WO2013103983A1

WO2013103983A1 - High throughput screening of live cells

Info

Publication number: WO2013103983A1
Application number: PCT/US2013/020569
Authority: WO
Inventors: Suzanne SCARLATTA; Peter Brink; Ira Cohen; Guo YUANJIAN; Michael Rosen
Original assignee: The Research Foundation Of State University Of New York
Priority date: 2012-01-06
Filing date: 2013-01-07
Publication date: 2013-07-11
Also published as: WO2013103983A9

Abstract

The invention features a high throughput method for removing a population of cells from a heterogeneous population of living cells using one or more molecular beacons and fluorescence activated cell sorting. In another embodiment, the invention features a high throughput method for screening and isolating a population of living cells.

Description

HIGH THROUGHPUT SCREENING OF LIVE CELLS

FEDERAL FUNDING

[0001] This invention was made with government support under grant numbers

GM053132, GM071558, and HL094410 awarded by the National Institute of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0002] The invention features a high throughput method for removing a population of cells from a heterogeneous population of living cells. In another embodiment, the invention features a high throughput method for screening and isolating a population of living cells.

BACKGROUND OF THE INVENTION

[0003] Many diseases and defects due to injury result in loss of cells thereby reducing organ or tissue function. Examples include the loss of dopaminergic neurons in Parkinson's disease and damage to cardiac tissue from a myocardial infarction. Cell-based therapies offer the potential to treat such conditions by repairing tissues and organs through the introduction of cells with the ultimate goal to regenerate and restore normal function. Cell-based therapies involve obtaining a heterogeneous population of cells and introduction of one or more cell types from this population into a subject. Such heterogeneous cell populations can contain cells that are not desired for that purpose and should be removed from the population.

[0004] Molecular beacons are dual-labeled oligonucleotide probes typically with a fluorescent dye and a fiuorescent quencher attached at the 5' and 3' ends of the oligonucleotide. In addition, molecular beacons have at either end (i.e., the 5' and 3' ends) complementary sequence such that they form a stem-loop structure when not bound to a target sequence. The loop region of the molecular beacon contains sequence that is complementary to a target (generally an m NA). When in the stem- loop formation, the dye and quencher are held in close proximity and fluorescence from the dye is quenched. However, when the molecular beacon hybridizes to its target, the dye and quencher are physically separated allowing a fiuorescent signal to be emitted upon excitation.

[0005] Fluorescence-activated cell sorting (FACS) is a type of flow cytometry used to separate cells on the basis of each cell's specific light scattering and fiuorescent characteristics. SUMMARY OF THE INVENTION

[0006] In a first aspect, the invention provides a high-throughput method for obtaining a population of living cells comprising: (a) providing a heterogeneous population of cells; (b) introducing a molecular beacon that targets an RNA expressed by a non-desired cell type; (c) detecting a change in fluorescence from the molecular beacon hybridizing its target RNA in the non-desired cell type; and (d) substantially removing the non-desired cell type from the population.

[0007] In another aspect, the invention provides a population of cells with a non-desired cell type removed by the steps comprising: (a) providing a heterogeneous population of cells; (b) introducing a molecular beacon that targets an RNA expressed by a non-desired cell type; (c) detecting a change in fluorescence from the molecular beacon hybridizing its target RNA in the non-desired cell type; and (d) substantially removing the non-desired cell type from the population.

[0008] In one embodiment, the invention provides a kit for performing the method for removing the non-desired cell type from the heterogeneous population of cells that includes a transfection agent; the molecular beacon that targets an RNA expressed by a non-desired cell type; and instructions for performing the method.

[0009] In one embodiment, the heterogeneous population of cells is selected from the group consisting of pluripotent stem cells; induced pluripotent stem cells; multipotent stem cells; myocytes; sperm; cells transfected with siRNA; and cells transfected with an expression construct.

[0010] In one embodiment, the molecular beacon comprises an RNA polynucleotide. In another embodiment, the molecular beacon comprises a fluorophore and a quencher. In a further embodiment the molecular beacon, in addition to a fluorophore and a quencher contains an additional fluorophore for tracking the cells that contain the molecular beacon. In one embodiment the molecular beacon comprises a FRET pair. In a further embodiment, the FRET pair comprises a donor molecule and an acceptor fluorophore that will fluoresce by FRET from the donor to the acceptor when the beacon is not hybridized to a target RNA. In another embodiment, the FRET pair comprises two fluorophores. In one embodiment, the FRET pair comprises Dabcyl as the FRET donor. [0011] In one embodiment, the transfection method used to introduce the molecular beacon into the heterogeneous population of cells is selected from the group consisting of calcium phosphate precipitation; Lipofectamine; FuGene 6; electroporation; and streptolysin O. In a further embodiment the transfection method is Lipofectamine. In another embodiment the transfection method is electroporation.

[0012] In one embodiment, the non-desired cell type is removed from the population of cells using fluorescence activated cell sorting (FACS).

[0013] In another aspect, the invention provides a high throughput method for screening and selecting a population of living cells expressing a gene of interest comprising: (a) providing a heterogeneous population of cells; (b) introducing a molecular beacon that targets an RNA expressed by the cells of interest; (c) detecting a change in fluorescence from the molecular beacon hybridizing to its target RNA in the cells of interest; and (d) isolating the cells of interest.

[0014] In another aspect, the invention provides a high throughput method for screening and selecting a population of living cells expressing a gene of interest comprising: (a) incorporating into the gene of interest a target sequence that is not expressed in the population of living cells; (b) introducing the gene of interest containing the target sequence from step (a) into the population of living cells; (c) introducing into the population of cells a molecular beacon comprising sequence that is complementary to the target sequence in step (a); (d) detecting a change in fluorescence from the molecular beacon hybridizing its target RNA in the population of living cells; and (e) isolating the cells expressing the gene of interest. In a further

embodiment, the target sequence that is incorporated into the gene of interest is not present in the genome of the population of living cells. In one embodiment, the molecular beacon comprises a probe sequence that is complementary to the target sequence and is selected from the group consisting of: CGTACGCGTCGGAGAT (SEQ ID NO: 1); CGGTACGATCTGGA (SEQ ID NO: 2); AATCTCCGACGCGTACG (SEQ ID NO: 3); CCGTACTCCGACGTACG (SEQ ID NO: 4); TAGACCCGCCCCGTTGG (SEQ ID NO: 5); and

GCCAACGCGCAGGCATA (SEQ ID NO: 6).

[0015] In one embodiment, the invention provides kit for high throughput screening and selecting a population of living cells expressing a gene of interest comprising: a transfection agent; the RNA beacon that targets an RNA expressed by the cells of interest; and instructions for performing the method. [0016] In one embodiment, the molecular beacon comprises an RNA polynucleotide. In another embodiment, the molecular beacon comprises a fluorophore and a quencher. In a further embodiment the molecular beacon, in addition to a fluorophore and a quencher contains an additional fluorophore for tracking the cells that contain the molecular beacon. In one embodiment the molecular beacon comprises a FRET pair. In a further embodiment, the FRET pair comprises a donor molecule and an acceptor fluorophore that will fluoresce by FRET when the beacon is not hybridized to a target RNA. In another embodiment, the FRET pair comprises two fiuorophores. In one embodiment, the FRET pair comprises Dabcyl as the FRET donor.

