNA transcript of the first epitope tag;
d) isolating cells that fluoresce;
e) generating a cell line expressing at least one preselected antisense RNA molecule by culturing the isolated cells;
Is provided.

  This cell line can also be directed to express at least one second preselected epitope-tagged antisense RNA. That is, the process comprises transfecting said cell line with a second DNA construct encoding a second preselected epitope-tagged antisense RNA molecule and a second drug resistance marker; Selecting a cell that expresses the marker; exposing the cell to a second molecular beacon that fluoresces when hybridized to an RNA transcript of a second epitope tag; and at least one mRNA transcript and a second mRNA transcript It further includes isolating cells that exhibit both fluorescence of the object. These steps may be performed simultaneously with the first or over time. Selection by co-transfection based on both constructs with the same drug resistance marker can be performed using the higher concentration of drug. Additional steps can be performed to introduce more than two epitope-tagged antisense molecules.

  The foregoing methods are used to prepare cells that express all types of RNA molecules, including antisense RNA molecules or structural RNAs including but not limited to hRNA and snRNA in addition to rRNA. Is possible. In addition, it should be noted that in all methods in which an epitope tag is provided for detection by a molecular beacon of the transfected construct, the epitope is as long as the epitope tag sequence is detectable by the molecular beacon. It is immaterial whether the tag is within the frame of the encoded protein or not.

In yet another aspect of the invention, a method for isolating a cell that expresses at least one protein or RNA comprising:
a) providing a cell expected to express at least one protein;
b) exposing the cell to at least one molecular beacon that fluoresces when hybridized to an mRNA transcript encoding the at least one protein;
c) isolating fluorescent cells;
Is provided.

  Fluorescence activated cell sorter technology can be used to isolate cells that fluoresce. As a non-limiting example, the protein may be a cell surface localized protein or an intracellular protein. Any other RNA can be detected. This method can also be used to identify cells that express a second protein or RNA. That is, the cells are exposed to a second molecular beacon that emits fluorescence when hybridized with the target RNA, and cells having the respective fluorescence of the first and second molecular beacons are isolated. Simultaneous expression of more than two proteins is also feasible.

The present invention is also a method for quantifying the level of at least one RNA transcript in a biological sample comprising:
a) exposing the biological sample to a first molecular beacon that fluoresces when hybridized to the RNA transcript;
b) quantifying the fluorescence level in the biological sample; and
c) correlating the fluorescence level with the level of said at least one RNA transcript;
Directed to a method comprising:

  The biological sample may be a cell sample or a tissue sample. This sample may be fixed. The RNA transcript may be, but is not limited to, RNA encoding a protein, structural RNA or antisense RNA. Fluorescence can be quantified by fluorescence microscopy or fluorescence activated cell sorter technology. The level of at least one second RNA transcript expression in the biological sample can also be quantified using a second molecular beacon that fluoresces when hybridized to the second RNA transcript.

In yet another aspect of the invention, a method for generating a cell line that overexpresses at least one protein comprising:
a) at least two DNA sequences, wherein one sequence has a portion encoding said protein, at least one epitope tag, and at least one drug resistance marker, and a second sequence comprises said protein Transfecting the cell with at least two DNA sequences having a portion encoding at least one second epitope tag and at least one second drug resistance marker;
b) selecting cells transfected with said at least two DNA sequences by expression of first and second drug resistance markers;
c) Exposing selected cells to first and second molecular beacons that fluoresce when hybridized to nucleotide sequences of RNA transcripts of the first and second epitope tags, respectively. Process;
d) isolating cells exhibiting fluorescence of the first and second molecular beacons respectively; and
e) generating a cell line overexpressing said at least one preselected protein by culturing the isolated cells;
Is provided.

  In the foregoing aspect of the invention, at least two DNA sequences may be localized on the same construct. The first and second epitope tags may be independently within or outside the frame of the protein. By using FACS to isolate cells that express the strongest fluorescence, it is possible to isolate cells that express both constructs. Similar to the methods described herein above, transfection may be performed simultaneously or sequentially. If the same drug resistance marker is present in both constructs and co-transfection is performed, selection can be achieved by increasing the drug level.

In yet another aspect of the invention, a method for generating a cell line that overexpresses at least one antisense RNA molecule comprising:
a) at least two DNA sequences, wherein one sequence has a portion encoding said protein, at least one epitope tag, and at least one drug resistance marker, and a second sequence comprises said protein Transfecting the cell with two such DNA sequences having a portion encoding at least one second epitope tag and at least one second drug resistance marker;
b) selecting cells transfected with said at least two DNA sequences by expression of first and second drug resistance markers;
c) Exposing selected cells to first and second molecular beacons that fluoresce when hybridized to nucleotide sequences of RNA transcripts of the first and second epitope tags, respectively. Process;
d) isolating cells exhibiting fluorescence of the first and second molecular beacons respectively; and
e) generating a cell line overexpressing said at least one preselected antisense RNA molecule by culturing isolated cells;
Is provided.

  The at least two DNA sequences may be localized on the same construct. The first and second epitope tags may each independently be within or outside the frame of the antisense RNA molecule. Conditions and alternative methods for realizing the desired product are as described above. Furthermore, any form of RNA may be supplied.

  In another aspect of the invention, a method of generating a cell that is functionally zero for the expression of at least one preselected protein or RNA, wherein the cell is directed against said preselected protein or RNA. Supplying a plurality of antisense RNAs, wherein a plurality of antisense RNAs against said at least one preselected protein or RNA bind to and remove virtually all RNA transcripts consisting of mRNA or other RNA There is provided a method comprising the steps comprising: If the cell is zeroed for a preselected protein, the preselected protein may be the only unique protein or alternatively spliced for a gene product. It may be a subset of a plurality of forms.

