Multiplex receptor-ligand interaction screening

Abstract

Nucleic acids, vectors, cells, viral particles, and methods that can be used to determine specific receptor activation are provided. [Solution] An embodiment of the present disclosure relates to a population of cells, each cell comprising (i) a heterologous receptor gene and (ii) an inducible reporter comprising a receptor-responsive element, wherein expression of the reporter depends on activation of the activity of a receptor encoded by the receptor gene, the reporter comprising a barcode comprising an index region unique to the heterologous receptor gene, and the cells express different heterologous receptors, with each single cell expressing one or more copies of one particular heterologous receptor and one or more copies of one particular reporter.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 528,833, filed July 5, 2017, which is incorporated by reference herein in its entirety.

[0002] This invention was made with Government support under 1555952 awarded by the National Science Foundation. The Government has certain rights in this invention.

[0003] 1. Technical field to which the invention pertains The present disclosure relates to the fields of medicine and drug discovery. [Background technology]

[0004] 2. Description of Related Technology G protein-coupled receptors (GPCRs) are one of the most important classes of drug targets, with approximately one-third of currently marketed drugs having their effect through GPCRs. G protein-coupled receptors (GPCRs) account for 50-60% of current drug targets. This family of membrane proteins plays a key role in current drug discovery. Classically, a large number of drugs based on GPCRs have been developed for various indications, such as cardiovascular, metabolic, neurodegenerative, psychiatric, and oncological diseases.

[0005] Furthermore, there are currently few, if any, methods that allow for effective and efficient large-scale screening of thousands, or even tens of thousands, of receptors in a single assay platform. There is a great need in the art for improved receptor and ligand interaction screening. Summary of the Invention

[0006] The present disclosure relates to nucleic acids, vectors, cells, viral particles, and methods that can be used to determine specific receptor activation. Thus, certain embodiments relate to nucleic acids that include (i) a heterologous receptor gene; and (ii) an inducible reporter that includes a receptor-responsive element; the expression of the reporter depends on the activation of the activity of the receptor encoded by the receptor gene, and the reporter includes a barcode that includes an index region that is uniquely identifiable for the heterologous receptor gene. Further aspects relate to vectors that include the nucleic acids of the present disclosure. Further aspects relate to vectors that include a heterologous receptor gene. The term "heterologous" in the context of polynucleotides refers to a gene or polynucleotide that has been introduced into a cell by a gene transfer method known in the art or described herein; the progeny of such cells may also be said to include a heterologous nucleic acid sequence if the exogenously introduced sequence remains in the progeny cell. The cell may already contain an endogenous gene that is identical to the heterologous receptor gene, or the cell may be missing any endogenous genes that are related to or identical to the heterologous gene. The term "heterologous cell" or "host cell" refers to a cell that intentionally contains a heterologous nucleic acid sequence.

[0007] The term "encode," as it applies to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide when, in its natural state, or when manipulated by methods well known to those of skill in the art, it can be transcribed and / or translated to produce mRNA for the polypeptide and / or fragments thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0008] In some embodiments, the vector further comprises an inducible reporter, expression of which is dependent on activation of the activity of a receptor encoded by the receptor gene, and the reporter comprises a barcode that includes an index region unique to the heterologous receptor gene. A further aspect relates to a vector comprising an inducible reporter that comprises a barcode.

[0009] Further embodiments relate to a population of cells, wherein each cell comprises (i) a heterologous receptor gene; (ii) an inducible reporter comprising a receptor responsive element; wherein expression of the reporter is dependent on activation of the activity of a receptor encoded by the receptor gene, and wherein the reporter comprises a barcode comprising an index region that is unique to the heterologous receptor gene; and wherein the cells express different heterologous receptors, and wherein each single cell expresses one or more copies of one particular heterologous receptor and one or more copies of one particular reporter. For example, a population of cells can include at least a first cell having a first receptor gene and a first inducible reporter, a second cell having a second receptor gene and a second inducible reporter, a third cell having a third receptor gene and a fourth inducible reporter, a fourth cell having a fourth receptor gene and a fourth inducible reporter, ...as well as a 1000th cell having a 1000th receptor gene and a 1000th inducible reporter, etc. A population of cells can include cells, each of which contains only one receptor and an associated inducible reporter that includes a barcode that contains an index region that can be used to identify heterologous receptors that are activated in the same cell. The population of cells may be at least or at most 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3500, 3600, 0, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10A population of cells (or any range of inducible therein) may contain a number of different receptor genes and their associated inducible reporters. Furthermore, in some embodiments, the inducible reporter produces an expressed nucleic acid that uniquely identifies the heterologous receptor gene expressed in the cell. The different receptor genes may be receptors belonging to a class of receptors, such as olfactory receptors, hormone receptors, adrenoceptors, drug-responsive receptors, and the like. Thus, a population of cells may contain cells that express only one receptor gene (although there may be multiple copies of the same gene expressed) and only one associated inducible reporter (although there may be multiple copies of the inducible reporter). In some embodiments, the cells each express one variant of the same receptor gene. It is intended that a single screen may contain the number of cells / receptors discussed herein. This is on a different scale than other screens, which may include using the screens sequentially, to have the magnitude of some embodiments provided by the present disclosure.

[0010] A further embodiment relates to a cell comprising (i) a heterologous receptor gene; and (ii) an inducible reporter comprising a receptor responsive element; where expression of the reporter is dependent on activation of the activity of a receptor encoded by the receptor gene, and the reporter comprises a barcode that includes an index region that is unique to the heterologous receptor gene. In some embodiments, expression of the heterologous gene is "sustainable," meaning that expression of the heterologous gene remains at a level within about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the expression level of the cells at or before the later cells for 1, 2, 3, 4, 5, 6, 7 days and / or 1, 2, 3, 4, 5 weeks and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months (or any range inducible therein) to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more passages (or any range inducible therein). In certain embodiments, the cells exhibit sustainable expression of the receptor being tested. In some embodiments, the cells express the receptor at levels within 2-fold of the level initially measured after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more passages (or any inducible range therein).

[0011] In some embodiments, the receptor gene encodes a G protein-coupled receptor (GPCR). In some embodiments, the reporter is induced upon signaling by an activated receptor protein. In some embodiments, activation of the receptor protein comprises binding of the receptor to a ligand. In some embodiments, the receptor gene further comprises one or more additional polynucleotides encoding auxiliary polypeptides. In some embodiments, the auxiliary polypeptide comprises a selectable or screenable protein. In some embodiments, the auxiliary polypeptide comprises a protein or peptide tag. In some embodiments, the auxiliary polypeptide comprises a transcription factor. In some embodiments, the auxiliary polypeptide comprises one or more trafficking tags. In some embodiments, the auxiliary polypeptide comprises two trafficking stages. In some embodiments, the auxiliary polypeptide comprises at least, at most, or exactly 1, 2, 3, 4, or 5 (or any inducible range therein) trafficking tags. In some embodiments, the trafficking tag comprises a Lucy and / or Rho trafficking tag. In some embodiments, the trafficking tag comprises a signal peptide. In some embodiments, the signal peptide is a cleavable peptide that is cleaved in vivo by an endogenous protein. Exemplary auxiliary polypeptides are described herein. In some embodiments, the receptor gene encodes a fusion protein comprising the receptor gene and the auxiliary polypeptide. In some embodiments, the fusion protein comprises a protease site between the receptor gene and the auxiliary polypeptide.

[0012] In some embodiments, the reporter is induced by signal transduction upon activation of the GPCR. In some embodiments, the receptor responsive element comprises one or more of a cAMP response element (CRE), a nuclear factor of activated T cells responsive element (NFAT-RE), a serum response element (SRE), and a serum response factor response element (SRF-RE). In some embodiments, the receptor responsive element comprises a DNA element bound by an auxiliary polypeptide transcription factor. In some embodiments, the auxiliary polypeptide transcription factor comprises a reverse tetracycline-controlled transactivator (rtTA) and the receptor responsive element comprises a tetracycline responsive element (TRE).

[0013] In some embodiments, the receptor responsive element comprises a CRE. In some embodiments, the CRE comprises at least 5 repeats of tgacgtca (SEQ ID NO: 1). In some embodiments, the CRE comprises at least, at most, or exactly 3, 4, 5, 6, 7, 8, 9, or 10 repeats of SEQ ID NO: 1 (or any range derivable therein). In some embodiments, the CRE comprises TIFF0007671316000001.tif26160 or SEQ ID NO: 2 or a fragment thereof, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250 , 275, 300, 301, 302, 304, 305, 306, 307, 308, 309, 310, 312, 313, 314, or 315 contiguous nucleic acid fragments (or any range derivable therein).

[0014] In some embodiments, the GPCR is an olfactory receptor (OR). ORs are known in the art and are further described herein. In some embodiments, the receptor gene comprises a nuclear hormone receptor gene. In some embodiments, the receptor gene comprises a receptor tyrosine kinase gene. In some embodiments, the receptor comprises an adrenergic receptor. In some embodiments, the adrenergic receptor comprises a beta-2 adrenergic receptor. In some embodiments, the receptor comprises a receptor described herein. In some embodiments, the receptor is a transmembrane receptor. In some embodiments, the receptor is an intracellular receptor.

[0015] In some embodiments, the vector is a viral vector. In further embodiments, the vector is one known in the art and / or described herein. In some embodiments, the vector comprises a lentiviral vector.

[0016] In some embodiments, the receptor gene comprises a constitutive promoter. Exemplary constitutive promoters include CMV, RSV, SV40, and the like. In some embodiments, the receptor gene comprises a conditional promoter. As used herein, the term "conditional promoter" refers to a promoter that can be induced by the addition of an inducer and / or can be switched from an "off" state to an "on" state or from an "on" state to an "off" state by a change in condition, such as a change in temperature or the addition of a molecule, such as an activator, coactivator, or ligand. Examples of conditional promoters include "Tet-on" or "Tet-off" systems that can be used to induce expression of a protein in a cell.

[0017] In some embodiments, the reporter comprises an expressed RNA. In some embodiments, the gene comprises a barcode of at least 10 nucleic acids. The barcode can be 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more, or at least or at most 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleic acids in length (or any range derivable therein). In some embodiments, the reporter comprises or further comprises an open reading frame (ORF) that comprises a 3' untranslated region (UTR). In some embodiments, the barcode is located in the 3'UTR of a gene, reporter, or other nucleic acid segment, such as a gene encoding a fluorescent protein. In some embodiments, the ORF encodes a selectable or screenable protein. In some embodiments, the ORF encodes a fluorescent protein. In some embodiments, the ORF encodes a luciferase protein.

[0018] In some embodiments, the reporter gene is flanked by an insulator sequence at the 5' and / or 3' end. In some embodiments, the reporter is flanked by an insulator sequence at the 5' and / or 3' end. In some embodiments, the reporter gene is flanked only at the 5' end or only at the 3' end. In some embodiments, the reporter gene is not flanked by an insulator at the 3' end. In some embodiments, the reporter gene is not flanked by an insulator at the 5' end. In some embodiments, the reporter gene is flanked only at the 5' end or only at the 3' end. In some embodiments, the reporter gene is not flanked by an insulator at the 3' end. In some embodiments, the reporter gene is not flanked by an insulator at the 5' end.