[0017] In one embodiment, the transfection method used to introduce the molecular beacon into the population of cells is selected from the group consisting of calcium phosphate precipitation; Lipofectamine; FuGene 6; electroporation and streptolysin O. In a further embodiment the transfection method is Lipofectamine. In another embodiment the transfection method is electroporation.

[0018] In one embodiment, the population of living cells is selected and screened using fluorescence activated cell sorting (FACS).

DESCRIPTION OF THE FIGURES

[0019] Fig. 1. Comparison of methods for introducing a nanog beacon into human mesenchymal stem cells. Images were obtained 3 hours after transfection.

[0020] Fig. 2. Human Aortic Adventitial Fibroblast (AOAF) cells transfected with nanog-red for 48hr cells (middle) were injected with 50 μΜ nanog beacon2 (right) mixed with 5 μΜ DAPI (left).

[0021] Fig. 3. FACS selection of cells expressing the nanog gene using a nanog beacon. Data collected for cells containing the nanog beacon. Two negative controls (AOAF cells and NHDF cells, the left and middle panels, respectively) do not express nanog and green

fluorescence is not observed. The right panel shows data for Human Mesenchymal Stem Cells (HMSC) which express nanog. The results are summarized in Table 1.

[0022] Fig. 4. Fluorescence microscopy images of HMSC transfected with a nanog beacon after three hours (top panels) and 24 hours (bottom panels).

[0023] Fig. 5. Detection of alternate genes by microscopy. Top panels: canine ventricular cardiomyocytes were micro-injected with the Oct4 beacon and DAPI as a tracer (left). No fluorescence from the Oct4 beacon could be detected in the cells (middle). The right panel is the corresponding phase contrast image of the cells. Bottom panels: Oct4 expression was detected in cell lines that express the gene, i.e., AOAF (left panel) and HMSC (right panel).

[0024] Fig. 6. Improved selection using multiple beacons. To achieve higher selectivity, beacons to multiple genes were transfected. AOAF cells were untransfected (left) or transfected (right) with an Oct4 beacon containing a Cy5/BHQ2a probe pair (fluoresces red) and a nanog beacon containing a 6-FAM/BHQla probe pair (fluoresces green). Using FACS, a 93.99% selection for Oct4 and 82.02% for nanog was obtained. Thus, very high selectivity can be obtained by using two beacons to different RNA targets simultaneously.

[0025] Fig. 7. Alternate detection methods. Wild type HEK293 (Human Embryonic Kidney) cells (top panels) and HEK293 cells that stably express nanog (bottom panels) were microinjected with a nanog beacon having a Cy5/Dabcyl fluorescence pair and Alexa as a marker. The left panels display the Alexa signal. The middle panels display the signal from Cy5, and the right panels are the merged image.

[0026] Fig. 8. Alternate detection methods. Wild type HEK293 cells (left panels) and HEK293 cells that stably express nanog (right panels) were transfected with a nanog beacon having a Cy5/Dabcyl fluorescence pair using calcium phosphate co-precipitation. 1.48 μg of the beacon was transfected into the cells at 70 to 80% confluence in 100 mm dishes. Three hours post transfection the cells were imaged for Cy5 fluorescence (top panels) or by the amount of FRET by exciting at the Dabcyl peak and monitoring Cy5 intensity (bottom panels).

[0027] Fig. 9. Sensitivity of beacons to gene expression. HEK293 cells were serum starved for 24 hours and transfected with beacon 2 (top panels). HEK293 cells grown in complete media (i.e., not serum starved) and transfected with beacon 2 (bottom panels).

[0028] Fig. 10. A nanog beacon containing a tracking dye (beacon 5 from Example 8) was transfected into HEK293 cells and the fluorescent signal tracked by fluorescent microscopy at 3, 24 and 48 hours. The first (i.e., left) and second columns display respectively the bright field images of control HEK 293 cells and HEK 293 cells transfected with beacon 5. The third and fourth columns display the fluorescent images for the Alexa 488 (fluoresces green upon beacon hybridization to target) and TF5 (the tracking dye, which fluoresces red).

[0029] Fig. 11. FACS separation of HMSCs transfected with a nanog beacon without a tracking dye (a), and with the tracking dye (b, c). No red fluorescence was seen for cells transfected with the 2 color beacon seen in (a). DETAILED DESCRIPTION

[0030] The invention features a high-throughput method of screening for, and removing, a cell-type from a heterogeneous population of cells. A heterogeneous population of cells is a collection of cells containing more than one cell-type where the cell-type is distinguished from the population based on the expression of one or more genes. The heterogeneous population of cells may be obtained directly from a subject, for example from peripheral blood, bone marrow, adipose tissue, or other tissue containing a population of cells of interest. Such populations will typically contain a certain amount of cells of a type that are not desirable for the given purpose and must be removed from the population.

[0031] In other embodiments, the heterogeneous population of cells is obtained by transfecting a cell population with an expression construct (e.g., a plasmid containing a gene of interest from which the gene may be expressed in the target cells). Such a population will often contain a proportion of cells that did not receive the construct or express the gene of interest from the construct at low levels. It is often desirable to remove the cells that do not express the gene of interest, or express it at low levels, from the heterogeneous population.

[0032] In one embodiment, the invention provides using a molecular beacon to identify an undesired cell-type in a heterogeneous population of cells. Molecular beacons are oligonucleotide (DNA or RNA) probes designed to hybridize to specific sequences (typically mR A). Molecular beacons typically have fluorophore and quencher molecules on opposing ends of the oligonucleotide. In the absence of a complementary RNA target, the beacon forms a stem-loop hairpin structure causing the fluorophore and quencher to be in close proximity, which allows the transfer of excitation energy from the fluorophore to the quencher, preventing fluorescence emission. Hybridization of the beacon with the target RNA opens the hairpin, which physically separates the fluorophore from the quencher, and allows the fluorophore to fluoresce upon excitation.

[0033] The stem of the molecular beacon is formed by two complementary short-arm sequences. The base pairing of the stem region is designed to have lower interaction energy than the loop-target pairing, but must be strong enough to remain paired until hybridization with the target. The stem is designed so that it will keep the fluorophore and quencher (or other FRET pair) in close proximity without significant shifting, and will generally have a high GC content. In one embodiment the GC content of the stem is greater than about 70%. In one embodiment, the GC content is greater than about 75%. In one embodiment, the GC content is greater than about 80%. In one embodiment each stem arm is about 3 to about 10 nucleotides in length. In one embodiment, each stem arm is about 4 to about 8 nucleotides. In one

embodiment, each stem arm is about 4 to about 6 nucleotides.