  In yet another aspect of the present invention, a method for generating a transgenic animal that is a functional zero-expressing mutant for at least one preselected protein, wherein the aforementioned steps are performed using embryonic stem cells. There is provided a method comprising quantifying the viability of stem cells that functionally zero-express the preselected protein and generating a transgenic animal using viable embryonic stem cells.

  Further, a method of generating a cell line that is functionally zero-expressed for at least one protein and over-expressed for at least one other protein, wherein the method previously described herein is performed on the same cell A method is provided. In addition, a cell line expressing lethal antisense RNA under the control of an inducible promoter is generated by performing the method described above, wherein step (a) is performed in the presence of a very small amount of inducer. A method is provided.

A method for identifying a genetic recombination event in a living cell, comprising:
a) exposing a cell to a molecular beacon that fluoresces when hybridized with an RNA sequence selected from the group consisting of an RNA sequence transcribed from a recombinant sequence and an RNA sequence transcribed from a non-recombinant sequence;
b) detecting or selectively extracting the cells;
It is a method including.

  The detection and / or selective extraction of the cells can be performed by FACS or microscopy.

  In another aspect, the present invention is directed to a single hybridization probe that generates proteolytic activity, referred to herein as a probe protease. This probe protease works in the same way as a molecular beacon, but is not a fluorescent change that occurs when the molecular beacon interacts with the target nucleic acid sequence, and the protease is proteolytic in the presence of the target. The probe protease composition is provided with a protease at one end of a molecular beacon type oligonucleotide and a complementary protease inhibitor at the other end. When this oligonucleotide is not hybridized to the target sequence, both ends of the oligonucleotide are close to each other, so that it is possible for the protease and the protease inhibitor to interact with each other, thereby suppressing the proteolytic activity of the protease. To do. However, when the target sequence of the oligonucleotide hybridizes to the target, the protease and its inhibitor are separated and activate the protease. The activation of this protease can be easily measured, and a protease having activity in the presence of a specific nucleic acid target sequence can be used not only for detection purposes but also for therapeutic purposes. That is, for therapeutic purposes, for example, when a specific gene, for example, a gene related to cell transformation, carcinogenicity, abnormal growth, etc. is transcribed, the cell into which the probe protease has been sent is proteolyzed, I can't live.

  The aforementioned probe may have a protease inhibitor that is a peptide or another molecule capable of reversibly inactivating the protease. Non-limiting examples of enzymes and inhibitors include aminopeptidase and amastatin, trypsin-like cysteine protease and antipain, aminopeptidase and bestatin, chymotrypsin-like cysteine protease and chymostatin, aminopeptidase and diprotin A or B, carboxypeptidase A and EDTA, elastase-like serine protease and elastinal, and thermolysin or aminopeptidase M and 1,10-phenanthrin.

  These and other aspects of the invention will be understood from the detailed description below.

Detailed Description of the Invention The invention herein relates to the ability of a molecular probe to allow identification of RNA sequences in living cells and cells that exhibit a certain level or levels of fluorescence at one or more wavelengths. Based on the use of fluorescent cell sorters or related techniques to identify and / or isolate It is now known that EDAC can suppress the emission of various strongest fluorescent molecules normally used. The fact that it is possible to detect not only mRNA but also other RNAs stably and efficiently in living cells means that, for example, using a fluorescence activated cell sorter (FACS) based on desired characteristics. Thus, in order to identify cells, it has become possible to use their RNA to isolate such cells if necessary. The present application greatly simplifies conventional known processes and provides a number of methods for significantly reducing the time required for execution thereof, as well as a new method. Conventional tests using beacons and live cells did not show any significant differences from controls. The problem is that by using a more powerful fluorescent molecule, the beacon can be made longer by increasing the number of nucleotides forming the inner stem portion of the stem-loop molecule longer than the conventional 5 or 6 nucleotide length. Removed by qualifying. This modification reduces the background because the stem loop molecule can more reliably maintain a non-fluorescent state in the absence of the target sequence.

  According to the methodology described above, RNAs of various roles, i.e., that do not encode proteins or act as antisense RNA, but are expressed by cells, RNA that act as messenger RNA, and those already mentioned It will be possible to stably generate transfected cell lines that express RNA that is antisense RNA to two classes of RNA. Examples of the other RNAs described above include structural RNAs such as ribosomal RNA, hnRNA, and snRNA.

  1. Generation of protein-expressing cell lines. Some of the most tedious processes involved in cell line generation are eliminated by operating molecular beacons as described herein. Following drug selection of cells transfected with a DNA construct that also encodes drug resistance in addition to the desired gene, a molecular beacon designed to recognize the message of the gene of interest is introduced into the cell . Cells that have transcribed the gene emit light. Fluorescent cells are then isolated by FACS analysis and then expanded to produce a cell line that expresses the selected gene.

  If the beacon designed to recognize the message of interest can detect the endogenously present target sequence, the endogenous sequence compared to the percentage of the target sequence produced by the transfected cells The ratio allows the sorter to distinguish between the two cell types. In another embodiment, the intrinsic fluorescence problem is eliminated by tagging the gene of interest with an epitope tag and designing the beacon to recognize the nucleotide sequence encoding this tag. These nucleotides may or may not match the reading frame of the gene message, depending on whether it is necessary to tag the protein to be produced. Good.

  Furthermore, the expression level of the target gene in any given cell may vary. In such cases, FACS is applied to assess expression levels. FACS is used to differentially select individual cells that express the same gene.

  2. Generation of antisense-expressing cell lines. There are several studies that describe the generation of cell lines that express RNA that is the antisense of a gene or part of a gene rather than the RNA encoding the protein. These methods aim to reduce the amount of specific protein in a given cell. The steps described above for the generation of protein-expressing cell lines are equally applicable in this case, and are almost identical except that the beacon is designed to detect RNA that is antisense RNA. It is.