[0019] In some embodiments, the insulator comprises a cHS4 insulator. TIFF0007671316000002.tif34160 or SEQ ID NO: 3 or a fragment thereof, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 205, 210, 215 , 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, or 231 contiguous nucleic acid fragments (or any range derivable therein).

[0020] In some embodiments, the insulator is a CTCF repressor found in the Drosophila gypsi retrotransposon or a CTCF insulator regulated by the gypsi insulator.

[0021] In some embodiments, the vector comprises a second, third, fourth, or fifth barcode. In some embodiments, at least one of the second, third, or fourth barcodes comprises an index region that is specific to one or more of the assay conditions or locations on the microplate. The assay conditions may include the addition of a specific ligand, the addition of a specific concentration of a ligand, or a variant of a ligand, or a concentration or variant of a metabolite, small molecule, polypeptide, inhibitor, repressor, or nucleic acid. In some embodiments, an additional barcode can be used to identify where the cells were placed on the microplate, so that the assay conditions at that particular location can be identified and connected to the barcode.

[0022] A further aspect of the present disclosure relates to a viral particle comprising one or more vectors or nucleic acids of the present disclosure. Yet another aspect of the present disclosure relates to a cell comprising the nucleic acid, vector or viral particle of the present disclosure. Further embodiments relate to a cell comprising multiple copies of the vector of the present disclosure. In some embodiments, the cell comprises at least three copies of the vector. In some embodiments, the cell comprises at least four copies of the vector. In some embodiments, the cell comprises at least, at most, or exactly 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or 20 copies (or any range inducible therein) of the vector.

[0023] In some embodiments, the one or more cells of the present disclosure further comprise one or more genes encoding one or more accessory proteins. In some embodiments, the one or more accessory proteins comprise one or more of Gα subunits, Ric-8B, RTP1L, RTP2, RTP3, RTP4, CHMR3, and RTP1S. In some embodiments, the one or more accessory proteins comprise an arrestin protein. In some embodiments, the one or more accessory proteins comprise a Gi or Gq protein. In some embodiments, the arrestin protein is fused to a protease. In some embodiments, the one or more accessory proteins comprise one or more of a chaperone protein, a G protein, and a guanine nucleotide exchange factor. In some embodiments, the accessory protein is integrated into the genome of the cell. As shown in the examples of the present application, stable integration of accessory factors provides surprisingly good results compared to transient expression. In some embodiments, the accessory protein is expressed transiently. In some embodiments, the cells comprise stable integration of one or more exogenous nucleotides encoding one or more accessory factor genes, including RTP1S, RTP2, Gα subunit (NCBI gene ID: 2774), or Ric-8b (NCBI gene ID 237422).

[0024] In some embodiments, the cell further comprises a receptor protein expressed from a heterologous receptor gene. In some embodiments, the receptor protein is localized intracellularly. In some embodiments, the cell lacks an endogenous gene encoding a protein that is at least 80% identical to the heterologous receptor gene. In some embodiments, the cell lacks an endogenous gene encoding a protein that is at least, at most, or exactly 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical (or any inducible range therein) to the heterologous receptor gene. In some embodiments, the receptor gene is integrated into the genome of the cell. In some embodiments, an inducible reporter is integrated into the genome of the cell. In some embodiments, the receptor gene and / or the inducible reporter are transiently expressed.

[0025] In some embodiments, the receptor gene and the inducible reporter are genetically linked. In some embodiments, the receptor gene and the inducible reporter are not genetically linked. In some embodiments, the receptor gene and the inducible reporter are inserted into the genome of the cell and are within or separated by at least 10, 50, 100, 200, 500, 1000, 2000, 3000, 5000, or 10000 base pairs (bp) of each other (or any inducible range therein). In further embodiments, the receptor gene and the inducible reporter are on separate genetic elements (e.g., separate chromosomes and / or extrachromosomal molecules).

[0026] In some embodiments, the integrated receptor gene and / or inducible reporter is integrated into the cell genome by targeted integration. In some embodiments, the integrated receptor gene and / or inducible reporter is randomly integrated into the genome. In some embodiments, the random integration comprises transposition of the receptor gene and / or inducible reporter. In some embodiments, the cell comprises at least two copies of the receptor gene and / or inducible reporter. In other methods of random integration, DNA can be introduced into the cell and randomly integrated by recombination. In some embodiments, the integration is into the H11 safe harbor locus. In some embodiments, the integration is into the H11 safe harbor locus by targeted integration.

[0027] In some embodiments, the receptor gene comprises a constitutive promoter. In some embodiments, expression of the receptor is constitutive. In some embodiments, the receptor gene comprises a conditional promoter. In some embodiments, expression of the receptor is conditional or inducible. In some embodiments, the heterologous receptor gene is operably linked to an inducible promoter. In some embodiments, the inducible or conditional promoter is a tetracycline responsive element.

[0028] In some embodiments, the expression level of the heterologous receptor is a physiologically relevant expression level. The term "physiologically relevant expression level" refers to an expression level similar or equivalent to the endogenous expression level of the receptor in the cell. In other embodiments, the expression level may be less than the physiologically relevant level. In some embodiments, it is contemplated that the sensitivity of sequencing the barcode allows for lower expression levels than would be required for a less sensitive assay. In some embodiments, the concentration of RNA transcripts is about 10, 10 2 , 10 3 , 10 4 , 10 5 , 106 , 10 7 , 10 8 , 10 9 , or 10 10 , or at least or at most about 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 , or any derivable range therein.

[0029] In some embodiments, the cells are frozen. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human embryonic kidney 293T (HEK293T) cells.

[0030] Further aspects relate to assay systems comprising the cells or populations of cells described herein.

[0031] Further aspects relate to methods for screening for ligand and receptor binding, comprising contacting a cell of the invention with a ligand; detecting one or more reporters; and determining the identity of the one or more reporters; wherein the identity of the reporter indicates the identity of the bound receptor. The method may include screening several receptors and / or several ligands within a certain period of time. In some embodiments, a single screen may screen about, at least about, or at most about 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10A variety of cells and / or receptors of a species (or any range inducible therein) can be expressed by about, at least about, or at most about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 3000, 3100, 3200, 3300, 340 , 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 The method includes assaying for a species ligand or potential ligand (or any range inducible therein) for 2, 3, 4, 5, 6, 7 days and / or 1, 2, 3, 4, 5 weeks and / or 1, 2, 3, 4, 5, or 6 months (and any range inducible therein), where screening begins when a candidate ligand is contacted with the cells and ends when a receptor is identified by the sequenced barcode.

[0032] In some embodiments, at least 300 different heterologous receptors are expressed in the population of cells. In some embodiments, at least 2, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, or more receptors are expressed in the population of cells. In some embodiments, the population of cells is at least or at most 10 4 , 10 5 , 10 6 , 10 7 , 108 , 10 9 , 10 10 , 10 11 , or 10 12 In some embodiments, the population of cells is co-mixed in one composition. The composition may be a suspension composition of cells or a plate composition of cells. In some embodiments, the population of cells is adhered to a substrate, such as a cell culture dish. In some embodiments, the population of cells is contained within one well of a substrate or within one cell culture dish.

[0033] In some embodiments, determining the identity of the reporter comprises isolating nucleic acid from the cells. In some embodiments, the nucleic acid comprises RNA. In some embodiments, the method further comprises performing a reverse transcriptase reaction on the isolated RNA to generate cDNA. In some embodiments, the method further comprises amplifying the isolated nucleic acid. In some embodiments, the method further comprises sequencing the isolated nucleic acid. In some embodiments, the reverse transcriptase reaction is performed in the lysate. In some embodiments, detecting the one or more reporters comprises detecting a level of fluorescence from the one or more cells. In some embodiments, the method further comprises plating the cells. In some embodiments, the cells are plated on a 96-well cell culture plate. In some embodiments, the cells are frozen and the method further comprises thawing the frozen cells.

[0034] A particular embodiment of the present invention relates to a method for screening for ligand and receptor binding, comprising the steps of: contacting a population of cells with a ligand, wherein each cell of the population of cells comprises (i) a heterologous receptor gene and (ii) an inducible reporter comprising a receptor responsive element, expression of the reporter being dependent on activation of the activity of a receptor encoded by the receptor gene, the reporter comprising a barcode comprising an index region unique to the heterologous receptor gene, the population of cells expressing at least two different receptors derived from the heterologous receptor genes, and each single cell has one or more copies of one particular heterologous receptor and one or more copies of one particular reporter; detecting the one or more reporters; and determining the identity of the one or more reporters, wherein the identity of the reporter indicates the identity of the bound receptor.

[0035] The method further includes expressing any receptors identified in the screen in a cell. The receptors may be purified or isolated. One or more of the identified receptors may also be cloned. They can then be transfected into a different host cell for expression.

[0036] Further embodiments relate to vector libraries comprising at least two different vectors, wherein the vectors comprise different heterologous receptor genes and different inducible reporters. The vectors can be vectors described herein. Further embodiments relate to cell libraries comprising a population of cells of the present disclosure. Further embodiments relate to virus libraries comprising at least two virus particles of the present disclosure, wherein the virus particles comprise different heterologous receptor genes and different inducible reporters.

[0037] A further aspect relates to a method for generating a library of cells containing receptor proteins, comprising (i) expressing a nucleic acid or vector of the present disclosure in the cells, or (ii) infecting the cells with a viral particle of the present disclosure; wherein the cells express different heterologous receptors, and each single cell has one or more copies of one particular heterologous receptor and one or more copies of one particular reporter. Each cell may have at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies (or any inducible range therein) of a heterologous receptor gene and / or an inducible reporter. In certain embodiments, the cells contain at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies (or any inducible range therein) of a nucleic acid encoding a receptor gene and / or an inducible reporter.

[0038] Further aspects relate to kits comprising the vectors, cells, nucleic acids, libraries, primers, probes, sequencing reagents and / or buffers described herein.

[0039] Further aspects relate to a nucleic acid comprising: (i) a heterologous receptor gene operably linked to an inducible promoter; and (ii) a reporter comprising a receptor responsive element, expression of the reporter being dependent on activation of an activity of a receptor encoded by the heterologous receptor gene, the reporter comprising a barcode that includes an index region that is unique to the heterologous receptor gene. In some embodiments, the nucleic acid comprises at least 2 copies to at least 6 copies of the nucleic acid.

[0040] The term "equivalent nucleic acid" refers to a nucleic acid having a nucleotide sequence with a degree of homology with the nucleotide sequence of a nucleic acid or its complement. A homolog of a double-stranded nucleic acid is intended to include a nucleic acid having a nucleotide sequence with a degree of homology with its complement. In one embodiment, a homolog of a nucleic acid can hybridize to a nucleic acid or its complement. The nucleic acids of the present invention also include equivalent nucleic acids.

[0041] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) may have at least, at most, or exactly 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% (or any range derivable therein) "sequence identity" or "homology" to another sequence, meaning that, when aligned, the percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and homology or percent sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology.

[0042] Biologically equivalent polynucleotides are those which have a certain percentage of homology and which encode polypeptides having the same or a similar biological activity.