[0034] In one embodiment, the molecular beacon is designed such that one arm of the stem of the molecular beacon contains part of the sequence complementary to the target such that the stem arm participates in either stem formation by hybridizing to the complementary stem arm, or target hybridization.

[0035] In one embodiment, the beacon comprises a quencher at one end of the molecule and a fluorophore on the other end. In another embodiment, the beacon comprises a quencher at one end of the polynucleotide and two fluorophores at the other end - one fluorophore (the donor) that absorbs strongly in the wavelength range of the light source, and a second fluorophore (the acceptor) that emits at the desired emission wavelength due to Forster resonance energy transfer (FRET) from the first fluorophore to the second fluorophore.

[0036] In one embodiment, the beacon comprises a FRET pair, which can be, for example, two fluorophores, one at the 5' end and the other at the 3' end. The FRET pair is selected such that when the beacon is in the stem-loop configuration (not hybridized to target), one fluorophore (the donor) absorbs strongly in the wavelength range of the light source, and a second fluorophore (the acceptor) emits due to FRET from the first fluorophore to the second fluorophore. Upon hybridization to the target, the FRET pair is physically separated preventing FRET to the second fluorophore and the first fluorophore will emit allowing detection of the target. The donor molecule of the FRET pair can also be a non- fluorescent molecule such as, for example, Dabcyl.

[0037] In one embodiment, two molecular beacons are used such that the two beacons hybridize to adjacent regions on the same R A target. In this scenario, one RNA beacon, the donor beacon, has a donor dye/quencher pair and the other, the acceptor beacon, has an acceptor dye/quencher pair. The donor beacon and acceptor beacon are designed to hybridize to adjacent regions of the target such that the donor fluorophore is adjacent to the acceptor fluorophore. When the two beacons hybridize to the target, the donor fluorophore, which absorbs in the wavelength range of the light source, is adjacent to the acceptor fluorophore, which will then emit due to FRET from the donor fluorophore to the acceptor fluorophore. Thus, emission from the acceptor fluorophore due to FRET will only occur upon hybridization of both probes to the target reducing false positive signals.

[0038] In one embodiment, the molecular beacon includes, in addition to a

fluorophore/quencher pair or a fluorophore FRET pair, an additional fluorophore. In one embodiment, the additional fluorophore is located between the probe (loop) and the stem sequences of the beacon. Incorporation into the beacon of an additional fluorophore that fluoresces at a different wavelength allows for selection of the cells that contain the beacon. Such a tracking fluorophore provides, for example, a means to select cells based on the presence of the beacon thereby distinguishing between cells that do not express the target, but received the beacon from cells that did not take up the beacon. Tracking fluorophores should allow for detection of the beacon without interference from, or with, the dye/quencher pair. For example, the tracking fluorophore should emit at a wavelength that is distinguishable from the wavelength of the dye in the dye/quencher pair. This approach is valuable in situations where false negatives create a significant barrier to implementation.

[0039] For example, a beacon containing a tracking fluorophore can be used to identify undifferentiated pluripotent stem cells from those that have been differentiated to a specific lineage. In this approach, a beacon containing a tracking fluorophore and targeting a pluripotent gene that has been down-regulated in the differentiated cell type is used to identify and select out the pluripotent stem cells, thus removing an oncogenic risk. The tracking fluorescent tag integrated in the beacon provides the identifying signal that a cell was loaded with the beacon. From the population of cells that contain the beacon, the pluripotent cells can be identified by the change in fluorescence due to the beacon hybridizing to its target. FACS, for example, can be used to select the cells containing the beacon and remove the cells in which the beacon has hybridized to its target.

[0040] In one embodiment, the length of complimentary (probe) sequence in the loop region of the beacon is about 10 to about 30 nucleotides in length. In one embodiment the complementary sequence is about 15 to about 25 nucleotides. Probe sequence (i.e., the portion of the beacon complementary to the target) is selected to ensure specificity for the target, and to have good target accessibility. Target accessibility can be determined by analyzing the secondary structure of the target RNA for regions with open, single-stranded sequence. Target site accessibility can be determined using software including, for example, Mfold

(http://mfold.rna.albany.edu/?q=mfold) (Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406-3415, 2003). [0041] Probe sequence should also be analyzed for specificity in order to reduce or prevent hybridization with, e.g., a non-specific RNA using, for example, Basic Local Alignment Search Tool (BLAST) analysis (National Center for Biotechnology Information).

[0042] In addition, the sequence of the loop region should generally have

complementarity to the target RNA and not to other regions of the beacon. The structure of the beacon can be analyzed using, e.g., the software found on http://kinefold.curie.fr/ and

http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi.

[0043] An additional consideration in the design of the molecular beacon is the choice of nucleotide(s) adjacent to the fluorophore(s). For example, it has been reported that guanosine nucleotides adjacent to the fluorophore should be avoided when designing oligonucleotides that contain a fluorescent reporter molecule (Nazarenko I, et al. (2002) Effect of Primary and

Secondary Structure of Oligodeoxyribonucleotides on the Fluorescent Properties of Conjugated dyes. Nuc Acids Res, 30: 2089-2195).

[0044] Fluorophores that are useful as fluorescent markers on molecular beacons are known in the art and include dyes that can be excited with one of the filters contained on a FACS instrument, for example: 6-FAM (6-carboxyfluorescein), HEX, TET, TYE 563, TYE 665, JOE, Oregon Green, Cy3, Cy3.5, Cy5, Cy5.5, ROX, TAMRA, Texas Red, CR6G, TF (Tide Fluor) 1, TF2, TF3, TF4, TF5, TF6, TF7, and TF8.

[0045] Quenchers that are useful on molecular beacons are known in the art and include, for example: Iowa Black FQ; Black Hole Quencher 1 (BHQ 1), Black Hole Quencher 2 (BHQ 2), Black Hole Quencher 3 (BHQ 3), Iowa Black FQ; Iowa Black RQ-Sp, TQ (Tide Quencher) l; TQ2; and TQ3.

[0046] Fluorophore and quencher combinations include, for example, BHQ 1 /6-FAM; BHQ 1/HEX; BHQ 1/JOE; BHQ 1/Oregon Green; BHQ 1/TET; BHQ 2/Cy3; BHQ 2/Cy3.5; BHQ 2/Cy5; BHQ 2/Cy5.5; BHQ 2/HEX; BHQ 2/ROX; BHQ 2/TAMRA; BHQ 2/Texas Red; BHQ 3/Cy5; BHQ 3/Cy5.5; TQ1/TF1; TQ2/6-FAM; TQ2/TF2; TQ3/Cy3; and TQ3/TF3.