  Not all attempts to create a stably transfected antisense-expressing cell line result in a cell line in which the expression of the target protein is sufficiently affected. This difficulty reduces the value of continuing production of this type of cell line. On the other hand, since the process described herein is simple, it is easy to assay potency for a number of different gene sequences for the ability to generate highly active antisense-expressing cell lines.

  3. Differentiation of cells based on cell surface localized antigens. Immunologists and other researchers have long used FACS to sort cells. According to the present invention, in order to detect the presence of a cell surface localized protein, a beacon having an mRNA encoding the target protein as a target is produced. This beacon is introduced into the cells by transfection, and then the cells that gave a positive score are isolated using a cell sorter.

  In addition, FACS is recently used to sort cells based on the expression of up to three cell surface localized proteins. This property of FACS is a combination of multiple beacons, each of which uses a combination of multiple beacons, each targeting one mRNA of the protein of interest and each labeled differently. This is achieved by using molecular beacons as described in. To sort cells based on more than three expressed RNAs, sorting is performed multiple times.

  4). Assay cells for specific RNA expression and quantify the level of RNA expression in the cells. When a molecular beacon target RNA introduced into a cell is present, the cell fluoresces. This information is quantitatively evaluated by using a fluorescence microscope or FACS, which can be quantified by either method. For example, in situ reverse transcription polymerase chain reaction (RT-PCR) has been performed on tissue slices to determine the expression pattern for a particular RNA, but this is not done and the beacon is used instead You can experiment. In addition, the presence or amount of several RNAs of interest are assayed in one step by using multiple beacons, each of which emits different fluorescence, each targeting multiple specific RNAs. .

  5. Antigen localization in combination with RNA detection in cells and tissues. Use fixed cells or tissue slices to describe the localization of recognized protein antigens by utilizing immunocytochemistry and target RNA in the same sample by using molecular beacons that target specific RNAs At the same time. Molecular beacons targeted against RNA have also been shown to function in fixed cells.

  6). Generation of cell lines that express multiple proteins. Using the method of the present invention, transformed cell lines expressing any number of proteins can be stabilized very quickly without the need to maintain those cells in the presence of a mixture of a number of selective agents. Generated automatically. After gene transfection and drug selection, a combination of multiple molecular beacons, each for each protein message, is introduced into the cell. Hybridize the loop sequence of each beacon to the mRNA of only one of the genes, or the nucleotide sequence of only one of the epitope tags (see above), to which the message may be associated. By design, each beacon is designed to recognize mRNA encoded by only one of a plurality of genes. Next, the cells are sorted by FACS. By selecting one, two or all three fluorescent signals, various cell lines are generated in a single operation.

  It may be necessary to produce a cell line that expresses more than three RNAs of interest. For example, if we could have a cell line in which all of the protein and RNA sequences thought to be involved in the formation of a particular complex could be overexpressed, it would yield very rich information. To achieve this, cells that already express a combination of multiple RNAs are used as host cells, and additional constructs encoding additional RNAs are transfected into these cells to Repeat the process.

  If multiple RNAs to be expressed are all cloned in a construct that confers resistance to the same drug on the cell, FACS is used to isolate cells that express all the desired RNA. Since these sequences are stably integrated into the genome, cells do not lack expression of any sequence. However, one or more of the sequences can be lost. In such a case, increasing the concentration of the selective agent in the culture medium in which the cells grow will reduce this possibility. Alternatively, the cells are maintained in a suitable drug mixture using multiple constructs that each confer resistance to separate drugs. Also, a subset of constructs that are stably transfected into the cell is selected to encode a resistance gene for one drug and another subset that encodes a resistance gene for another drug.

  Furthermore, when some cells lose the target RNA in a certain cell line, as a remedy, the aforementioned first experiment conducted for isolating the cell line is repeated to obtain new cells. Alternatively, the aforementioned cell mixture is analyzed by FACS in order to reisolate cells that express all of the desired RNA. This is a very useful treatment. This is because cells are again obtained which produce a cell line with the same genetic makeup as the original selected cell line.

  The method described above provides an unlimited supply of cells expressing any combination of multiple protein and RNA sequences that can be applied to almost infinite analytical methods. Nonetheless, overexpressed proteins may be toxic to the cell, but as will be discussed later, this possibility can be addressed quickly.

  The ability to easily re-isolate cells that express all of the desired RNA from cell line clones that contain some cells that no longer express all of the desired RNA. Note that it can be maintained in the absence of a drug or in the presence of a minimal concentration of drug.

  7). Generation of cell lines that markedly overexpress one or more proteins. To highly overexpress each gene, for example, two or more sequences of the same gene are first cloned into a DNA construct that confers drug resistance. The multiple sequences of each gene are each designed to include sequences encoding different epitope tags. After this DNA construct is transfected into cells and then drug selection is performed, multiple molecular beacons are targeted to a single epitope tag, each of which is separately fluorescently labeled. And isolate cells that are positive for their signal using a cell sorter. Since these cells incorporate at least one copy of each distinct epitope sequence tagged gene in their genome, expression of the subject sequence results from increasing copies of the same subject sequence in nature. . This method is used in combination with the use of FACS to select the cells that gave the highest score for each fluorescent molecule signal.

  8). Generation of cell lines that express multiple antisense RNAs. A stably transfected cell line that emits multiple antisense messages is formed as follows. This antisense message targets mRNA or other RNA. Cells that are expressed at various levels for any one of the plurality of transfected antisense sequences are selected. By repeating stable transfection, cells that can produce a stably transfected cell line that expresses an unlimited number of RNA antisense messages are easily selected.

  Of course, cells that express RNA other than antisense RNA can also be prepared by the methods described herein. Such RNA includes, but is not limited to, mRNA, rRNA, other structural RNA, hnRNA or snRNA.