[0043] "About" and "approximately" are generally intended to mean an acceptable degree of error for the quantity measured, given the nature or precision of the measurement. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, particularly in biological systems, the terms "about" and "approximately" can mean values ​​that are within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a given value. In some embodiments, it is contemplated that the numerical values ​​discussed herein can be used with the terms "about" or "approximately".

[0044] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude others. When used to define compositions and methods, "consisting essentially of" is intended to mean excluding other elements that have an essential significance to the combination for the described purpose. "Consisting essentially of" in the context of pharmaceutical compositions of the present disclosure is intended to include all recited active agents, excluding any additional unrecited active agents, but not excluding other components of the composition that are not active ingredients. Thus, a formulation consisting essentially of the elements defined herein does not exclude trace contaminants from isolation and purification methods, as well as pharmacy-acceptable carriers such as phosphate buffered saline, preservatives, etc. "Consisting of" is intended to mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of the present invention, or process steps for producing the compositions or achieving the intended results. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0045] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein when referring to gene products or functional proteins.

[0046] The terms "contacted" and "exposed," as applied to cells, are used herein to describe the process by which an agent is delivered to a target cell or is brought into direct juxtaposition with a target cell or molecule.

[0047] In the claims and / or this specification, the use of the words "a" or "an" when used in conjunction with the term "comprising" may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0048] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0049] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer only to alternatives or where the alternatives are mutually exclusive, however, the present disclosure supports a definition that refers only to alternatives, similar to "and / or." As used herein, "another" may mean at least a second or more.

[0050] As used in this specification and the claims, the terms "comprising" (and with any formation such as "comprises" and "comprise"), "having" (and with any formation such as "having" and "having"), "including" (and with any formation such as "include" and "includes") or "containing" (and with any formation such as "contain" and "contains") are inclusive or open-ended and do not exclude additional, unrecognized elements or method steps. It is contemplated that any embodiment indicated with the term "comprising" may also have the word "consisting" substituted in place of "comprising."

[0051] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different embodiments can be combined.

[0052] The use of one or more compositions can be used according to the method described herein. The use of one or more compositions can be used in the preparation of a drug for treatment according to the method described herein. Other embodiments are discussed throughout this application. Any embodiment discussed with respect to one aspect of this disclosure also applies to other aspects of this disclosure, and vice versa. It is understood that the embodiments in the examples section are applicable to all aspects of the technology described herein.