[0047] FRET donor and acceptor pairs where the acceptor fluoresces due to FRET from the donor to the acceptor upon excitation of the donor are known in the art and include, for example, Dabcyl/6-FAM; Dabcyl/Oregon Green; Dabcyl/CR6G; Dabcyl/Cy3; Dabcyl/Cy3.5; Dabcyl/Cy5; Dabcyl/Cy5.5; Dabcyl/HEX; Dabcyl/ROX; Dabcyl/TAMRA; Dabcyl/TET; and Dabcyl/Texas Red. [0048] Dual fluorophores useful for FRET are known in the art and include, for example, Tamra/FAX; Tamra/HEX; and Tamra/TET.

[0049] In one embodiment, the fluorescent marker is a quantum dot. Quenchers of quantum dots are known in the art and include, for example, Iowa Black and 1.4nm nanogold (Cady et al. Molecular and Cellular Probes (2007) 21 : 116-124; Kim et al. Sensors Actuators B (2004) 102:315-9).

[0050] In addition to a DNA or RNA backbone, the molecular beacons used in the present invention may comprise non-standard nucleic acids including 2-OMe-modified RNA, peptide nucleic acids (PNAs), and lock nucleic acids (LNAs). LNAs are a conformationally restricted nucleic acid analogue, in which the ribose ring is locked into a rigid C3'-endo or northern-type conformation by a 2'-0, 4'-C methylene bridge.

[0051] In one embodiment, the cell population is trans fected with two or more molecular beacons. In a further embodiment, the cell population is transfected with two molecular beacons, which target different RNAs expressed by the non-desired cell type. In one

embodiment, the molecular beacon directed to the target gene is co-transfected with a second beacon to a commonly expressed gene, for example a housekeeping gene as a control for transfection.

[0052] In one embodiment of the invention, the probe sequence in the beacon is designed to be complementary to a unique target sequence. Preferably, the unique target sequence is not present in the genome of the target cell. Even if present in the genome of the target cell, the unique target sequence is preferably not present in an expressed RNA in the target cell at detectable levels. The DNA sequence encoding the unique target sequence can be incorporated into the gene of interest by a variety of methods so that a beacon containing probe sequence complementary to the unique target can be used to detect expression of the gene of interest. Thus, a beacon containing the probe for a unique target sequence can be used to detect any RNA target engineered to contain the unique target sequence. The unique target sequence may be incorporated into the genome of the cells or may be incorporated into extrachromosomal DNA. The gene of interest containing the sequence encoding the unique target may be expressed stably or transiently in the target cell. In one embodiment, the unique target sequence is incorporated into a non-coding sequence of the gene of interest, for example, the 5' untranslated region (5' UTR) or the 3' untranslated region (3' UTR). In one embodiment, the unique target sequence is incorporated into a coding sequence of the gene of interest. The unique target sequence may be incorporated into the gene by, for example, restriction endonuclease cleavage followed by ligation, by recombination, or other techniques known in the art. In one embodiment, the target sequence is incorporated into the gene of interest by site- specific mutagenesis of the gene sequence. The use of a unique target sequence allows a single beacon with probe complementary to the unique target to be used in the detection of multiple targets, each of which having the unique target sequence incorporated. In addition, the target sequence can be designed to be particularly effective for the cell selection method, for example, FACS. Cells carrying the target sequence that is transcribed to mR A can then by selected using the beacon technology described above. The unique target/probe sequence is designed using the above criteria for designing beacons. However, the use of a unique target/probe allows additional flexibility in the design of the probe sequence because the target sequence is not limited to the sequence in the target of interest.

[0053] Molecular beacons may be purchased from a variety of commercial sources including, for example, Eurofins MWG Operon (www.operon.com).

[0054] The delivery of the molecular beacon(s) to the cell population can be

accomplished by any high-throughput and efficient method for the delivery of such molecules known in the art including, for example, chemical-based transfection methods, liposomal-based transfection methods and non-chemical transfection methods. In one embodiment the molecular beacon is delivered to greater than about 70%, greater than about 80%>, greater than about 90%>, and greater than about 95% of the cells in the heterogeneous population. In one embodiment, the molecular beacon is delivered to the heterogeneous population of cells by a transfection method selected from the group consisting of calcium phosphate precipitation; Lipofectamine; FuGene 6; and electroporation. In another embodiment, the transfection method is a toxin-based membrane permeabilization, including for example, streptolysin O. In one embodiment, the molecular beacon is delivered to the heterogeneous population of cells by electroporation. In another embodiment the molecular beacon is delivered to the heterogeneous population of cells by Lipofectamine. In one embodiment, the transfection method results in low cell death in the heterogeneous population of cells. In one embodiment, the transfection method results in less than about 30%, less than about 20%, less than about 10%, and less than about 5% cell death in the heterogeneous population of cells.

[0055] In one embodiment, the methods of the invention can be used for lineage selection from pluripotent or multipotent stem cells or from other differentiated lineages. Stem cells express a number of messages that are down-regulated. Further, if multiple differentiated lineages are possible and only one is desired, particular R As up-regulated in the alternative lineage selections, but not in the desired lineage, can be chosen (e.g., Kvl .5 is not expressed in ventricular myocytes, but is expressed in atrial myocytes). If ventricular myocytes are desired, atrial myocytes can be removed by using a beacon for the mR A generated from transcribing the Kvl .5 gene.

[0056] In one embodiment, the invention provides a method for the selection of gametes for the absence of an abnormal gene by creating a beacon that targets the R A encoding the abnormal protein and removing those sperm/eggs that fluoresce. For example, sperm from a carrier of a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be screened using the methods of the invention and those spermatozoa carrying the mutated gene can be removed from the population prior to fertilization. Thus, in one embodiment, sperm are transfected with a beacon that targets the mRNA encoding CFTR containing the Δ508 mutation and the sperm that do not fluoresce are retained for use in fertilization.

[0057] In one embodiment, the invention provides a method to enhance selection of induced pluripotent stem (IPS) cells that may be pluripotent. In the creation of an induced pluripotent stem cell, a terminally differentiated source cell is manipulated to dedifferentiate into pluripotency. This dedifferentiation requires the loss of expression of proteins key to the terminally differentiated cell type, for example, vimentin, desmin, FSPl and others referred to in Kalluri, R. and Zeisberg, M. (2006) Nature Reviews Cancer 6:392-401. Beacons that target the RNA encoding these proteins can be used to identify and remove terminally differentiated cells.

[0058] In one embodiment, the invention provides a method for the removal of cancer cells from a heterogeneous population of cells obtained from a subject's blood or marrow. Beacons are targeted to proteins present in the cancer cells and not present in the normal cells in the blood or marrow. Cells that do not fluoresce may then be selected. This method may be used to identify and remove, for example, prostate cancer cells (J. Urology (1997) 158:861-864 and lung cancer cells {Clinical Cancer Res. (2010) 16:3976-3987). In another embodiment, molecular beacons can be used to identify circulating cancer cells as a predictor of prognosis.

[0059] In one embodiment, the heterogeneous population of cells is autologous stem cells including, for example, mesenchymal stem cells derived from, for example, bone marrow, blood, or adipose tissue. In one embodiment, the invention provides a method for removing differentiated cells from a heterogeneous cell population leaving stem cells for future use. Molecular beacons are designed to target RNA that encodes proteins present in the differentiated cells but not present in the desired stem cell. Cells that do not fluoresce may then be selected.