  9. Generation of a functionally knocked out cell line for one or more proteins. The methods of the present invention provide a means of preparing functional knockouts in cultured cells. This eliminates the need to decipher the role of the human protein by examining the knockout characteristics of the human protein in yeast or mice. By generating cells that express almost the same antisense RNA to unique RNA sequences from multiple loci, a cell line in which any protein of interest is functionally knocked out is generated. For example, a plurality of constructs, each of which encodes an antisense RNA of a particular gene, are stably transfected into the cell. In this case, each antisense RNA sequence differs only in that each is tagged with a unique epitope tag nucleotide sequence. Cells expressing all of the variously tagged antisense RNAs are selected. As described above, when the fluorescence is quantified using FACS, it is possible to select a cell that highly expresses any one or more of the antisense sequences by this advantageous benefit.

  Importantly, this method uses different antisense sequences that target the same gene. For example, some of the plurality of antisense RNAs that have been selected for their expression using molecular beacons and FACS are designed to target specific regions of the messenger RNA of the gene, , Another antisense RNA is designed to target another part of the same messenger. In order to generate a cell line that is a functional knockout of a protein of interest, the formation of a cell line that does not express the protein of interest at a detectable level or alternatively at an acceptable low level. As many of the necessary gene sequences encoding similar or different antisense RNAs to the same gene of interest are stably transfected into the cells.

  In addition, multiple proteins are functionally knocked out by repeating the process described above while targeting any number of sequences that are cell lines that are functionally knocked out by antisense. Generate multiple cell lines. For example, in order to examine the function of a complex composed of a plurality of proteins, all or any combination of the plurality of proteins constituting the complex is knocked out.

  10. Generation of a cell line that is a functional knockout of a single selected form of one or more genes or alternatively spliced forms. Different spliced variants of a single gene are often translated into proteins with different functions. Using the methods of the present invention, a cell line is generated that is a functional knockout of only one of a plurality of alternatively spliced forms of one or more proteins. For example, by designing an antisense that targets only the alternative splicing variants of the messenger RNA of a gene that you want to remove from the cell, you can select for the target gene All of the splicing RNA is functionally knocked out.

  11. Generation of a cell line that expresses one or more proteins and on the other hand functionally knocked out the expression of one or more other proteins. Using the methodology described above, multiple RNAs with different roles, ie, some RNAs do not encode proteins or act as antisense RNAs, but are expressed by cells while others are Acts as a messenger RNA, and another produces a stably transfected cell line that expresses multiple such RNAs that are antisense RNAs to the two previously mentioned. It is possible. Such multiple RNAs include, but are not limited to, structural RNAs such as ribosomal RNA, hnRNA, snRNA and other non-mRNAs.

  For example, if there is a group of proteins that are thought to interact with each other, a stably transfected cell line in which one or more of the proteins of interest is functionally knocked out by cellular expression of antisense RNA By generating, it is possible to examine their interaction. Next, it becomes possible to investigate the function of the remaining target protein in the cell more interestingly. That is, the cells can now be further manipulated to overexpress the remaining one or more proteins of interest. Such a series of thoughts can be advanced indefinitely, which makes it possible to express another protein in the cell or to remove its expression.

  Such methods alter the science of mammalian cells and other cells that can be maintained in tissue culture to allow a number of genetic manipulations that were not possible previously. For the first time, it has become possible to generate functional knockouts in human cells and to overexpress proteins in human cells. Again, overexpression of certain proteins or functional knockout of certain proteins can be fatal to the cell. This is a problem that will be addressed below.

  12 Generation of transgenic mice. Depending on the purpose, research on cultured cells may not be sufficient. However, the methodology described above also applies to the manipulation of embryonic stem cells. By complying with the above process, it is possible to obtain embryonic stem cells that express multiple proteins or functionally knock out multiple proteins, or a subset of alternative spliced forms of multiple proteins. Is possible. Next, this embryonic stem cell is used as a formation base of a knockout animal. This process is used to determine if the experiment results in a lethal phenotype prior to performing an attempt to form a knockout animal. For example, if an essential protein is functionally knocked out in embryonic stem cells, the cells cannot survive after isolation by FACS.

  13. To generate an inducible, stably transfected cell line. In cells, overexpression or underexpression of certain proteins or RNAs can be fatal. Nevertheless, it may be critical to examine cells that overexpress harmful proteins or RNA, or cells that have been functionally knocked out of proteins or RNA that cannot survive without it. For this purpose, a stably transfected cell, in which the selected RNA with a detrimental effect on the cell is used for such cells under the control of an inducible promoter. Generate. To isolate such cell lines, transfected and drug-selected cells are first subjected to minimal induction, affecting inducible gene transcription and then recognizing the appropriate RNA. FACS analysis is performed after the cells are transfected with a molecular beacon designed as such. The resulting cells are maintained such that no toxic RNA is induced and only transcribed when needed.

  Induction systems are also advantageous for applications other than the expression of toxic RNA. For example, expression of gene sequences stably transfected into cells is induced at some point in the cell cycle of the synchronized cell line. Alternatively, if one set of one or more stably transfected gene sequence expression products is expected to affect another set of expression products, the first set of gene sequences Are of interest if they are cloned under the control of one inducible promoter and the second set is cloned under the control of a second inducible promoter. By altering induction, the expression product encoded by either set of gene sequences is examined in the absence or presence of the other set of expression products.

  14 Detection of genetic recombination events in living cells and subsequent isolation of non-recombinant or various recombined cells. In parallel with the use of molecular beacons for detection of recombination events leading to stable cell lineages, to detect and isolate cells that have experienced different, specific recombination events from a mixture of various living cells A beacon is used. The same principle can be used, for example, in assays for VDJ recombination, translocation, and viral genome uptake.

  In a cell recombination event, for example, one sequence of genomic DNA is exchanged for another. When a DNA sequence encoding a region where a recombination event has occurred is transcribed into RNA, the presence of such an event will recognize RNA transcribed from the non-recombinant DNA sequence or RNA transcribed from the recombinant sequence. Detected by molecular beacons designed for This assay is also performed by fluorescence microscopy. When it is desired to separate recombined cells and cells that have not undergone recombination, these cells are applied to FACS and sorted. In addition, FACS is used to sort cells based on the presence or absence of multiple recombination events in the cells.