[0053] [The present invention 1001] (i) with a heterologous receptor gene; (ii) an inducible reporter comprising a receptor responsive element, Expression of the reporter is dependent on activation of the activity of the receptor encoded by the receptor gene; The reporter comprises a barcode that includes an index region that is unique to the heterologous receptor gene. Inducible reporters and A nucleic acid comprising: [The present invention 1002] A vector comprising the nucleic acid of the present invention. [The present invention 1003] A vector containing a heterologous receptor gene. [The present invention 1004] The vector of the present invention 1003, further comprising an inducible reporter, the expression of which depends on activation of the activity of a receptor encoded by said receptor gene, and said reporter comprising a barcode comprising an index region unique to said heterologous receptor gene. [The present invention 1005] A vector comprising an inducible reporter, the reporter comprising a barcode. [The present invention 1006] The vector of any one of claims 1002 to 1004, wherein the receptor gene encodes a G protein-coupled receptor (GPCR). [The present invention 1007] The vector of any one of claims 1002 to 1006, wherein the receptor gene further comprises one or more additional polynucleotides encoding auxiliary polypeptides. [The present invention 1008] 1007. The vector of claim 10, wherein the auxiliary polypeptide comprises a selectable or screenable protein. [The present invention 1009] The vector of any one of claims 1007 to 1008, wherein the auxiliary polypeptide comprises a protein tag. [The present invention 1010] The vector of any one of claims 1007 to 1008, wherein the auxiliary polypeptide comprises a transcription factor. [The present invention 1011] The vector of the present invention, wherein the receptor gene encodes a fusion protein comprising the receptor gene and an accessory polypeptide. [The present invention 1012] The vector of the present invention, wherein the fusion protein comprises a protease site between the receptor gene and the accessory polypeptide. [The present invention 1013] The vector of any one of claims 1002 to 1012, wherein the auxiliary polypeptide comprises one or more transport tags. [The present invention 1014] The vector of the present invention, wherein said auxiliary polypeptide comprises two transport tags. [The present invention 1015] The vector of claim 1013 or 1014, wherein the transport tag comprises a Lucy and / or Rho tag. [The present invention 1016] The vector of any one of claims 1002 to 1015, wherein the reporter is induced by signal transduction upon activation of a GPCR. [The present invention 1017] The vector of any of claims 1002 to 1016, wherein the receptor responsive element comprises one or more of a cAMP response element (CRE), a nuclear factor of activated T-cell response element (NFAT-RE), a serum response element (SRE), and a serum response factor response element (SRF-RE). [The present invention 1018] The vector of the present invention, wherein the receptor responsive element comprises CRE. [The present invention 1019] The vector of the present invention, wherein the CRE comprises at least five repeats of SEQ ID NO:1. [The present invention 1020] The vector of any of claims 1010 to 1019, wherein the receptor responsive element comprises a DNA element bound by the auxiliary polypeptide transcription factor. [The present invention 1021] The vector of the present invention 1020, wherein said auxiliary polypeptide transcription factor comprises a reverse tetracycline-controlled transactivator (rtTA) and said receptor responsive element comprises a tetracycline responsive element (TRE). [The present invention 1022] The vector of any one of claims 1006 to 1021, wherein the GPCR is an olfactory receptor (OR). [The present invention 1023] The vector of any one of claims 1002 to 1022, wherein the receptor comprises an adrenergic receptor. [The present invention 1024] The vector of the present invention, wherein the adrenergic receptor comprises a beta-2 adrenergic receptor. [The present invention 1025] The vector of any one of claims 1002 to 1022, wherein the receptor gene comprises a nuclear hormone receptor gene. [The present invention 1026] The vector of any one of claims 1002 to 1022, wherein the receptor gene comprises a receptor tyrosine kinase gene. [The present invention 1027] The vector of any one of claims 1002 to 1026, wherein the receptor is a transmembrane receptor. [The present invention 1028] The vector of any one of claims 1002 to 1026, wherein the receptor is an intracellular receptor. [The present invention 1029] The vector of any one of claims 1002 to 1028, comprising a viral vector. [The present invention 1030] 1029. A vector of the present invention, including a lentiviral vector. [The present invention 1031] The vector of any of claims 1002 to 1029, wherein the receptor gene comprises a constitutive promoter. [The present invention 1032] The vector of any of claims 1002 to 1029, wherein the receptor gene comprises a conditional promoter. Invention 1032.1 The vector of any of claims 1002 to 1032, wherein the heterologous receptor gene is operably linked to a conditional promoter. Invention 1032.2 The vector of the present invention 1032.1, wherein said conditional promoter is a tetracycline response element. [The present invention 1033] The vector of any one of claims 1002 to 1032, wherein the barcode is at least 10 nucleic acids. [The present invention 1034] The reporter comprises, or further comprises, an open reading frame (ORF); The gene comprises a 3' untranslated region (UTR), The vector of any one of 1002 to 1032 of the present invention. [The present invention 1035] The vector of the present invention, wherein the barcode is located in the 3'UTR of a gene for a fluorescent protein. [The present invention 1036] The vector of any one of claims 1034 to 1035, wherein the ORF encodes a selectable or screenable protein. [The present invention 1037] The vector of the present invention 1036, wherein the ORF encodes a luciferase protein. [The present invention 1038] The vector of any of claims 1002 to 1037, wherein the receptor gene is adjacent to an insulator sequence at the 5' end and / or the 3' end. [The present invention 1039] The vector of any of claims 1002 to 1037, wherein the reporter is flanked by insulator sequences at the 5' and / or 3' ends. [The present invention 1040] The vector of the present invention 1038 or 1039, wherein the insulator comprises a cHS4 insulator. [The present invention 1041] The vector of any one of 1002 to 1040, comprising a second, third, or fourth barcode. [The present invention 1042] The vector of the present invention 1041, wherein at least one of said second, third, or fourth barcodes comprises an index region that is unique to one or more of an assay condition or a location on a microplate. [The present invention 1043] A viral particle comprising the vector of any one of the present inventions 1002 to 1040. [The present invention 1044] A cell comprising the vector of any one of the present inventions 1003 to 1039 or the viral particle of the present invention 1043. Invention 1044.1 A cell comprising multiple copies of any one of the vectors of the present invention 1002 to 1042. Invention 1044.2 The cell of the present invention 1044.1, comprising at least three copies of said vector. [The present invention 1045] A population of cells, Each cell is (i) with a heterologous receptor gene; (ii) an inducible reporter comprising a receptor responsive element, Expression of the reporter is dependent on activation of the activity of the receptor encoded by the receptor gene; The reporter comprises a barcode that includes an index region that is unique to the heterologous receptor gene. Inducible reporters and Including, The cells express different heterologous receptors, and each single cell has one or more copies of one particular heterologous receptor and one or more copies of one particular reporter. A population of cells. [The present invention 1046] (i) with a heterologous receptor gene; (ii) an inducible reporter comprising a receptor responsive element, Expression of the reporter is dependent on activation of the activity of the receptor encoded by the receptor gene; The reporter comprises a barcode that includes an index region that is unique to the heterologous receptor gene. Inducible reporters and comprising a cell. [The present invention 1047] The cell of any of claims 1044.2 to 1046, wherein the receptor gene encodes a GPCR. [The present invention 1048] The cell of claim 1047, wherein said reporter is induced by signal transduction upon activation of a GPCR. [The present invention 1049] The cell of any of claims 1044.2 to 1048, wherein the receptor gene further comprises one or more additional polynucleotides encoding accessory polypeptides. [The present invention 1050] The cell of claim 1049, wherein the auxiliary polypeptide comprises a selectable or screenable protein. [The present invention 1051] The cell of any one of claims 1049 to 1050, wherein the auxiliary polypeptide comprises a protein tag. [The present invention 1052] The cell of any of claims 1049 to 1051, wherein the auxiliary polypeptide comprises a transcription factor. [The present invention 1053] The cell of any of claims 1049 to 1052, wherein the receptor gene encodes a fusion protein comprising the receptor gene and the auxiliary polypeptide. [The present invention 1054] The cell of claim 1053, wherein the fusion protein comprises a protease site between the receptor gene and the accessory polypeptide. [The present invention 1055] The cell of any of claims 1047 to 1054, wherein the inducible reporter comprises one or more of a cAMP response element (CRE), a nuclear factor of activated T cells response element (NFAT-RE), a serum response element (SRE), and a serum response factor response element (SRF-RE). [The present invention 1056] The cell of any one of claims 1047 to 1055, wherein the GPCR is an olfactory receptor (OR). [The present invention 1057] The cell of any of claims 1044 to 1056, further comprising one or more genes encoding one or more accessory proteins. [The present invention 1058] 1057. The cell of claim 1057, wherein the one or more accessory proteins comprise one or more of a Gα subunit, Ric-8B, RTP1L, RTP2, RTP3, RTP4, CHMR3, and RTP1S. Invention 1058.1 The cell of any of claims 1044.2 to 1058, wherein the cell comprises a stable integration of one or more exogenous nucleotides encoding one or more accessory factor genes, the accessory factor genes including RTP1S, RTP2, Gα subunit, and Ric-8b. [The present invention 1059] The cell of claim 1057, wherein the one or more accessory proteins comprises an arrestin protein. [The present invention 1060] The cell of claim 1059, wherein the arrestin protein is fused to a protease. [The present invention 1061] The cell of any of claims 1044.2 to 1060, wherein the receptor gene comprises a nuclear hormone receptor gene. [The present invention 1062] The cell of any of claims 1044.2 to 1061, wherein the receptor gene comprises a receptor tyrosine kinase gene. [The present invention 1063] The cell of any of claims 1044.2 to 1062, wherein the receptor is a transmembrane receptor. [The present invention 1064] The cell of any of claims 1057 to 1063, wherein the one or more accessory proteins comprise one or more of a chaperone protein, a G protein, and a guanine nucleotide exchange factor. [The present invention 1065] The cell of any of the present inventions 1044.2 to 1064, further comprising a receptor protein expressed from the heterologous receptor gene. g [Invention 1066] The cell of the present invention, wherein the receptor protein is localized intracellularly. [The present invention 1067] The cell of any of claims 1044.2 to 1066, which lacks an endogenous gene encoding a protein that is at least 80% identical to the heterologous receptor gene. [The present invention 1068] The cell of any one of claims 1044.2 to 1067, wherein the receptor gene is integrated into the genome of the cell. [The present invention 1069] The cell of any one of 1044.2 to 1068, wherein the inducible reporter is integrated into the genome of the cell. [The present invention 1070] The cell of claim 1068 or 1069, wherein said receptor gene and said inducible reporter are genetically linked. [The present invention 1071] The cell of claim 1068 or 1069, wherein said receptor gene and said inducible reporter gene are not genetically linked. [The present invention 1072] The cell of any of claims 1068 to 1071, wherein the integrated receptor gene and / or the inducible reporter gene is integrated by targeted integration. [The present invention 1073] The cell of claim 1072, wherein the integration is within the H11 safe harbor locus. [The present invention 1074] The cell of any of claims 1068 to 1071, wherein the integrated receptor gene and / or the inducible reporter are randomly integrated into the genome. [The present invention 1075] The cell of claim 1074, wherein said random integration comprises rearrangement of said receptor gene and / or an inducible reporter gene. [The present invention 1076] The cell of any of claims 1044.2 to 1075, comprising at least two copies of the receptor gene and / or the inducible reporter gene. [The present invention 1077] The cell of any of claims 1044.2 to 1076, wherein the receptor gene comprises a constitutive promoter. [The present invention 1078] The cell of any of claims 1065 to 1077, wherein the expression of the receptor is constitutive. [The present invention 1079] The cell of any of claims 1044.2 to 1076, wherein the receptor gene comprises a conditional promoter. [The present invention 1080] The cell of any one of claims 1065 to 1076 or 1079, wherein expression of the receptor is conditional. [The present invention 1081] The cell of any of claims 1044.2 to 1080, wherein the barcode and / or index region is at least 10 nucleic acids. [The present invention 1082] The cell of any of the present inventions 1044.2 to 1081, wherein the reporter comprises or further comprises a gene for a fluorescent protein, the gene comprising a 3' untranslated region (UTR). [The present invention 1083] The cell of the present invention, wherein the barcode is located in the 3'UTR of the gene for the fluorescent protein. [The present invention 1084] The cell of claim 1082 or 1083, wherein said gene encodes a luciferase protein. [The present invention 1085] The cell of any of claims 1068 to 1084, wherein the receptor gene is flanked by insulator sequences at the 5' end and 3' end. [The present invention 1086] The cell of any of claims 1068 to 1085, wherein the reporter is flanked by insulator sequences at the 5' and 3' ends. [The present invention 1087] The cell of any of claims 1044.2 to 1086 of the present invention, wherein the expression level of the heterologous receptor is a physiologically relevant expression level. [The present invention 1088] A cell of any one of 1044.2 to 1087 of the present invention, which is frozen. [The present invention 1089] The cell of any one of 1044.2 to 1088 of the present invention, which is a mammalian cell. [The present invention 1090] The cell of the present invention, which is a human embryonic kidney 293T (HEK293T) cell. [The present invention 1091] An assay system comprising any one of the cells of the present invention 1044 to 1090. [The present invention 1092] 1. A method for screening ligand and receptor binding, comprising: A step of contacting any one of the cells of the present invention 1044 to 1090 with a ligand; detecting one or more reporters; determining the identity of one or more reporters; Including, The identity of the reporter indicates the identity of the bound receptor. method. [The present invention 1093] The method of claim 1092, wherein the step of determining the identity of the reporter comprises isolating nucleic acid from the cell. [The present invention 1094] The method of claim 1093, wherein the nucleic acid comprises RNA. [The present invention 1095] The method of claim 1094, further comprising the step of performing a reverse transcriptase reaction on the isolated RNA to produce cDNA. [The present invention 1096] The method of any one of claims 10 to 15, wherein said RT is performed in said lysate. [The present invention 1097] The method of any of claims 1093 to 1096, further comprising the step of amplifying the isolated nucleic acid. [The present invention 1098] The method of any one of claims 1093 to 1097, further comprising a step of sequencing the isolated nucleic acid. [This invention 1099] The method of any one of claims 1092 to 1098, wherein the step of detecting said one or more reporters comprises detecting a level of fluorescence from said cells. [The present invention 1100] The method of any of claims 1092 to 1099, wherein at least two different heterologous receptors are expressed in said cell. [The present invention 1101] The method of any of claims 1092 to 1100, wherein the populations of cells are co-mixed in one composition. [The present invention 1102] The method of any one of claims 1092 to 1101, wherein the population of cells is adherent to a substrate. [The present invention 1103] The method of any of claims 1092 to 1102, wherein the population of cells is contained within a single well of the substrate or within a single cell culture dish. [The present invention 1104] The method of any one of claims 1092 to 1103, further comprising the step of plating the cells. [The present invention 1105] The method of claim 1104, wherein said cells are plated on a 96-well cell culture plate. [The present invention 1106] The method of any of claims 1092 