[0060] In one embodiment, the heterogeneous population of cells is a cell population expressing or transfected with an siRNA. Thus, in one embodiment, the invention provides a method selecting cells that have been silenced by siRNA. The beacon is designed to target the same RNA that the siRNA targets. Cells that do not fluoresce may then be selected.

[0061] In one embodiment, the heterogeneous population of cells comprises a tissue- specific differentiated autologous cell population harvested from an individual for reintroduction into a second site in order to, for example, repair damaged tissue. The use of autologous cells is advantageous because such cells will not evoke an immune response requiring

immunosuppressive drug therapy. Examples of tissue-specific differentiated autologous cells include chondrocytes for cartilage repair, myocytes for myocardial repair, keratinocytes or dermal fibroblasts for burn and wound repair, retinal pigment epithelial cells for the treatment of age-related macular degeneration, and Schwann cells to restore myelin in CNS lesion.

[0062] In one embodiment, the heterogeneous cell population comprises allogeneic stem cells for use in a bone marrow transplant to treat hematologic disorders and cancer.

[0063] The present invention provides a high-throughput method for removing an undesired cell-type from the cell population prior to its use in patients. Such undesired cell- types include, for example, malignant or dysplastic cells, pluripotent cells, senescent cells, dedifferentiated cells, and stem cells.

[0064] In one embodiment, the methods of the invention are used to identify and remove pluripotent cells from a population of differentiated cells to avoid the formation of teratomas after the cell population is introduced into a subject. Pluripotent cells can be removed from a population of differentiated cells by introducing into the population molecular beacons that target RNA from genes that are hallmarks of pluripotency (e.g., OCT4 and NANOG). Those cells that fluoresce or demonstrate a change in fluorescence due to the beacon hybridizing to its target are discarded and the remaining cells are selected for further use.

[0065] The invention provides a method for substantially removing the non-desired cell type from the heterogeneous population of cells. In one embodiment, greater than about 90% of the non-desired cells are removed from the heterogeneous population of cells. In another embodiment, greater than about 95%, greater than about 97%, and greater than about 99% of the non-desired cells are removed from the heterogeneous population of cells. [0066] In one embodiment of the invention, fluorescence-activated cell sorting (FACS) is used to identify and remove an undesired cell type from a heterogeneous population of cells. In another embodiment, two or more undesired cell types are removed from the heterogeneous population using FACS. FACS is a type of flow cytometry used to separate cells on the basis of each cell's specific light scattering and fluorescent characteristics is commercially available. In another embodiment, FACS is used to identify and select a desired cell type from a

heterogeneous population of cells. In one embodiment, the heterogeneous population of cells is screened by FACS in less than about 5 hours of being transfected with the molecular beacon(s). In another embodiment, the heterogeneous population of cells is screened by FACS in less than about 4 hours; less than about 3 hours; or less than about 2 hours of being transfected with the molecular beacon(s).

[0067] It is to be understood and expected that variations in the principles of the invention disclosed herein may be made by one skilled in the art and it is intended that such modifications are to be included within the scope of the present invention.

[0068] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application. The following examples further illustrate the invention, but should not be construed to limit the scope of the invention in any way.

EXAMPLES

[0069] EXAMPLE 1: Beacon Delivery into Cells.

[0070] Several methods for introducing RNA beacons into cultured and primary cells were studied to determine an optimum delivery method with high efficiency and low cell death. A beacon that targets nucleotides 217- 237 of the nanog mRNA (5' 6-FAM- GGTGCGACAAGCTGGATCCACACTGCACC-BHQ1 3') (SEQ ID NO: 7) was designed and used for these experiments. Beacons were purchased from Eurofins MWG Operon

(www.operon.com). Transfection methods included calcium phosphate precipitation;

Lipofectamine; FuGene 6; electroporation; and microinjection. Cell lines tested included the following:

HEK 293: Human Embryonic Kidney cell ATCC # CRL-1573; Macrophage cells: mouse ATCC# TIB-67; CHO: Chinese Hamster Ovary ATCC# CCL-61; FRT: Rat Thyroid ATCC# CRL-1468;

AOAF: Human Aortic Adventitial Fibroblast Lonza CC -7014;

NHDF: Normal Human adult Dermal Fibroblast Lonza CC-2511

HMSC: Human Mesenchymal Stem Cells Lonza PT-2501; and

CMSC: Canine Mesenchymal Stem Cells.

[0071] Cell Culture and Isolation: Human Embryonic Kidney cells (HEK293), SK-N- SH cells, mouse macrophages, and Chinese Hamster Ovary (CHO) cells were obtained from ATCC (CRL-1573, TIB-67, CCL-61, CRL-146). HEK293, macrophage cells, and SK-NSH cells were maintained in Dulbecco's modified Eagle's medium (DMEM, GIBCO 11965) supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin sulfate. Fischer rat thyroid (FRT) cells were a gift from Dr. Deborah Brown (Stony Brook University). FRT and CHO cells were cultured in Ham's F12 medium (Sigma F6636) with 10% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin sulfate. Human Aortic Adventitial Fibroblast (AOAF), Normal Human adult Dermal Fibroblast (NHDF), and Human Mesenchymal Stem cells (HMSC) were grown in medium purchased from Lonza Cell Culture Services (CC -7014, CC-2511, and PT-2501). All cells were incubated at 37°C with 5% C0₂.

[0072] Canine Mesenchymal Stem cells (cMSC) were isolated by Ficoll-Paque Plus density gradient centrifugation from aspirated bone marrow. Primary cultures of cMSC were maintained at 37°C in 5% C0₂, 95% air with an initial medium for 48 hours. Medium was changed every 3-4 days. Cell colonies with spindle-like morphology were transferred 7days after initial plating. After confluence, cells were harvested with 0.25% trypsin-EDTA, and replated. Isolated cells were characterized at passages 2-4 by flow cytometric analysis of specific surface antigens with fluorescein isothiocyanate-(FITC) conjugated rat anti-canine CD44, FITC- conjugated rat anti-canine CD4S unconjugated rat anti-canine CD90, and phycoerythrin-(PE) conjugated mouse anti-canine CD34. A large majority of the cells were CD44 (99.61%) and CD90 (93%>) positive and CD34 or CD45 were 98%> negative; suggesting a significant majority were MSC. To further validate the cMSC properties of the isolated cells, we subjected subsets of the cells to osteogenic, apdipogenic and chondrogenic differentiation protocols. For adipogenic and osteogenic differentiation, the cells were plated in 6-well or 12-well plates. Adipogenic and osteogenic induction was initiated using kits available from Lonza. For adipogenesis, three to five cycles of the following media changes were performed: 2-3 days of exposure to adipogenic induction medium followed by 2-3 days of exposure to maintenance medium. Osteogenic induction was carried by feeding the cells with osteogenic induction medium every 3-4 days for 2-3 weeks. Chondrogenic induction was performed by pelleting 2.5xl0⁵ cells in chondrogenic induction medium containing TGF-P3. Complete media changes were performed every 2-3 days for 3-4 weeks. At the end of the induction protocols, the cells were rinsed with PBS and fixed with 10% formalin. Adipogenesis was assayed using Oil Red O staining. Osteogenesis was assayed by staining for calcium deposition using Alizarin Red staining. Chondrogenic pellets were embedded in cryogenic cutting medium, sectioned for histology, and glycosaminoglycans were stained by Safranin O.