  15. Sorting of cells based on expressed RNA. The use of molecular beacons described earlier in this specification makes it possible to sort cells not only based on the expression of internally localized proteins but also for proteins that cannot form antibodies. For example, starting from a mixed population of various cells, cells expressing the subject internal localization protein are isolated by designing a molecular beacon that recognizes the mRNA that results in that protein. This molecular beacon is transfected into the cell mixture and the cells are sorted appropriately using FACS. Sorting may be performed multiple times.

  In addition, researchers may be interested in isolating cells that are stimulated with, for example, cytokines and expressed to express one or more specific protein mRNAs from cells that are not induced in a similar manner. There is. To this end, the cell mixture is first induced with cytokines and then transfected with a plurality of beacons, each beacon designed to recognize mRNA that yields one of the proteins of interest. . Next, the cell which gave the positive score with respect to object mRNA is isolated using FACS. In another embodiment, for example, cells infected with a virus are also assayed for the presence or absence of expression of a particular gene.

  Using the methodology described hereinabove, multiple RNAs with different roles, ie, certain RNAs do not encode proteins or act as antisense RNAs, but are expressed by cells while Some others act as messenger RNAs, and others can detect cells that are positive for the presence of multiple RNAs that are antisense RNAs to the two mentioned above. It is. Such multiple RNAs include, but are not limited to, structural RNAs such as ribosomal RNA, hnRNA, snRNA and other non-mRNAs.

  16. In vivo detection of nucleic acids in combination with subsequent cell selection. Since the required chemical details are well elucidated, molecular beacons can be modified in various ways to create new possibilities. For example, cells that express certain specific mRNAs whose expression results in malignant transformation of the cells are selectively destroyed, while cells that do not express this mRNA are left to a greater or lesser extent as described below. . Molecular beacons are selected to consist of oligonucleotides designed to recognize and hybridize to a specific sequence of nucleic acid contained in a gene of interest that is transcribed in a portion of the cell mixture. . The oligonucleotide is covalently linked with a protease at one end and one or more protease inhibitors, eg, peptide inhibitors, at the other end. Note that for this oligonucleotide used, it is important to synthesize a beacon with both ends capable of hybridizing to each other, as in the case of molecular beacons. As long as the molecule described above maintains its stem-loop structure, the peptide inhibitor at one end of the molecule is sufficiently close to the protease at the other end to perform the suppression. For ease of discussion, the above molecule will be called a probe protease.

  When a probe protease is transfected into a cell that expresses RNA recognized by the probe protease, the probe protease hybridizes to the target. This activates the protease. This is because in the hybridized state, the protease is no longer in the vicinity of the peptide inhibitor. Cells in which such probe protease targets are present and recognized are destroyed and are therefore decimated.

  Furthermore, the probe protease is useful in various other applications. For example, if there is a cell population containing cells in which some of the cells are infected by a particular virus, a probe protease that targets the particular viral mRNA is introduced into the cell. A cell having such mRNA activates the proteolytic activity of its own probe protease, thereby destroying the cell.

  Using the methodology described hereinabove, multiple RNAs with different roles, ie, certain RNAs do not encode proteins or act as antisense RNAs, but are expressed by cells while Some others act as messenger RNAs, and some others are stably transfected cell lines that express multiple RNAs that are antisense RNAs to the two mentioned above. Can be generated.

  Based on the above description, the following method can be implemented.

A method of generating a cell line expressing at least one preselected protein comprising:
a) transfecting a cell line with at least one DNA construct encoding said at least one preselected protein and at least one drug resistance marker;
b) a step of selecting a cell that has been given resistance by a marker and has become resistant to a drug, wherein the cell has transcribed at least one DNA construct and at least one drug resistance marker. Process;
c) exposing selected cells to a first molecular beacon, wherein the first molecular beacon fluoresces when hybridized to an RNA transcript of said at least one preselected protein;
d) isolating said cells that fluoresce;
e) generating a cell line expressing at least one preselected protein by growing said isolated cells;
Including methods.

  In this method, the isolation of the cells that fluoresce may be performed using fluorescence activated cell sorter technology. The cell line may further be prepared to express the second preselected protein simultaneously or sequentially. That is, the additional step comprises transfecting the cell line with a second DNA construct encoding a second preselected protein and a second drug resistance marker; cells expressing the second marker Exposing the cell to a second molecular beacon that fluoresces when hybridized to an RNA transcript of a second preselected protein; and the at least one mRNA transcript and the second mRNA transcript. Isolating cells that exhibit fluorescence in each object. At present, it is possible to detect up to 3 different fluorophores in the sorting process in the latest technology, and at present, the expression of up to 3 different proteins can be achieved by repeating the above process simultaneously. It is possible to obtain If necessary, cell lines expressing three different proteins may then be used as a starting point for the introduction of more proteins after performing this process.

  The DNA construct used for transfection may encode a single gene and a drug resistance marker, or may encode a further gene with a corresponding marker. Successful transfection of each gene can be achieved by selection with the corresponding agent. As described above, the number of expression messages that can be detected at a time, whether simultaneously introducing new genes or being transferred over time, may be limited by fluorescence technology. It is possible to iterate to add more genes. If up to three fluorophores are detectable, it is possible to introduce three genes at a time.

Related methods are disclosed in which epitope tags that bind to the transfected gene are used as targets for molecular beacons. This method allows the selection of cells that express proteins that may be difficult to identify from the background, such as when molecular beacons detect closely related RNA species. Become. Accordingly, a method for generating a cell line expressing at least one preselected protein comprising:
a) transfecting a cell line with the at least one preselected protein, at least one drug resistance marker, and at least one DNA construct encoding at least one first epitope tag;
b) selecting a cell that is tolerated by the marker and that is resistant to the drug, wherein the cell has transcribed at least one DNA construct and at least one drug resistance marker;
c) exposing the cell to a first molecular beacon, wherein the first molecular beacon fluoresces when hybridized to an RNA transcript of a first epitope tag;
d) isolating the cells that fluoresce; and e) generating a cell line expressing the at least one preselected protein by growing the isolated cells;
Is provided.