to 1105, wherein the cells are frozen, and the method further comprises the step of thawing the frozen cells. [The present invention 1107] A method for screening ligand and receptor binding, comprising the steps of: Contacting the population of cells with a ligand, Each cell in the population of cells (i) with a heterologous receptor gene; (ii) an inducible reporter comprising a receptor responsive element, Expression of the reporter is dependent on activation of the activity of the receptor encoded by the receptor gene; The reporter comprises a barcode that includes an index region that is unique to the heterologous receptor gene. Inducible reporters and Including, The population of cells expresses at least 300 different receptors derived from the heterologous receptor genes, and each single cell has one or more copies of one particular heterologous receptor and one or more copies of one particular reporter. The process and detecting one or more reporters; Determining the identity of one or more reporters, which identity indicates the identity of the bound receptor. [The present invention 1108] A vector library comprising at least two different vectors, said vectors comprising different heterologous receptor genes and different inducible reporters. [The present invention 1109] A cell library comprising a population of any one of cells 1045 to 1090 of the present invention. [The present invention 1110] A viral library comprising at least two viral particles of the invention 1043, wherein said viral particles comprise different heterologous receptor genes and different inducible reporters. [The present invention 1111] 1. A method for generating a library of cells containing a receptor protein, comprising: (i) expressing the nucleic acid of the present invention 1001 or any of the vectors of the present inventions 1002 to 1039 in a cell; or (ii) Infecting the cells with the virus of the invention 1043 Including, The cells express different heterologous receptors, and each single cell expresses one or more copies of one particular heterologous receptor and one or more copies of one particular reporter. [The present invention 1112] A kit comprising any one of the libraries of the present invention Nos. 1108 to 1110. [The present invention 1113] (i) a heterologous receptor gene operably linked to an inducible promoter; (ii) a reporter comprising a receptor-responsive element, Expression of the reporter is dependent on activation of the activity of a receptor encoded by a heterologous receptor gene, The reporter comprises a barcode that includes an index region that is unique to the heterologous receptor gene. With reporter A nucleic acid comprising: [The present invention 1114] A cell comprising at least 2 copies to at least 6 copies of the nucleic acid of the present invention. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0054] The accompanying drawings form a part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0055] [Figure 1] Overview of the multiplexed reporter scheme. Detailed diagram of the multiplexed scheme. Detailed diagram of the barcoding strategy for the OR library. Each OR is linked to a unique barcode in the 3'UTR of a reporter gene. Mukku3a cells are clonally integrated with each OR, pooled, and plated for odorant induction. After induction, barcoded transcripts are sequenced and quantified to determine the relative affinity for each odorant-receptor pair. [Diagram 2] Individual cell line Luc / RNA and pilot screen. a) Individual Luc for stable cell lines. b) Individual RNA for stable cell lines. a) Individual stable OR activation with known ligands measured via cAMP-responsive luciferase gene reporter in Mukku3a cells. b) Individual stable OR activation with known ligands measured via Q-RTPCR of barcoded genetic reporter in Mukku3a cells. [Diagram 3] Combined vs separate gene reporters. a) Combined vs separate schematic. b) Separate vs combined transient data. a) Plasmid constructs to encode ORs and reporters separately and together. b) Transient OR activation (MOR42-3 and MOR9-1) versus known ligands was measured via a cAMP-responsive luciferase gene reporter in separate and combined constructs. [Figure 4A]Landing pad. a) Schematic diagram of Bxb1, b) integration efficiency, c) B2 and OR integrated luciferase. a) Schematic diagram of Bxb1 recombination into the landing pad. HEK293T cells were pre-engineered to contain a single copy of the landing pad of the safe harbor locus H11 (Mukku1a cells). The landing pad contains the Bxb1 recombinase recognition site attp. Co-expression of the recombinase with a plasmid containing the corresponding attb recognition site results in a single irreversible site-specific integration event. This integration strategy allows for clonal integration of a heterogeneous library in a single pot. b) Assessment of integration efficiency of the Bxb1 landing pad using flow cytometry. Cells were co-transfected with a plasmid expressing the recombinase and a plasmid conditionally expressing mCherry upon integration, as well as the mCherry plasmid alone. After multiple passages, as much as 7-8% of cells transfected with the recombinase were fluorescent, whereas cells without the recombinase were not fluorescent. c) A combined genetic reporter encoding an OR (MOR42-3) and a beta-2 adrenergic receptor (ADRB2) was engineered into the landing pad. Both were induced with known agonists and genetic reporter activation was measured by luciferase assay. Dose-dependent activation was observed for ADRB2 but not for MOR42-3. [Figure 4B] Please see the legend to FIG. 4A. [Figure 4C] Please see the legend to FIG. 4A. [Diagram 5]Inducible scheme. a) Overview, b) Transient and integrated inducibility. a) Mukku1a cells were transduced to constitutively express the reverse tetracycline transactivator (m2rtTA), replacing the constitutive promoter driving OR expression with a tetracycline-regulated promoter. (Tetracycline-responsive GFP was integrated to confirm expression in the landing pad by addition of doxycycline) b) The inducible combined gene reporter was transiently screened for OR activation and integrated into the landing pad of Mukku2a cells. Transient activation of MOR42-3 was observed in the presence of dox when stimulated with odorants, but not when integrated into the landing pad. The bars above each concentration in part b represent -Dox (left bar) and +Dox (right bar). [Figure 6] Copy number. a) Transposon method, b) Constitutive transposon, c) Inducible transposon, d) QPCR. a) Schematic of transposon. PiggyBac transposase excises the combined genetic reporter flanked by the intermediate terminal repeat. Multiple copies of this sequence are then inserted into TTAA loci across the genome. b) When transposed under constitutive expression in Mukku1a cells, MOR42-3 shows no dose-responsive luciferase production to ligand. c) When transposed into Mukku2a under inducible expression, MOR42-3 shows strong dose-responsive luciferase production to ligand in the presence of doxycycline. The bars above each concentration in part c represent -Dox (left bar) and +Dox (right bar). d) The copy number of the transposon was determined for the transposons of three different ORs by QPCR of genomic DNA. Absolute copy numbers were determined by comparing the Cq of the transposon relative to the clonally integrated gene reporter in the landing pad. Bars in part d represent (from left to right) control, MOR203-1, MOR9-1, and Olfr62. [Figure 7]a) Trans-AF, b) Clonal selection. a) Comparison of transient OR activation (Olfr62 and MOR30-1) with known ligands measured via combined luciferase gene reporters in the presence or absence of accessory factors RTP1S and RTP2. b) Mukku2a cells were transfected with four accessory factors (RTP1S, RTP2, Gαolf, and Ric8b) regulated by inducible expression. Individual clones were isolated and functionally assessed for accessory factor expression. Clones were assayed for transient OR activation (Olfr62 and OR7D4) with known ligands via separate luciferase gene reporters. A clone (Mukku3a) that showed strong activation for both typical morphology and growth rate was selected for downstream applications. [Figure 8] Includes landing pad. [Figure 9] Genomically integrated synthetic circuits allow screening of mammalian olfactory receptor activation. a) Schematic of synthetic circuits for stable OR expression and function in engineered HEK293T cell lines. b) MOR42-3 reporter activation expressing transiently or genomically integrated receptors at various copy numbers and under constitutive or inducible expression. c) Olfr62 reporter activation with / without accessory factors and transiently expressed / integrated in engineered cell lines. d) Dose-response curves of OR reporter activation integrated in engineered cell lines. [Figure 10]Large-scale multiplex screening of olfactory receptor-odorant interactions. a) Schematic for the generation and multiplex screening of a library of OR reporter cell lines. b) Comparison of MOR30-1 and Olfr62 reporter activation when tested in transient or genome-integrated luciferase assays or pooled RNA-seq assays. c) Heatmap of all interactions from the screen clustered by odorant and receptor response similarity and colored by the lowest concentration that induced reporter activity. d) Hits identified for the four ORs (black) were mapped onto the PCA projection of the chemical space of our odorant panel (grey). [Figure 11] Engineering HEK293 cells for stable functional OR expression. a) Comparison of MOR42-3 activation from inducibly driven receptor expression, either transiently transfected or integrated in single copy at the H11 genomic locus. B. Activation from cells with MOR42-3 integrated in multiple copies in the genome under either constitutive or inducible expression. c) Relative receptor / reporter DNA copy numbers determined using qPCR for three transposed ORs compared to single copy integrants. d) MOR30-1 and Olfr62 activation (stimulated with decanoic acid and 2-coumaranone, respectively) were co-transfected with or without accessory factors (AFs) Gαolf, Ric8b, RTP1S, and RTP2. e) Cell line generation for stable accessory factor expression. Following transfection, clones were isolated and screened for activation of ORs that require accessory factors to functionally express, Olfr62 and OR7D4. Dark grey bars represent clones selected for further experiments. [Figure 12]Design of multigene reporters for OR activation. a) Schematic of vectors containing OR expression cassettes and gene reporters for integration. b) MOR42-3 reporter activation in cells transiently co-expressing the reporter cassettes on separate plasmids or together. c) Fold activation of engineered CRE enhancers compared to Promega's pGL4.19 CRE enhancer. d) Basal activation of gene reporters upon induction of inducible OR promoters with or without DNA insulators upstream of the CRE enhancer. [Figure 13] Schematic of the synthetic organic activation circuit in engineered cell lines. Full graphical representation of the expressed components for expression / signaling of ORs and barcoded reporter systems as shown in Figure 9 and described in Example 2. Receptor expression is controlled by the Tet-On system. After doxycycline induction, ORs are expressed on the cell surface with the help of two exogenously expressed chaperones, RTP1S and RTP2. Upon odorant activation, g protein signaling induces cAMP production. Signaling is augmented by transgenic expression of the native OR G alpha subunit, Golf, and its corresponding GEF, Ric8b. cAMP leads to activation of the kinase PKA, which phosphorylates the transcription factor CREB, resulting in expression of the barcoded reporter. [Figure 14A] Pilot-scale replication of odorant responses in multiplex. a) Heatmap showing 40 pooled receptors responding to 9 odorants and 2 mixtures. Interactions are colored by log2 fold activation of the genetic reporter. Previously identified odorant interactions (Saito et al., 2009) are boxed in yellow. b) Dose-response curves of odorants or forskolin (an adenylate cyclase stimulator) screened against the OR library at 5 concentrations. Curves of ORs known to interact with odorants are colored. Stimulation with forskolin shows no substantial differential activity between ORs in our assay. [Figure 14B]Please see the legend to Figure 14A. [Figure 15] Library representation. Representation of individual ORs in the OR library. a) Frequency of each OR as a fraction of the library determined by the relative activation of each reporter incubated with DMSO. b) Relationship between the frequency of each OR in the library and the average coefficient of variation among biological replicate measurements of reporter activation for all conditions. [Figure 16] Reproducibility of large-scale multiplex screens. a) Histogram showing distribution of coefficient of variation of the OR library when stimulated with DMSO. b) Histogram showing distribution of coefficient of variation of the OR library for all conditions assayed. c) Dose-response curves of control odorants included in each 96-well plate assayed. Each color represents a different plate. [Figure 17] Significance and fold change of high-throughput assay data, a) False discovery rate (FDR) – calculated from a generalized linear model using negative binomial assumptions, followed by multiple hypothesis correction, plotted against the fold change of each OR-odorant interaction. The dashed line represents 1% FDR, a conservative cutoff used to identify interactions. b) The subset of interactions selected for individual orthogonal luciferase assay colorimetry indicates whether an interaction was detected. Twenty-one of the 28 interactions above 1% FDR also showed interactions in the orthogonal follow-up assay. [Figure 18-1] Reproduction of the screen in a temporal orthogonal system. Secondary screening of chemicals against cell lines expressing a single olfactory receptor with luciferase readout. Each plot shows the behavior of a negative control cell line not expressing the OR but treated with odorant (black line), as well as a cell line expressing a specific OR. Additionally, data from a high-throughput sequencing screen (labeled Seq) are plotted for reference. [Figure 18-2] This figure shows a continuation of Figure 18-1. [Figure 18-3] This figure shows the continuation of Figure 18-2. [Figure 18-4] This figure shows a continuation of Figure 18-3. [Figure 19] Concordance of the assay with previously screened odorant-receptor pairs. a) FDR plotted against fold induction for 540 odor-OR interactions previously tested by Saito et al. The points are colored by the EC50 of interactions identified by Saito et al. (2009). Grey points represent interactions not identified in the previous screen. Comparing the transient and integrated luciferase assays revealed that in some cases the integrated system required a higher concentration of odorant to achieve significant activation, likely due to lower DNA copy numbers of the CRE-driven luciferase and receptor. As the highest odorant concentration assayed was 1 mM, low affinity interactions may not have been detectable in this screen. b) FDR in the assay was related to the EC50 of hits from the previous screen colored by fold activation from the multiplexed screen. [Figure 20] Clustering of odorant responses to receptors. Here, we plot the position of any hits (black) against other chemicals tested (grey) on the same coordinates as in Figure 20. This provides a visualization of the breadth of activity of a given OR relative to the larger chemical space. [Figure 21] 1 shows an overview of deep mutation scanning. [Figure 22] Distribution of library activities. [Figure 23] β2 variant activity landscape at 0.625uM isoproterenol. [Figure 24] Comparison with mutants assayed individually [Diagram 25] Ligand interaction sites. [Figure 26] k stands for clustering. [Figure 27]A) Diagram of how Bxb1 recombination works in the context of a test to ensure that only one construct is inserted per cell (cells are only red or only green). B) Results of the two color test streams. C) Activity of the reporter when stimulated with the B2 agonist, isoproterenol, in KO or wild type cells. D) Addition of transgenic B2 to a single copy locus can restore the ability to read out B2 activity E) It can also be reduced at the RNA level, improving fold activation with an insulator element. [Figure 28] Diagram of the B2 construct inserted into the H11 locus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] Description of exemplary embodiments Brute force chemical screening has significant financial costs, scaling problems, and in the case of some receptors, such as olfactory receptors, the screen also suffers from unreliable functional expression. Recently, a large-scale effort to perform a comprehensive olfactory screen of human receptors assayed 394 ORs across 73 odorants. In combination with transient transfection, researchers constructed cell lines that allow expression of all factors required for functional OR expression. Activation of the transiently transfected ORs results in luciferase reporter expression, which can be assayed in multiwell plates. This screen required over 50,000 individual measurements and took many years. This work alone doubled the known number of ligand-receptor binding pairs and mapped 27 human OR receptors to their chemical ligands. Despite the success of this approach, the scale required to perform this relatively small chemical screen was enormous, as every compound had to be tested at a range of concentrations across hundreds of ORs, with each test requiring a separate transient transfection. Therefore, such methods have little opportunity to scale to the types of methods disclosed herein.