[0073] Cardiomyocytes were isolated from canine ventricle using a method described previously (Valiunas et al. J. Physiol. (2009) 587:5211-5226; Potapova et al. Circ. Res. (2004) 94:952-959). The primary cardiac myocytes were plated on 10 μg/ml laminin-coated 35-mm glass bottom MatTek (Ashland, MA) dishes in KB buffer (K-reversal Tyrode buffer: 35 mmol/L of HEPES, 140 mmol/L of KCI, 8 mmol/L of KHC0₃, 2 mmol/L of MgCl₂, and 0.4 mmol/L of KH₂PO₄, pH 7.5). The buffer was gradually changed to M199 medium supplemented with 15%> fetal bovine serum, 1%> streptomycin, and 0.5%> gentamicin and incubated at 37°C in 5%> C0₂.

[0074] Lipofectamine: Beacon trans fections were carried out using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Briefly, ΙΟΟηΜ beacon, 400 nM lipofectamine agent and serum free medium mixture were added to 2xl0⁶ suspended cells. The cells were then plated onto 100mm dish and incubated for 3 hours at 37°C in 5%> C0₂ before cell sorting.

[0075] Calcium Phosphate Precipitation: Cells that were about 50%> confluent cells in 100 mm dish were fed with 7 ml fresh medium before transfection. Four to ten μg of the beacon were incubated with 120 μΜ CaCl₂ and HEPES buffer containing NaCl, NaHP0₄ and HEPES pH 7.1 for 10 min on ice. After mixing, the mixture was immediately added to the surface of the cultured cells, swirled gently and returned to the incubator at 37°C with 5%> C0₂ until needed.

[0076] F11GENE6: Transfections were performed according to the manufacturer's instruction (Roche Molecular Biochemicals). Briefly, cells were grown to 50-80% conf uency in 35 mm culture dishes. FuGENE 6 transfection agent (μΐ) to beacon ^g) were added in a 3: 1 ratio in serum-free medium. After 3 to 8 hours, serum was added to the medium or the medium was changed to one containing serum.

[0077] Electroporation: Beacons were introduced into cells by electroporation using a protocol adapted from Lonza Cell Culture. Briefly, the medium was aspirated, and cells were harvested by adding 1 ml of trypsin/EDTA. Cells were then spun down for 5 min at 1500 x g and resuspended with 2-4 μg beacon and mixed. For electroporation of HMSC cells, beacons were mixed with 100 μΐ HMSC nucleofector obtained from Lonza. The mixture was transferred into an electroporation cuvette using a 200 μΐ pipette being careful to avoid generating air bubbles. Electroporation was carried out in a BioRad Xcell electroporation instrument using program "C- 17" or "U-23."

[0078] Microinjection: Cells were cultured in glass bottom MatTek wells for 24 hours. Before microinjection, the media was changed to phenol- free Leibovitz-15. For primary cardiomyocytes, the media was first changed to phenol-free Leibovitz-15 with 14 mM EGTA. 50 μΜ beacon was injected with trace amounts of DAPI to identify microinjected cells. The control cells were injected with the dye tracer alone. Injection was carried out using self-pulled needles and an InjectMan NI2 with a FemtoJet pump from Eppendorf mounted on an Axiovert 200 M (Carl Zeiss) equipped with a long working distance 40x phase 2 objective. Solutions were microinjected into the cytoplasm. Typical settings were as follows: the injection pressure was Pi = 40 hPa and the compensation pressure Pc = 20 hPa for all cells except cardiomyocytes. For cardiomyocytes, the injection pressure was Pi = 90 hPa and the compensation pressure Pc = 45 hPa. The injection time was t = 0.7 s. Typically, about 10-25 cells were injected within a 10 to 20 min period. The microinjected cells were examined under the phase microscope to select viable cells. Although microinjection cannot be used for high throughput screening, it is useful in identifying the cells that contained beacon because a tracing dye can be included in the injection solution.

[0079] Cell Sorting: 3-5xl0⁶ cells/ml in serum- free medium were sorted by flow cytometry using the FACS instruments as Stony Brook University as described at their website (http://www.stonybrookmedicalcenter.org/pathology/research/flowcyto).

[0080] Immunostaining: Immunostaining of fixed cells to visualize proteins was performed as previously described (Guo et al, (2011) Biophysical J. 100:1599-1607).

[0081] Both lipofectamine and electroporation had very high (over 90%) efficiency but electroporation was more lethal {see Fig. 1). Subsequent studies were carried out using lipofectamine (except where noted). [0082] EXAMPLE 2: FACS selection of cells expressing nanog

[0083] The fluorophore/quencher and FRET pair were chosen to maximize the change in intensity or other fluorescence properties, and to allow detection on the particular instrument. The change in fluorescence was determined using a nanog beacon (5' 6-FAM- GGTGCGACAAGCTGGATCCACACTGCACC-BHQ1 3') treated with RNAse. The change in fluorescence was measured using a commercial fluorometer (ISS PCI). The largest change in intensity occurred using a fluorescein-based probe (5' 6'-FAM) paired with a BlackHole quencher (BHQ 1 3'). In aqueous solution, this beacon will give a 29 fold increase in intensity with RNAse treatment. After 1 year storage, the change diminishes to 22.

[0084] EXAMPLE 3: Detection of alternate genes.

[0085] Beacons to Oct4 (5' 6-FAM-GAGACGGAGGGGGCGAGAAGGGTCTC-BHQl 3') (SEQ ID NO: 8) and Oct2 were individually microinjected into canine ventricular cardiomyocytes with DAPA as a tracer and fluorescence was determined by microscopy. No fluorescence from the Oct4 beacon was detected in these cells, but was detected in cells that express Oct4 (AOAF and HMSC) (Fig. 5). Similar studies were carried out using a beacon for Sox2 and Kfl4.

[0086] EXAMPLE 4: Beacon degradation.

[0087] Measurements were taken within 3 hours following transfection. The fluorescent signal gradually declined and dispersed from the nucleus to the cytosol after 12 hours in all cell types. No signal was seen after 24 hours in a fast growing cell line (SK-H-SH) and after 48 hours in slow growing cells (AOAF) (Fig. 2). Fig. 4 shows the change in signal by fluorescence microscopy of HMSC transfected with a nanog beacon after three hours (top panels) and 24 hours (bottom panels).