  This method is virtually the same as described above, except that the molecular beacon used is designed to recognize the epitope tag rather than the RNA transcript of the desired transprotein. The advantage of this process over the previous one is that it requires only a few molecular beacons, corresponding to the number of different epitope tags, to prepare a large number of different cell lines expressing one or more proteins. It is a point that is not done. Currently, fluorescent sorter technology is limited to three fluorescent molecules, so this method requires only three different beacons. This requirement may be increased if the technology enables the simultaneous detection of a larger number of fluorophores in the future. However, even when up to three transduction proteins can be added at a time, cells that have been successfully transfected with the three genes can be used as starting points for the addition of additional genes. It is.

  Examples of epitope tags that can be used in the present invention against which beacons can be prepared include HA (influenza hemagglutinin protein), myc, his, protein C, VSV-G, FLAG or FLU. However, it is not limited to these. The above and other epitope tags are known to those skilled in the art. As used herein, an epitope tag is a unique nucleic acid sequence that is recognized by a molecular beacon. It is not critical whether this nucleic acid sequence is in reading frame with the encoded protein of the construct. It is not critical. That is, molecular beacons can be recognized in any case. Thus, transcribed RNA having an epitope tag nucleic acid sequence need not be translated just because of the detection of transcripts by molecular beacons.

  Of course, one or more proteins may be introduced into cells using the methods described above. That is, additional execution steps are performed simultaneously or sequentially: with a second DNA construct encoding a second preselected protein, a second drug resistance marker, and a second epitope tag. Transfecting a cell line; selecting a cell that expresses the second marker; exposing the cell to a second molecular beacon that fluoresces when hybridized to an RNA transcript of the second epitope tag; And isolating said cells exhibiting fluorescence of each of said first and second epitope tags. The second epitope tag may or may not match the reading frame and the portion of the DNA sequence encoding the second protein.

  Selection of successfully transfected cells is performed by standard methods. That is, in the presence of a drug, the cells to which the drug resistance marker is supplied are proliferated to counter it. If the selection is made on two different markers, the cells can be selected simultaneously or sequentially. When using the same drug resistance marker for two different transfected constructs, the higher of the dose effects should be considered to select cells transfected with both constructs. Drug concentration is required.

The methods described above for preparing a cell line that expresses one or more proteins can be readily applied to a method for generating a cell line that expresses one or more antisense RNA molecules. The method is
a) transfecting a cell line with at least one preselected antisense RNA molecule and a DNA construct encoding at least one drug resistance marker;
b) selecting a cell to which resistance is imparted by the marker and having resistance to a drug, wherein the cell has transcribed at least one DNA construct and at least one drug resistance marker. ;
c) exposing the cell to a molecular beacon that fluoresces when hybridized to the antisense RNA molecule;
d) isolating the cells that fluoresce; and e) generating a cell line expressing the at least one preselected RNA sequence by growing the isolated cells;
including.

All the details of these methods are as described above. If the beacon cannot detect a particular mRNA, the epitope tag method, i.e.
a) transfecting a cell line with at least one preselected antisense RNA molecule, at least one drug resistance marker, and a DNA construct encoding a first epitope tag nucleotide sequence;
b) selecting for cellular resistance to a drug such that the marker confers resistance, wherein the cell has transcribed at least one DNA construct and at least one drug resistance marker;
c) exposing the selected cell to a first molecular beacon that fluoresces upon hybridizing to the first epitope tag mRNA transcript;
d) isolating the cells that fluoresce; and e) generating a cell line that expresses at least one preselected antisense RNA molecule by growing the isolated cells. The method can be used.

  As already mentioned, the advantage of the epitope tag detection method is that it is not necessary to prepare different beacons for each antisense RNA. Since beacons are only required for a very small number of epitope tags, these tags can be used in a continuous process to introduce multiple antisense molecules, or overexpression of one molecule, into a cell line. Of course, the method may be repeated simultaneously or sequentially to introduce two or more antisense RNA molecules in a given cell line.

  In addition, cells adapted to express a specific protein or proteins may be used as a starting point for creating cells that express multiple proteins and antisense RNA molecules. Of course, cells expressing this plurality of antisense molecules can also be used as a starting point for adding expressed proteins using the methods herein. Co-transfection of proteins and antisense molecules may be performed with corresponding beacons and, if necessary, epitope tags. Various combinations of the foregoing processes are also encompassed herein.

Similarly, a method for isolating a cell expressing at least one protein comprising:
a) providing a cell expected to express the at least one protein;
b) exposing the cell to at least one molecular beacon that fluoresces when hybridized to an mRNA transcript encoding the at least one protein;
c) isolating the cells that fluoresce;
Is provided.

  This method is particularly useful for proteins that are cell surface localized proteins or intracellular proteins. This method does not require the use of a probe on the protein itself, but the use of this probe is more difficult or can affect the cells to the extent that no further experimentation is possible. Using multiple molecular beacons, more than one protein can be identified, up to a number that can be detected simultaneously with the techniques used for isolation.

Of course, the foregoing procedure can be used to quantify the level of at least one RNA transcript expression in a biological sample, the procedure comprising:
a) exposing the biological sample to a first molecular beacon that fluoresces when hybridized to the RNA transcript;
b) quantifying the fluorescence level in the biological sample; and
c) correlating the fluorescence level with said level of at least one mRNA transcript;
including.

  The biological sample can be a cell sample or a tissue sample. These can be fixed, for example, by any number of known cell fixatives that do not interfere with the detection of RNA using formaldehyde, glutaraldehyde, or molecular beacons.