[0057] The methods of the present disclosure describe the construction of large libraries of receptors contained within cell lines that can report on their activity in multiplexes using the detection methods described herein. With this automatable characterization platform, ligand and receptor binding can be investigated on a much larger scale than previously performed using current methods. The assays and methods can have numerous applications in drug discovery and testing.

[0058] I. Receptors and inducible reporter elements The current methods, nucleic acids, vectors, viral particles, and cells of the present disclosure relate to receptor proteins that induce transcription of a reporter via a receptor-responsive element upon ligand engagement. Thus, the reporter is either under the direct control of the receptor protein or indirectly controlled by the receptor protein. The term "receptor-responsive element" refers to an element in the promoter region of an inducible reporter that is bound by the receptor, or a downstream element of the receptor after engagement of the receptor and ligand. In some embodiments, the receptor protein is a G protein-coupled receptor (GPCR) or the receptor gene encodes a GPCR. G protein-coupled receptors (GPCRs) regulate a wide variety of normal biological processes and play a role in the pathophysiology of many diseases upon dysregulation of their downstream signaling activity. Ligands of GPCRs include neurotransmitters, hormones, cytokines, and lipid signaling molecules. GPCRs regulate a wide variety of biological processes, including vision, olfaction, the autonomic nervous system, and behavior. Besides its extracellular ligand, each GPCR binds to a specific intracellular heterotrimeric G protein consisting of G-alpha, G-beta, and G-gamma subunits that activate downstream signaling pathways. These intracellular signaling pathways include cAMP / PKA, calcium / NFAT, phospholipase C, protein tyrosine kinase, MAP kinase, PI-3-kinase, nitric oxide / cGMP, Rho, and JAK / STAT. Disruption of GPCR function or signaling contributes to pathological conditions that vary depending on their ligands and the processes they regulate, ranging from neurological to immune to hormonal disorders. GPCRs account for 30% of all current drug development targets. Developing drug screening assays requires investigation of both target and associated GPCR expression and function in selected cell-based model systems, as well as expression of associated GPCRs to evaluate both direct and potential off-target side effects.

[0059] Based on the extensive knowledge of receptor signaling and the transcriptional regulation exerted by receptors, it is within the skill of one in the art to construct receptor genes / receptor-responsive elements.

[0060] In GPCRs, inducible reporters contain response elements that direct transcriptional activity of the reporter upon activation of GPCR signaling by ligand engagement. GPCR response elements include cAMP response elements (CRE), nuclear factor of activated T cells response elements (NFAT-RE), serum response elements (SRE), and serum response factor response elements (SRF-RE). GPCRs further include G s , G i , G q , G 12 Examples of receptor genes / proteins and response elements are shown in the table below. TIFF0007671316000003.tif48161

[0061] G olf Or the G olfactory receptor is G S It is a GPCR whose signal transduction converts ATP to cAMP, which then directs transcription via the CRE response element. Exemplary olfactory receptors include those shown in the table below.

[0062] Olfactory receptors, family 1: TIFF0007671316000004.tif229164TIFF0007671316000005.tif243164TIFF0007671316000006.tif243164TIFF0007671316000007.tif47164

[0063] Olfactory receptors, family 2: TIFF0007671316000008.tif184164TIFF0007671316000009.tif244164TIFF000 7671316000010.tif237164TIFF0007671316000011.tif250164TIFF0007671316 000012.tif234164TIFF0007671316000013.tif245164TIFF0007671316000014. tif245164TIFF0007671316000015.tif245164TIFF0007671316000016.tif16164

[0064] Olfactory receptors, family 3: TIFF0007671316000017.tif115163

[0065] Olfactory receptors, family 4: TIFF0007671316000018.tif77164TIFF0007671316000019.tif245164TIFF00076713160 00020.tif245164TIFF0007671316000021.tif245164TIFF0007671316000022.tif245164 TIFF0007671316000023.tif237164TIFF0007671316000024.tif240164TIFF0007671316 000025.tif245164TIFF0007671316000026.tif245164TIFF0007671316000027.tif93164

[0066] Olfactory receptors, family 5: TIFF0007671316000028.tif123161TIFF0007671316000029.tif245161TIFF000 7671316000030.tif241161TIFF0007671316000031.tif245161TIFF0007671316 000032.tif242161TIFF0007671316000033.tif237161TIFF0007671316000034. tif245161TIFF0007671316000035.tif245161TIFF0007671316000036.tif93161

[0067] Olfactory receptors, family 6: TIFF0007671316000037.tif138161TIFF0007671316000038.tif245161TIFF0007671316000039.tif245161TIFF0007671316000040.tif169161

[0068] Olfactory receptors, family 7: TIFF0007671316000041.tif47161TIFF0007671316000042.tif236161TIFF00076713160 00043.tif245161TIFF0007671316000044.tif236161TIFF0007671316000045.tif236161 TIFF0007671316000046.tif216161TIFF0007671316000047.tif226161TIFF00076713160 00048.tif227161TIFF0007671316000049.tif227161TIFF0007671316000050.tif229161 http: / / www.genenames.org / cgi-bin / download?title=Genefam+data&submit=submit&hgnc_dbtag=on&preset=genefam&status=Approved&status=Entry+Withdrawn&status_opt=2&=on&format=text&limit=&.cgifields=&.cgifields=chr&.cgifields=status&.cgifields=hgnc_dbtag&where=gd_gene_fam_name%20RLIKE%20'(%5e|%20)OR7($|,)'&order_by=gd_app_sym_sort

[0069] Olfactory receptors, family 8: TIFF0007671316000051.tif184161TIFF0007671316000052.tif237161TIFF0007671316000053.tif245161TIFF0007671316000054.tif138161

[0070] Olfactory receptors, family 9: TIFF0007671316000055.tif77162TIFF0007671316000056.tif245162TIFF0007671316000057.tif62162

[0071] Olfactory receptors, family 10: TIFF0007671316000058.tif146162TIFF0007671316000059.tif245162TIFF00076713160 00060.tif245162TIFF0007671316000061.tif245162TIFF0007671316000062.tif191162

[0072] Olfactory receptors, family 11: TIFF0007671316000063.tif31163TIFF0007671316000064.tif245161TIFF0007671316000065.tif108161

[0073] Olfactory receptors, family 12: TIFF0007671316000066.tif62161

[0074] Olfactory receptors, family 13: TIFF0007671316000067.tif31161TIFF0007671316000068.tif245161TIFF0007671316000069.tif93161

[0075] Olfactory receptors, family 14: TIFF0007671316000070.tif123161

[0076] Olfactory receptors, family 51: TIFF0007671316000071.tif237161TIFF0007671316000072.tif229161TIFF0007671316000073.tif237161TIFF0007671316000074.tif47161

[0077] Olfactory receptors, family 52: TIFF0007671316000075.tif184161TIFF0007671316000076.tif245161TIFF0007671316000077.tif245161TIFF0007671316000078.tif93161

[0078] Olfactory receptors, family 55: TIFF0007671316000079.tif31161

[0079] Olfactory receptors, family 56: TIFF0007671316000080.tif77161TIFF0007671316000081.tif77161TIFF0007671316000082.tif111161

[0080] Further exemplary receptor genes / proteins useful as heterologous receptors according to the disclosed methods and compositions include receptors such as those listed in the table below.

[0081] GPCR Receptors TIFF0007671316000083.tif243170TIFF0007671316000084.tif243170TIFF0007671316000085.tif243170TIFF0007671316 000086.tif242170TIFF0007671316000087.tif242170TIFF0007671316000088.tif243170TIFF0007671316000089.tif68170

[0082] Nuclear Hormone Receptors TIFF0007671316000090.tif156169TIFF0007671316000091.tif60169

[0083] Catalytic Receptors TIFF0007671316000092.tif152168TIFF0007671316000093.tif244168TIFF0007671316000094.tif238168 TIFF0007671316000095.tif242168TIFF0007671316000096.tif236168TIFF0007671316000097.tif166168

[0084] The ligand may be a known ligand for the receptor or test compound.For example, in the case of olfactory receptor, the ligand may be an odorant.Exemplary odorants include geranyl acetate, methyl formate, methyl acetate, methyl propionate, methyl propanoate, methyl butyrate, methyl butanoate, ethyl acetate, ethyl butanoate, ethyl butanoate, isoamyl acetate, pentyl butanoate, pentyl pentanoate, octyl acetate, benzyl acetate, and methyl anthranilate.

[0085] In some embodiments, the ligand comprises a small molecule, a polypeptide, or a nucleic acid ligand. The method of the present invention relates to a screening procedure to detect ligand binding to a receptor. Thus, the ligand may be a test compound or a drug. The method of the present invention may be utilized to determine the engagement of a ligand and a receptor for the purpose of determining ligand / pharmaceutical efficacy and / or off-target effects. The polypeptide ligand may be a peptide less than 100 amino acids in length.

[0086] Chemical agents are "small molecule" compounds, which are organic non-peptide molecules with a molecular weight typically less than 10,000 Da. In some embodiments, they are less than 5,000 Da, less than 1,000 Da, or less than 500 Da (and any range derivable therein). This class of modulators includes chemically synthesized molecules, such as compounds from combinatorial chemical libraries. Synthetic compounds can be rationally designed or identified from the screening methods described herein. Methods for generating and obtaining small molecules are well known to those skilled in the art (Schreiber, Science 2000; 151:1964-1969; Radmann et al., Science 2000; 151:1947-1948, incorporated herein by reference).

[0087] II. Reporter Systems A. Nucleic Acid Reporters The reporter comprises a barcode region that includes an index region that can identify an activated receptor. The index region can be a polynucleotide that is at least, at most, or exactly 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200 or more nucleotides in length (or any range derivable therein). The barcode can include one or more universal PCR regions, adaptors, linkers, or combinations thereof.

[0088] The index region of the barcode is a polynucleotide sequence that is unique to a particular heterologous receptor in the context of the screening utilized and can therefore be used to identify heterologous receptors that are activated and / or expressed in the same cells as the barcode. In embodiments relating to a population of cells, determining the identity of the barcode is done by determining the nucleotide sequence of the index region to identify which receptors are activated in the population of cells. As discussed herein, the method can include sequencing one or more index regions or having such index regions sequenced.

[0089] The nucleic acid constructs are generated by any means known in the art, including the use of polymerases and solid-state nucleic acid synthesis (e.g., on columns, multi-wall plates, or microarrays). The present invention provides the inclusion of barcodes to facilitate the determination of activity of specific nucleic acid regulatory elements (i.e., receptor response elements) that can be indexes of activated receptors. These barcodes are contained in an expression vector that includes the nucleic acid construct and the nucleic acid regulatory elements. Each index region of the barcode is unique to the corresponding heterologous receptor gene (i.e., a particular nucleic acid regulatory element may have more than one barcode or index region (e.g., 2, 3, 4, 5, 10, or more), but each barcode indicates activation of a single receptor). These barcodes are oriented in the expression vector so that they are transcribed in the same mRNA transcript as the associated open reading frame. The barcodes can be oriented in the mRNA transcript 5' to the open reading frame, 3' to the open reading frame, immediately 5' to the terminal polyA tail, or anywhere in between. In some embodiments, the barcode is in the 3' untranslated region.

[0090] The unique portions of the barcode may be contiguous along the length of the barcode sequence, or the barcode may include stretches of nucleic acid sequence that are not unique to any one barcode. In one application, the unique portions of the barcode (i.e., index regions) may be separated by stretches of nucleic acid that are removed by the cellular machinery during transcription into mRNA (e.g., introns).

[0091] The inducible reporter comprises a regulatory element, such as a promoter, and a barcode. In some embodiments, the regulatory element further comprises an open reading frame. The open reading frame may encode a selectable or screenable marker, as described herein. The nucleic acid regulatory element may be 5', 3', or within the open reading frame. The barcode may be located anywhere within the region that is transcribed into mRNA (e.g., upstream of the open reading frame, downstream of the open reading frame, or within the open reading frame). Importantly, the barcode is located 5' of the transcription termination site.

[0092] The barcode and / or index regions are quantified or determined by methods known in the art, including quantitative sequencing (e.g., using an Illumina® sequencer) or quantitative hybridization techniques (e.g., using microarray hybridization techniques or the Luminex® bead system). Sequencing methods are further described herein.