[0088] In addition, a nanog beacon containing a tracking dye (beacon 5 from Example 8) was transfected into HEK293 cells. Fig. 10 shows the change in signal at 3, 24 and 48 hours by fluorescence microscopy of from the TF5 tracking dye (right column) and from the beacon hybridizing target (third column).

[0089] EXAMPLE 5: Improvement of selection using multiple beacons.

[0090] Multiple beacons targeting different RNAs were used to improve selectivity. The AOAF cells were transfected with both an Oct4 beacon containing a Cy5/BHQ2a probe pair, which is red in color and a nanog beacon containing the 6-FAM/BHQla probe pair, which is green. By FACS, 93.99% of cells were expressing Oct4 and 82.02% were expressing nanog. Thus, a two color process can be used when high selectivity is required.

[0091] EXAMPLE 6: Alternate detection methods.

[0092] Wild-type (wt) HEK293 cells and a sister line that was stably transfected with nanog were used to study FRET pairs for the detection of nanog expression. The same nanog beacon described above, but having a Cy5/Dabcyl fluorescence pair was microinjected into the two cell lines as described above (Fig. 7). Dabcyl molecules are not fluorescent, but when they absorb light they can transfer energy to Cy5 in a process called fluorescence resonance energy transfer (FRET). Thus, the beacon was monitored in two ways: (1) by Cy5 fluorescence, to observe cells that contain the beacon, and (2) by FRET to determine if the beacon is complexing with its target RNA. When the beacon binds to its target, a reduction in Cy5 fluorescence was observed due to a loss of FRET as Dabcyl separates from Cy5. In addition, the beacon was transfected into the wt HEK293 cells the HEK cell stably expressing nanog using calcium phosphate precipitation (Fig. 8). Beacon fluorescence was much higher in the cells that over- express nanog.

[0093] EXAMPLE 7: Sensitivity of the methods using molecular beacons.

[0094] HEK cells, which have endogenous nanog expression, were serum starved in order to arrest the cells in stationary phase where nanog expression is greatly diminished.

Control HEK cells (grown in medium with serum) and HEL cells grown in serum- free medium for 24 hours were transfected with a nanog beacon (beacon 2) and the fluorescence was measured using fluorescent microscopy (Fig. 9). [0095] EXAMPLE 8: Simultaneous assessment of beacon incorporation and response.

[0096] Design of a beacon with an internal tracking probe. As a more sensitive alternative to the use of a FRET pair for negative selection, we designed a nanog beacon containing a fluorescent tracker. The tracking dye, TF5, was placed between the loop and stem of a nanog beacon as follows: 5' A488-GGTGC-GACAAGCTGGATCCACACT-TF5-GCACC- BHQ1 3' (SEQ ID NO: 9) where TF5 refers to the probe, Tide Fluor™ 5 phosphoramidite [TF5 CEP] (AAT Bioquest, Inc.) and the underlined sequence denotes the stem. By simultaneously sorting the cells by fluorescence from the red tracking dye (TF5) and the green fluorescence from a positive beacon response (hybridization to target), cells expressing nanog RNA were identified and removed from those that were no longer expressing nanog RNA. This type of beacon reduces the importance of high transfection efficiency. Results for this beacon are provided in Fig. 11 and Table 2.

Table 2.

[0097] EXAMPLE 9: Design of unique targets and corresponding beacons

[0098] Unique target/beacon design: Freely available software (NIH BLAST) was used to search the human genome for sequences that do not match any human transcript mRNA. Additionally, it may be advantageous for the 'unique' target sequence to be unique not just to the transcriptome, but to the entire genome as well so that the corresponding probe does not cause the beacon to partition into the nucleus, hybridize with it the nonspecific site in the genome and create a false positive signal. Alternatively, where nuclear partitioning is a concern, it can be prevented by blocking nuclear pores upon transfection. Additional criteria in the design of the unique target polynucleotide included: (i) that the target sequence be at least 17 nucleotides long; and (ii) the Tm (temperature required to melt or separate the hybridized target and probe sequence) be about 65°C. Additional criteria in the design of the beacon included (i) the base pairing energy of the stem should be strong enough to insure the beacon remains closed under cellular conditions to avoid opening of the beacon and false positives; (ii) the loop region should have a G/C content of about 50% so that the energy of hybridization of the loop region to its target is strong enough to unzip the stem base pairs; and (iii) the beacon sequence should not have significant secondary structure, which may occur when portions of the loop structure hybridize with complementary sequences in the loop or stem since this would create an added stabilization energy of the beacon and reduce the net amount of hybridization energy given off when it binds to its target mRNA.

[0099] The unique probe sequence is sandwiched between 5 to 6 base complementary sequences (with G/C to A/T ratio of about 4) that constitute the beacon stem. The fluorophore 6FAM and its corresponded quencher BHQ1 are placed on the 5' and 3' ends of the beacon. Additionally, the tracking dye TF5 (Tide Fluor™ 5 phosphoramidite [TF5 CEP] (AAT Bioquest, Inc.) is inserted at between the probe and stem.

[00100] Using the above method, the following beacon was designed: 5 6FAM- CCGTAC-CGTACGCGTCGGAGAT-TF5-GTACGG-BHQ1 3 (SEQ ID NO: 10), where the underlined text denotes the stem sequences and TF5 is the tracking fluorophore. Additional probe sequences, complementary to unique target sequences have been designed and are listed below:

[00101] In addition, the probe sequence CGGTACGATCTGGA was identified.

[00102] Introducing the unique target sequence into target mRNA: There are many strategies to introduce the unique target sequence into the target mRNA while avoiding influence on the production of the transcript, or other function, due to the presence of the unique target sequence. Using nanog as a test case, a the target sequence is incorporated at the 5' UTR in a region that does not contain regulatory regions such as a CAAT or CCAAT box or a TATA box, which signal the binding of RNA transcription factors. The unique sequence is introduced through the mutation of several nucleotides in the -165 region of nanog using the Quikchange mutagenesis kit (Agilent, Inc).

[00103] In another case, a unique target sequence was placed after the stop codon in hHCN2 while avoiding an siRNA binding site

(www.broadinstitute.org/rnai/public/trans/candidates). Specifically, the 2766 to 2799 base region of hHCN2 was mutated to contain the unique target sequence described above. Because probe to this target appears to bind genomic DNA as well as the target, the more specific R-l sequence is being incorporated into the nanog and the hHCN2 plasmids, which will then be transfected into a series of positive and negative control cells, as well as test cells, and the mRNA expression will be detected.

Claims

[00104] Claims:

1. A method for obtaining a population of living cells comprising: a) providing a heterogeneous population of cells; b) introducing a molecular beacon that targets an RNA expressed by a non-desired cell type; c) detecting a change in fluorescence from the molecular beacon hybridizing its target RNA in the non-desired cell type; and d) substantially removing the non-desired cell type from the population.