  The detected RNA transcript can be RNA encoding protein, structural RNA, and antisense RNA. Fluorescence can be quantified by fluorescence microscopy or fluorescence activated cell sorter technology. Additional RNA species can be quantified simultaneously using a second molecular beacon that fluoresces when hybridized to the second RNA transcript.

Another application of the method is to generate a cell line that overexpresses at least one protein. This is accomplished by transfecting the cell with two or more sequences encoding the same protein, selecting and identifying expression by epitope tags associated with various copies of the transcript. This process
a) transfecting a cell with at least two DNA sequences, each of said sequences encoding a protein, at least one epitope tag, and at least one drug resistance marker; and the same protein, at least one Having a portion encoding two second epitope tags and at least one second drug resistance marker;
b) selecting cells transfected with said at least two DNA sequences by expression of first and second drug resistance markers;
c) exposing selected cells to first and second molecular beacons, wherein each molecular beacon hybridizes to the nucleotide sequence of the RNA transcript of the first and second epitope tags, respectively. Fluoresce in the process;
d) isolating cells exhibiting the respective fluorescence of the first and second molecular beacons; and
e) generating a cell line that overexpresses the at least one preselected protein by growing the isolated cells;
including.

  As already mentioned, higher levels of drug may be required to select cells transfected with both sequences when the same drug resistance marker is used for each copy of the gene. As in the method described above, the at least two DNA sequences can be in the same construct. The first and second epitope tags may or may not match the protein and the reading frame independently of each other.

The foregoing procedure can be readily applied to generate cells that are overexpressed with at least one antisense RNA molecule. The method is
a) transfecting with at least two DNA sequences, the sequences encoding said antisense RNA molecule, at least one epitope tag, and at least one drug resistance marker; and said antisense RNA Having a moiety encoding a molecule, at least one second epitope tag, and at least one second drug resistance marker;
b) selecting cells transfected with the at least two DNA sequences by expression of the first and second drug resistance markers;
c) exposing the selected cells to first and second molecular beacons, wherein each molecular beacon hybridizes to the nucleotide sequence of the RNA transcript of the first and second epitope tags, respectively. Fluoresce in the process;
d) isolating cells exhibiting the respective fluorescence of the first and second molecular beacons; and
e) generating a cell line that overexpresses the at least one preselected antisense RNA molecule by growing the isolated cells;
including.

  Details are the same as described above.

  Introducing an antisense RNA molecule into a cell is useful for functionally removing one or more proteins from the cell. In accordance with the foregoing method, there is provided a method of generating a cell that is functionally null for the expression of at least one preselected protein, the method comprising, in the cell, for the preselected protein, Providing a plurality of antisense RNAs, each antisense RNA being provided according to the method described above, wherein said plurality of antisense RNAs for said at least one preselected protein is said at least one preselected It binds to virtually all mRNA transcripts of the protein. This preselected protein may be an alternative spliced form of the gene product. In accordance with a similar outline, a method is provided for generating a transgenic animal that is functionally null-expressing variant with respect to at least one preselected protein, the method performing the foregoing steps using embryonic stem cells. Determining the survival rate of the stem cells that functionally null-express the preselected protein, and producing the transgenic animal using the viable embryonic stem cells.

  Similarly, a method is provided for generating a cell line that is functionally null-expressed for at least one protein and over-expressed for at least one other protein, the method being the same as that previously described herein. A method is provided that includes performing on a cell. By performing the methods herein in a similar manner, a method is provided for generating a cell line that expresses a lethal antisense RNA under the control of an inducible promoter, wherein the transfection step comprises Performed in the presence of a minimum amount of inducer.

A method for identifying a genetic recombination event in a living cell, comprising:
a) exposing a cell to a molecular beacon that fluoresces when hybridized with an RNA sequence selected from the group consisting of an RNA sequence transcribed from a recombinant sequence and an RNA sequence transcribed from a non-recombinant sequence;
b) detecting the cell expressing the RNA sequence;
Is provided.

  The present invention also relates to a new form of molecular beacon that differs from the fluorescent properties exhibited by the prior art in binding to target nucleic acids. Such proteolytic activity can be used not only for detection purposes, but also to denature specific protein sequences in cells where the mRNA encoding the protein should be present in the cell. For example, proteases that specifically cleave viral proteins can be activated when viral transcription is activated, as in the case of latent infections.

  Preferably, the proteolytic enzyme inhibitor is a peptide in order to provide reversible inhibition of the enzyme by interaction with the inhibitor, although other molecules including metals and metal chelators are equally useful. . Examples of useful pairs of proteolytic enzymes and proteolytic enzyme inhibitors include aminopeptidase and amastatin, trypsin-like cysteine protease and antipain, aminopeptidase and bestatin, chymotrypsin-like cysteine protease and chymostatin, aminopeptidase and diprotin A or B Carboxypeptidase A and EDTA, elastase-like serine protease and elastinal, and thermolysin or aminopeptidase M and 1,10-phenanthroline, but are not limited to these.

  The invention may be better understood by reference to the following, non-limiting examples provided for illustration of the invention. The following examples are presented in order to more fully illustrate the more preferred embodiments of the invention. These examples are in no way considered to limit the broad scope of the invention.

Example 1
Overall protocol. Starting material: Molecular beacons can be introduced into cells that do not express any RNA from the plasmid, or can be used to detect RNA messages encoded in the plasmid. The method of introducing a beacon into these two types of cells is the same. The protocol described below only requires that the cells to be analyzed are separable from each other and that the separation follows FACS analysis.

  1) As described in more detail herein, beacons can be used in combination with FACS to classify tissue cells based on the expression or lack of expression of specific RNA within the cells. To do this, the cells must first be separated from each other by standard and well-established methods such as homogenization and further chemical treatment. A suitable beacon can then be introduced into the cells according to the following protocol.