[0093] B. Sequencing Methods for Detecting Barcodes 1. Massively parallel signature sequencing (MPSS). The first of the next-generation sequencing technologies, massively parallel signature sequencing (or MPSS), was developed at Lynx Therapeutics in the 1990s. MPSS was a bead-based method that used a complex approach of adapter ligation followed by decoding the adapters and reading the sequence four nucleotides at a time. This method was prone to sequence-specific bias or loss of specific sequences. Because the technology was so complex, MPSS was only performed "in-house" by Lynx Therapeutics, and DNA sequencing machines were not sold to independent laboratories. Lynx Therapeutics merged with Solexa (later acquired by Illumina) in 2004, leading to the development of sequencing-by-synthesis, a simpler approach derived from Manteia Predictive Medicine, making MPSS obsolete. However, the essential characteristics of MPSS output were typical of later "next-generation" data types, containing hundreds of thousands of short DNA sequences. In the case of MPSS, these were typically used to sequence cDNA for the measurement of gene expression levels. Indeed, the powerful Illumina HiSeq2000, HiSeq2500, and MiSeq systems are based on MPSS.

[0094] 2. Polony Sequencing Polony sequencing, developed in the laboratory of George M. Church at Harvard, was one of the first next-generation sequencing systems and was used to sequence a complete genome in 2005. It combined in vitro paired-tag libraries with emulsion PCR, automated microscopy, and ligation-based sequencing chemistry to sequence the E. coli genome with >99.9999% accuracy and at approximately 1 / 9 the cost of Sanger sequencing. The technology was licensed to Agencourt Biosciences, which then spun out into Agencourt Personal Genomics, and eventually incorporated into the Applied Biosystems SOLiD platform, now owned by Life Technologies.

[0095] 3.454 Pyrosequencing A parallel version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics. The method amplifies DNA in droplets of water in an oil solution (emulsion PCR), with each droplet containing a single DNA template bound to a single primer-coated bead, which then forms clonal colonies. The sequencing machine contains many picoliter-volume wells, each containing a single bead and a sequencing enzyme. Pyrosequencing uses luciferase to generate light for the detection of individual nucleotides added to the nascent DNA, and the combined data is used to generate a sequence readout. This technology offers intermediate read lengths and price per base compared to Sanger sequencing on one end and Solexa and SOLiD sequencing on the other end.

[0096] 4. Illumina (Solexa) Sequencing Solexa, now part of Illumina, developed a sequencing method based on an internally developed reversible dye-terminator technology, and an engineered polymerase. The termination chemistry was developed internally at Solexa, and the concept of the Solexa system was invented by Balasubramanian and Klennerman from the Chemistry Department at the University of Cambridge. In 2004, Solexa acquired Manteia Predictive Medicine to obtain a massively parallel sequencing technology based on "DNA clusters," which involve clonal amplification of DNA on a surface. The cluster technology was jointly acquired with Lynx Therapeutics of California. Solexa Ltd. later merged with Lynx to form Solexa Inc.

[0097] In this method, DNA molecules and primers are first deposited on a slide and amplified with polymerase, resulting in the formation of local clonal DNA colonies (later, cast "DNA clusters"). To determine the sequence, four reversible terminator bases (RT-bases) are added and unincorporated nucleotides are washed away. An image of the fluorescently labeled nucleotides is taken with a camera, and then the dye is chemically removed from the DNA along with the terminal 3' blocker, starting the next cycle. Unlike pyrosequencing, the DNA strand is extended one nucleotide at a time, and image acquisition can be performed at delayed moments, allowing very large arrays of DNA colonies to be captured by successive images taken from a single camera.

[0098] The decoupling of enzymatic reaction and image capture allows optimal throughput and theoretically infinite sequencing capacity. Thus, in an optimal configuration, the final achievable instrument throughput is determined only by multiplying the camera analog / digital conversion rate by the number of cameras and dividing by the number of pixels per DNA colony required to optimally visualize them (approximately 10 pixels / colony). In 2012, with cameras operating at A / D conversion rates above 10 MHz and available optics, fluidics and enzymes, the throughput can be a multiple of 1 million nucleotides / sec, which roughly corresponds to one human genome equivalent / instrument at 1x coverage per hour and one human genome resequencing (approximately 30x) / instrument (equipped with a single camera) per day.

[0099] 5. SOLiD Sequencing Applied Biosystems' (now Life Technologies brand) SOLiD technology uses sequencing by ligation, where a pool of all possible oligonucleotides of fixed length is labeled according to the position to be sequenced. The oligonucleotides are annealed and ligated; preferential ligation by DNA ligase for matching sequences results in an informative signal of the nucleotide at that position. Prior to sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each containing a single copy of the same DNA molecule, are placed on a glass slide. The result is a quantity and length of sequence comparable to Illumina sequencing. This sequencing by ligation method has been reported to have some problems with sequencing palindromic sequences.

[0100] 6.Ion Torrent Semiconductor Sequencing Ion Torrent Systems Inc. (now owned by Life Technologies) has developed a system that uses standard sequencing chemistry but has a novel semiconductor-based detection system. This sequencing method, in contrast to the optical methods used in other sequencing systems, is based on the detection of hydrogen ions released during DNA polymerization. A microwell containing the template DNA strand to be sequenced is flooded with a single type of nucleotide. If the introduced nucleotide is complementary to the leading template nucleotide, it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, indicating that a reaction has occurred. If a homopolymeric repeat is present in the template sequence, a large number of nucleotides are incorporated in a single cycle. This releases a corresponding number of hydrogens, resulting in a proportionally high electronic signal.

[0101] 7. DNA Nanoball Sequencing DNA nanoball sequencing is a type of high-throughput sequencing technology used to determine the whole genome sequence of an organism. Complete Genomics uses this technology to sequence samples submitted by independent researchers. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. Non-stranded sequencing by ligation is then used to determine the nucleotide sequence. This DNA sequencing method allows a large number of DNA nanoballs to be sequenced per run and at a low reagent cost compared to other next-generation sequencing platforms. However, only a short sequence of DNA is determined from each DNA nanoball, which makes it difficult to map short reads to a reference genome. The technology has been used for several genome sequencing projects and is planned to be used for more.

[0102] 8. Heliscope Single Molecule Sequencing Helicos sequencing is a method of single molecule sequencing developed by Helicos Biosciences. It uses DNA fragments with polyA tail adapters attached to the flow cell surface. The next step involves extension-based sequencing by periodic washing of the flow cell with fluorescently labeled nucleotides (one nucleotide type at a time, similar to the Sanger method). The reads are performed by a Helicos sequencer. The reads are short, up to 55 bases per run, but recent improvements allow more accurate reading of stretches of one type of nucleotide. This sequencing method and device were used to sequence the genome of the M13 bacteriophage.

[0103] 9. Single Molecule Real-time (SMRT) Sequencing SMRT sequencing is based on a sequencing by synthesis approach. DNA is synthesized in a small well-like container with a zero-mode waveguide (ZMW) - a capture tool located at the bottom of the well. Sequencing is performed with an unmodified polymerase (attached to the ZMW bottom) and fluorescently labeled nucleotides that flow freely in solution. The wells are constructed in such a way that only the fluorescence generated by the bottom of the well is detected. The fluorescent label is separated from the nucleotide upon incorporation into the DNA strand, leaving behind an unmodified DNA strand. According to Pacific Biosciences (SMRT technology developer), this methodology allows the detection of nucleotide modifications (such as cytosine methylation). This occurs through the observation of polymerase kinetics. This approach allows for reads of more than 20,000 nucleotides, with an average read length of 5 kilobases.

[0104] C. Measuring Gene or Barcode Expression The embodiments of the present disclosure relate to determining the expression of reporter barcodes and / or reporter genes or open reading frames. The expression of reporters can be determined by measuring the levels of the RNA transcripts of the barcode or index region and any other polynucleotides expressed from the reporter construct. Methods suitable for this purpose include, but are not limited to, RT-PCR, Northern blot, in situ hybridization, Southern blot, slot blot, nucleic acid protection assay and oligonucleotide array.

[0105] In certain aspects, RNA isolated from cells can be amplified to cDNA or cRNA prior to detection and / or quantification. The isolated RNA can be either total RNA or mRNA. RNA amplification can be specific or non-specific. In some embodiments, the amplification is specific in that it specifically amplifies the reporter barcode or a region thereof (e.g., index region). In some embodiments, the amplification and / or reverse transcriptase step excludes random priming. Suitable amplification methods include, but are not limited to, reverse transcriptase PCR, isothermal amplification, ligase chain reaction, and Qbeta replicase. Amplified nucleic acid products can be detected and / or quantified by hybridization to a labeled probe. In some embodiments, detection can include fluorescence resonance energy transfer (FRET) or other types of quantum dots.

[0106] Amplification primers or hybridization probes for reporter barcodes can be prepared from the sequence of the expressed portion of the reporter. The terms "primer" or "probe" as used herein are meant to encompass any nucleic acid capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides 10-20 and / or 30 base pairs in length, although longer sequences can be used. Primers are provided in double-stranded and / or single-stranded form, although single-stranded form is preferred.

[0107] The use of probes or primers between 13-100 nucleotides, particularly 17-100 nucleotides in length, or in some embodiments 1-2 kilobases or more in length, allows the formation of duplex molecules that are stable and selective. Molecules with complementary sequences over a contiguous stretch of more than 20 bases in length can be used to increase the stability and / or selectivity of the resulting hybrid molecules. Nucleic acid molecules for hybridization can be designed with one or more complementary sequences of 20-30 nucleotides, or longer if desired. Such fragments can be readily prepared, for example, by direct synthesis of the fragments by chemical means or by introducing selected sequences into a recombinant vector for recombinant production.

[0108] In one embodiment, each probe / primer comprises at least 15 nucleotides. For example, each probe may comprise at least or at most 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400 or more nucleotides (or any range derivable therein). They may have these lengths and may have sequences identical or complementary to the genes described herein. In particular, each probe / primer has a relatively high degree of sequence complexity and does not have any ambiguous residues (undetermined "n" residues). The probe / primer may hybridize to the target gene (including its RNA transcript) under stringent or highly stringent conditions. In some embodiments, each of the biomarkers has more than one human sequence, so it is contemplated that probes and primers may be designed for use with each of these sequences. For example, inosine is a nucleotide that is frequently used in probes or primers to hybridize to two or more sequences. It is contemplated that a probe or primer may have inosine or other design implementations that accommodate the recognition of two or more human sequences for a particular biomarker.

[0109] In applications requiring high selectivity, it is typically desirable to use relatively high stringency conditions to form hybrids. For example, relatively low salt and / or high temperature conditions, such as those provided by about 0.02M to about 0.10M sodium chloride at a temperature of about 50°C to about 70°C. Such high stringency conditions tolerate little, if any, mismatch between the probe or primer and the template or target strand, and are particularly suitable for isolating a specific gene or detecting a specific mRNA transcript. It is generally understood that conditions can be made more stringent by increasing the amount of formamide added.