2. The method of claim 1 wherein the heterogeneous population of cells is selected from the group consisting of pluripotent stem cells; induced pluripotent stem cells; multipotent stem cells; myocytes; sperm; cells transfected with siRNA; and cells transfected with an expression construct.

3. The method of claim 1, wherein the molecular beacon comprises an RNA

polynucleotide.

4. The method of claim 1, wherein the molecular beacon comprises a fluorophore and a quencher.

5. The method of claim 4, wherein the molecular beacon further comprises an additional fluorophore for identifying the cells transfected with the molecular beacon.

6. The method of claim 1, wherein the molecular beacon comprises a FRET pair.

7. The method of claim 6, wherein the FRET pair comprises a donor molecule and an

acceptor fluorophore that will fluoresce by FRET from the donor to the acceptor when the beacon is not hybridized to a target RNA.

8. The method of claim 7, wherein the FRET pair comprises two fluorophores

9. The method of claim 7, wherein the FRET pair comprises Dabcyl as the donor molecule.

10. The method of claim 1, wherein the molecular beacon is introduced into the

heterogeneous population of cells by a transfection method selected from the group consisting of calcium phosphate precipitation; Lipofectamine; FuGene 6; electroporation and streptolysin O.

11. The method of claim 9, wherein the transfection method is Lipofectamine.

12. The method of claim 9, wherein the transfection method is electroporation.

13. The method of claim 1, wherein the non-desired cell type is removed from the population using FACS.

14. A kit for performing the method of claim 1 comprising a transfection agent; the RNA beacon; and instructions for performing the method.

15. A population of cells with a non-desired cell type removed by the steps comprising: a) providing a heterogeneous population of cells; b) introducing a molecular beacon that targets an RNA expressed by a non-desired cell type; c) detecting a change in fluorescence from the molecular beacon hybridizing its target RNA in the non-desired cell type; and d) substantially removing the non-desired cell type from the population

16. The population of cells of claim 15, wherein the molecular beacon comprises an RNA polynucleotide.

17. The population of cells of claim 15, wherein the molecular beacon comprises a

fluorophore and a quencher.

18. The method of claim 17, wherein the molecular beacon further comprises an additional fluorophore for identifying the cells transfected with the molecular beacon.

19. The population of cells of claim 15, wherein the molecular beacon comprises a FRET pair.

20. The population of cells of claim 19, wherein the FRET pair comprises a donor molecule and an acceptor fluorophore that will fluoresce by FRET from the donor to the acceptor when the beacon is not hybridized to a target RNA.

21. The population of cells of claim 20, wherein the FRET pair comprises two fluorophores.

22. The population of cells of claim 20, wherein the FRET pair comprises Dabcyl as the donor molecule.

23. The population of cells of claim 15, wherein the molecular beacon is introduced into the heterogeneous population of cells by a transfection method selected from the group consisting of calcium phosphate precipitation; Lipofectamine; FuGene 6; electroporation and streptolysin O.

24. The population of cells of claim 23, wherein the transfection method is Lipofectamine.

25. The population of cells of claim 23, wherein the transfection method is electroporation.

26. The population of cells of claim 15, wherein the non-desired cell type is removed from the population using FACS.

27. A high throughput method for screening and selecting a population of living cells

expressing a gene of interest comprising: a) providing a heterogeneous population of cells; b) introducing an molecular beacon complementary to an R A expressed by the cells of interest; c) detecting a change in fluorescence from the molecular beacon hybridizing its target RNA in the cells of interest; and d) isolating the cells of interest.

28. A kit for the method of claim 27 comprising a transfection agent; the molecular beacon; and instructions for performing the method.

29. The method of claim 27, wherein the molecular beacon comprises an RNA

polynucleotide.

30. The method of claim 27, wherein the molecular beacon comprises a fluorophore and a quencher.

31. The method of claim 30, wherein the molecular beacon further comprises an additional fluorophore for identifying the cells transfected with the molecular beacon.

32. The method of claim 27, wherein the molecular beacon comprises a FRET pair.

33. The method of claim 32, wherein the FRET pair comprises a donor molecule and an acceptor fluorophore that will fluoresce by FRET from the donor to the acceptor when the beacon is not hybridized to a target RNA.

34. The method of claim 33, wherein the FRET pair comprises two fluorophores.

35. The method of claim 33, wherein the FRET pair comprises Dabcyl as the donor

molecule.

36. The method of claim 27, wherein the molecular beacon is introduced into the heterogeneous population of cells by a transfection method selected from the group consisting of calcium phosphate precipitation; Lipofectamine; FuGene 6; electroporation and streptolysin O.

37. The method of claim 36, wherein the transfection method is Lipofectamine.

38. The method of claim 36, wherein the transfection method is electroporation.

39. The method of claim 27, wherein the cells of interest are isolated from the population of cell using FACS.

40. A high throughput method for screening and selecting a population of living cells

expressing a gene of interest comprising: a) incorporating into the gene of interest a target sequence that is not expressed in the population of living cells; b) introducing the gene of interest containing the target sequence from step (a) into a population of living cells; c) introducing into the population of cells a molecular beacon comprising sequence that is complementary to the target sequence in step a); d) detecting a change in fluorescence from the molecular beacon hybridizing its target RNA in the population of living cells; and e) isolating the cells expressing the gene of interest.

41. A kit for the method of claim 40 comprising a transfection agent; the molecular beacon; and instructions for performing the method.

42. The method of claim 40, wherein the molecular beacon comprises a probe sequence that is complementary to the target sequence wherein the probe sequence is selected from the group consisting of: CGTACGCGTCGGAGAT; CGGTACGATCTGGA;

AATCTCCGACGCGTACG; CCGTACTCCGACGTACG;

TAGACCCGCCCCGTTGG; and GCCAACGCGCAGGCATA.

43. The method of claim 40, wherein the molecular beacon comprises an RNA

polynucleotide.

44. The method of claim 40, wherein the molecular beacon comprises a fluorophore and a quencher.

45. The method of claim 44, wherein the molecular beacon further comprises an additional fluorophore for identifying the cells transfected with the molecular beacon.

46. The method of claim 40, wherein the molecular beacon comprises a FRET pair.

47. The method of claim 46, wherein the FRET pair comprises a donor molecule and an acceptor fluorophore that will fluoresce by FRET from the donor to the acceptor when the beacon is not hybridized to a target RNA.

48. The method of claim 46, wherein the FRET pair comprises two fluorophores.

49. The method of claim 46, wherein the FRET pair comprises Dabcyl as the donor

molecule.

50. The method of claim 40, wherein the molecular beacon is introduced into the population of cells by a transfection method selected from the group consisting of calcium phosphate precipitation; Lipofectamine; FuGene 6; electroporation and streptolysin O.

51. The method of claim 50, wherein the transfection method is Lipofectamine.

52. The method of claim 50, wherein the transfection method is electroporation.

53. The method of claim 40, wherein the cells expressing the gene from the expression construct are isolated from the population of cell using FACS.