  2) Secondly, beacons can be used to select for cells that express a particular RNA encoded by a plasmid transfected into a cell population. To do this, the cell culture must first be transfected with a single or multiple plasmids encoding the desired RNA. A beacon is then created to recognize these RNAs, as described in further detail herein. Transfection of plasmids into cells can be done either using proprietary reagents or using kits obtained from biochemical companies (Qiagen, Promega, Geneporter, Invitrogen, Stratagene, etc.) according to the manufacturer's instructions. Can be achieved by a wide variety of methods. The plasmids should be chosen so that each confers resistance to one antibiotic. After introducing these plasmids into the cells and collecting the cells in a short time (24 hours), the cells are subjected to an appropriate antibiotic so that only those cells that have acquired antibiotic resistance by the plasmid can survive. This generally takes 3 to 4 days, depending on both the cell type and the antibiotic used.

  As a result, a lump of cells remains, all of which are resistant to antibiotics, but only a small portion of them express the RNA of interest. In order to select cells expressing the desired RNA, the following protocol may be followed.

Example 2
Selection of cells with beacons 1) Transfect cells with beacons. A beacon must be designed to recognize the desired RNA either by hybridizing to a sequence endogenous to the RNA or by hybridizing to a tag added to the native RNA sequence. The beacon design is discussed in detail herein.

  Transfection can be performed by a variety of methods, similar to transfection of plasmid cells. The method employed should be selected based on the cell type used. This is because the transfection method that reacts better depends on the cell. Transfection should be performed according to the manufacturer's instructions for the transfection reagent used.

  Depending on the transfection reagent used, the beacon transfection of the cells may be carried out in suspension cells or in cells growing on a solid surface.

  2) After transfection of beacon cells, the cells are subjected to FACS analysis. FACS can be used to classify cells that are positive for any one or more of the multiple beacons used. FACS can also be used to sort cells based on the intensity of the beacon signal, which allows researchers to select cells that express RNA to varying degrees.

Example 3
Generation of cell lines expressing one or more RNAs After FACS selection, cells marked positive can be maintained in a suitable culture medium as described in more detail herein. . This cell becomes a cell line expressing the target RNA.

  Beacon concentration: The concentration of the beacon used depends on several factors. For example, the amount of RNA detected in the cell and the opportunity for contact of this RNA with a beacon must be considered. For example, if there is only a small amount of RNA to be detected, or if the RNA is present in an RNA portion that is not easily contacted by three-dimensional folding of RNA, a large amount of RNA will be detected. In some cases and in comparison to the case where the site recognized by the beacon is fully accessible, a larger amount of beacons must be used. The exact amount of beacons used must be determined empirically for each application.

  This introduces different amounts of beacons into different cell populations, under conditions where the background fluorescence is low and the signal is high (some but not all of the cells Can be achieved by selecting a condition for recording positive).

Claims (12)

A method of generating a cell line that expresses at least two RNAs of interest comprising:
a) introducing DNA encoding the first RNA of interest and DNA encoding the second RNA of interest into viable cells;
b) exposing the viable cell to a first molecular beacon that emits fluorescence when hybridized to the first RNA of interest c) a second molecular beacon that emits fluorescence when hybridized to the second RNA of interest Exposing to;
d) detecting cells exhibiting fluorescence of both the first molecular beacon and the second molecular beacon;
e) isolating the detected fluorescent cells using fluorescence activated cell sorter technology; and f) generating a cell line by growing the isolated cells;
Including methods. A method of generating a cell line that expresses at least one first RNA of interest comprising:
a) supplying a living cell expected to express at least the first RNA of interest;
b) exposing the surviving cells to at least one first molecular beacon that fluoresces when hybridized to the first RNA of interest;
c) detecting a cell exhibiting fluorescence of the first molecular beacon;
d) isolating the cells that fluoresce detected using fluorescence activated cell sorter technology; and
e) generating a cell line by growing the isolated cells
Including
The viable cells are expected to express at least one second RNA of interest, the method comprising:
f) exposing the surviving cells to at least one second molecular beacon that fluoresces when hybridized to the second RNA of interest; and g) detecting cells that exhibit fluorescence of the second molecular beacon;
The method further comprising: The method according to claim 1 or 2 , wherein any one of the step of exposing the viable cell to a molecular beacon and the step of detecting a fluorescent cell are performed simultaneously for the first RNA of interest and the second RNA of interest. The method described. The method according to any one of claims 1 to 3 , wherein the first molecular beacon and the second molecular beacon include separate fluorescent substances. The 1RNA of the end, the 2RNA when present the object, or both said purpose of the 1RNA and said purpose of the 2RNA when present is expressed from the endogenous gene, either Claim 1-4 The method according to claim 1. The first RNA of interest, the second RNA of interest when present, or the first RNA of interest and the second RNA of interest when present are structural RNA, antisense RNA, rRNA, hnRNA, snRNA and mRNA selected from the group consisting of the method according to any one of claims 1-5. 7. The method of claim 6 , wherein the first RNA of interest, the second RNA of interest, if present, or both the first RNA of interest and the second RNA of interest, when present, encode a cell surface protein. 7. The method of claim 6 , wherein the first RNA of interest and the second RNA of interest, if present, are involved in complex formation. The method of any one of claims 1 to 8 , further comprising: quantifying the fluorescence level of the first molecular beacon; and correlating the fluorescence level with the level of the target first RNA. The method described. And further comprising the step of quantitating the fluorescence level of the second molecular beacons, the step of correlating the fluorescence level as the level of the 2RNA of the target, according to any one of claims 1 to 9 the method of. The method according to any one of claims 1 to 10 , wherein the isolated viable cell is an embryonic stem cell. A method for generating a non-human transgenic animal expressing at least one first RNA of interest comprising:
a) supplying an isolated embryonic stem cell according to claim 1 1, comprising the step of producing non-human transgenic animals using and b) said embryonic stem cells.