[0110] In one embodiment, quantitative RT-PCR (e.g., TaqMan, ABI) is used to detect and compare the levels of RNA transcripts in samples. Quantitative RT-PCR involves reverse transcription (RT) of RNA into cDNA, followed by relative quantitative PCR (RT-PCR). The concentration of target DNA in the linear portion of the PCR method is proportional to the starting concentration of the target before PCR begins. By completing the same number of cycles and determining the concentration of the PCR products of the target DNA in PCR reactions that are in their linear range, the relative concentrations of specific target sequences in the original DNA mixture can be determined. If the DNA mixture is cDNA synthesized from RNA isolated from different tissues or cells, the relative abundance of the specific mRNA from which the target sequences are derived can be determined for each tissue or cell. This direct proportionality between the concentration of the PCR product and the amount of mRNA is true in the linear region of the PCR reaction. The final concentration of target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mixture and is independent of the original concentration of the target DNA. Thus, sampling and quantification of amplified PCR products can be performed when the PCR reactions are in the linear portion of their curves. Furthermore, the relative concentration of amplifiable cDNA can be normalized to some independent standard, which can be based on either internally present or externally introduced RNA species. The abundance of a particular mRNA species can also be determined relative to the average abundance of all mRNA species in the sample.

[0111] In one embodiment, PCR amplification utilizes one or more internal PCR standards.Internal standards can be abundant housekeeping genes in cells, or can be GAPDH, GUSB and β-2 microglobulin in particular.These standards can be used to normalize expression levels so that the expression levels of different gene products can be directly compared.Those skilled in the art know how to use internal standards to normalize expression levels.

[0112] A challenge inherent in some samples is that they are of varying quantity and / or quality. This problem can be overcome if RT-PCR is performed as relative quantitative RT-PCR, where the internal standard is an amplifiable cDNA fragment similar or larger than the target cDNA fragment, and the abundance of the mRNA encoding the internal standard is approximately 5-100 times higher than the mRNA encoding the target. This assay measures the relative abundance of each mRNA species, rather than the absolute abundance.

[0113] In another embodiment, relative quantitative RT-PCR uses an external reference protocol. In this protocol, PCR products are sampled in the linear part of their amplification curve. The optimal number of PCR cycles for sampling can be empirically determined for each target cDNA fragment. Furthermore, reverse transcriptase products of each RNA population isolated from various samples can be standardized for equal concentration of amplifiable cDNA.

[0114] A nucleic acid array may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more different polynucleotide probes that may hybridize to different and / or the same biomarkers. Multiple probes for the same gene may be used on a single nucleic acid array. Probes for other disease genes may also be included in the nucleic acid array. The probe density on the array may be in any range. In some embodiments, the concentration is 50, 100, 200, 300, 400, 500 or more probes / cm. 2 It could be.

[0115] Particularly contemplated are chip-based nucleic acid technologies such as those described by Hacia et al. (1996) and Shoemaker et al. (1996). Briefly, these technologies include quantitative methods for rapid and accurate analysis of a large number of genes. By tagging genes with oligonucleotides or using fixed probe arrays, target molecules can be separated as high-density arrays, and chip technology can be used to screen these molecules based on hybridization (see also Pease et al., 1994; and Fodor et al., 1991). It is contemplated that this technology can be used in conjunction with evaluating the expression level of one or more cancer biomarkers for diagnostic, prognostic, and treatment methods.

[0116] Certain embodiments may include the use of arrays or data generated from arrays. The data is readily available. Furthermore, arrays may be prepared to generate data that may be used in correlation experiments.

[0117] Array generally refers to an ordered macroarray or microarray of nucleic acid molecules (probes) that are perfectly or nearly complementary or identical to multiple mRNA or cDNA molecules and located on a support material in a spatially separated configuration. Macroarrays are typically sheets of nitrocellulose or nylon onto which the probes are spotted. Microarrays arrange the nucleic acid probes more densely, such that up to 10,000 nucleic acid molecules can fit in an area typically of 1-4 square centimeters. Microarrays can be made by spotting nucleic acid molecules (e.g., genes, oligonucleotides, etc.) onto a substrate or by fabricating oligonucleotide sequences in situ on a substrate. Spotted or fabricated nucleic acid molecules can be applied in a high density matrix pattern of up to about 30 or more non-identical nucleic acid molecules per square centimeter, for example up to about 100 or even 1000 non-identical nucleic acid molecules per square centimeter. Microarrays typically use coated glass as the solid support, as opposed to the nitrocellulose-based material of filter arrays. By having an ordered sequence of complementary nucleic acid samples, the location of each sample can be tracked and linked to the original sample. A variety of different array devices, in which a plurality of separate nucleic acid probes are stably associated with the surface of a solid support, are known to those skilled in the art. Useful substrates for arrays include nylon, glass and silicon. Such arrays can vary in many different ways, including average probe length, sequence or type of probe, nature of binding (e.g., covalent or non-covalent) between probe and array surface, and the like. The labeling and screening methods and arrays are not limited in their usefulness with respect to any parameter, except that the probe detects expression level; as a result, the methods and compositions can be used with a variety of different types of genes.

[0118] Representative methods and apparatus for preparing microarrays are described, for example, in U.S. Pat. Nos. 5,143,854, 5,202,231, 5,242,974, 5,288,644, 5,324,633, 5,384,261, 5,405,783, 5,412,087, 5,424,186, 5,429,807, 5,432,049, 5,436,327, 5,445,934, 5,468,613, 5,470,710, 5,472,672, 5, No. 492,806, No. 5,525,464, No. 5,503,980, No. 5,510,270, No. 5,525,464, No. No. 5,527,681, No. 5,529,756, No. 5,532,128, No. 5,545,531, No. 5,547,839 , No. 5,554,501, No. 5,556,752, No. 5,561,071, No. 5,571,639, No. 5,580,72 No. 6, No. 5,580,732, No. 5,593,839, No. 5,599,695, No. 5,599,672, No. 5,610;No. 287, No. 5,624,711, No. 5,631,134, No. 5,639,603, No. 5,654,413, No. 5,658,734, No. 5,661,028, No. 5, No. 665,547, No. 5,667,972, No. 5,695,940, No. 5,700,637, No. 5,744,305, No. 5,800,992, No. 5,807,522, Nos. 5,830,645, 5,837,196, 5,871,928, 5,847,219, 5,876,932, 5,919,626, 6,004,755, 6,087,102, 6,368,799, 6,383,749, 6,617,112, 6,638,717, 6,720,138, and WO 93 / 17126, WO 95 / 11995, WO 95 / 21265, WO 95 / 21944, WO 95 / 35505, WO 96 / 31622, WO 97 / 10365, WO 97 / 27317, WO 99 / 35505, WO 09923256, WO 09936760, WO0138580, WO 0168255, WO 03020898, WO 03040410, WO 03053586, WO 03087297, WO 03091426, WO03100012, WO 04020085, WO No. 04027093, EP 373 203, EP 785 280, EP 799 897 and UK 8 803 000, the disclosures of which are all incorporated herein by reference.

[0119] It is contemplated that the arrays may be high density arrays, such as containing 100 or more different probes. It is contemplated that they may contain 1000, 16,000, 65,000, 250,000 or 1,000,000 or more different probes. The oligonucleotide probes, in some embodiments, range in length from 5 to 50, 5 to 45, 10 to 40, or 15 to 40 nucleotides. In certain embodiments, the oligonucleotide probes are 20 to 25 nucleotides in length.

[0120] The location and sequence of each different probe sequence in the array is generally known. 2 The surface area of ​​the array can occupy a relatively small area providing a high density array having a probe density of generally greater than about 60, 100, 600, 1000, 5,000, 10,000, 40,000, 100,000, or 400,000 different oligonucleotide probes per square centimeter. 2 , or approximately 1, 1.6, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm 2 It may be less than.

[0121] Moreover, one skilled in the art can easily analyze the data generated using the array. Such protocols include the information found in WO9743450; WO030023058; WO03022421; WO03029485; WO03029485; WO03067217; WO03066906; WO03076928; WO03093810; WO03100448A1, all of which are incorporated herein by reference.

[0122] In one embodiment, nuclease protection assay is used to quantify RNA from cancer samples. There are many different versions of nuclease protection assays known to those skilled in the art. The common feature that these nuclease protection assays have is that they include hybridization of antisense nucleic acid with the RNA to be quantified. The resulting hybrid double-stranded molecule is then digested with a nuclease that digests single-stranded nucleic acid more efficiently than double-stranded molecules. The amount of antisense nucleic acid that survives digestion is a measure of the amount of the target RNA species to be quantified. An example of a commercially available nuclease protection assay is the RNase protection assay manufactured by Ambion, Inc. (Austin, TX).

[0123] III. Receptor gene and inducible reporter addition In certain embodiments, the reporter gene and / or inducible reporter system comprises one or more polynucleotide sequences encoding one or more auxiliary polypeptides. Exemplary auxiliary polypeptides include transcription factors, protein or peptide tags, and screenable or selectable genes.

[0124] A. Gene Selection and Screening In certain embodiments of the present disclosure, the inducible reporter and / or receptor gene may include or further include a selection or screening gene. Additionally, the cells, vectors, and viral particles of the present disclosure may further include a selection or screening gene. In some embodiments, the selection or screening gene is fused to the receptor gene such that there is one fusion protein in the cell that includes the receptor protein fused to the selection or screening protein. Such a gene confers an identifiable change to the cell, allowing easy identification of cells with activation of the heterologous receptor gene. In general, a selectable (i.e., selection gene) gene is a gene that confers a property that allows for selection. A positive selection gene is a gene whose presence of the gene or gene product allows for its selection, while a negative selection gene is a gene whose presence of the gene or gene product prevents its selection. An example of a positive selection gene is an antibiotic resistance gene.

[0125] Typically, the inclusion of a drug selection gene aids in the cloning and identification of cells with an activated receptor gene, for example, via successful ligand engagement. Selection genes can be genes that confer resistance to, for example, neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, G418, phleomycin, blasticidin, and histidinol. In addition to genes that confer a phenotype that allows for the identification of receptor activation based on the implementation of a condition, other types of genes are also contemplated, including screenable genes such as GFP, whose gene products provide colorimetric analysis. Alternatively, screenable enzymes (e.g., herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT)) can be utilized. Those skilled in the art will also know how to use screenable genes and their protein products, possibly in combination with FACS analysis. Further examples of selectable and screenable genes are well known to those skilled in the art. In certain embodiments, the gene produces a fluorescent protein, an enzymatically active protein, a luminescent protein, a photoactivatable protein, a photoconvertible protein, or a colorimetric protein. Fluorescent markers include, for example, GFP and variants such as YFP, RFP, and other fluorescent proteins such as DsRed, mPlum, mCherry, YPet, Emerald, CyPet, T-Sapphire, Luciferase, and Venus. Photoactive markers include, for example, KFP, PA-mRFP, and Dronpa. Photoconvertible markers include, for example, mEosFP, KikGR, PS-CFP2, and the like. Luminescent proteins include, for example, Neptune, FP595, and phiarisin.

[0126] B. Protein or Peptide Tags Exemplary protein / peptide tags include AviTag, a peptide that allows biotinylation by the enzyme BirA, allowing the protein to be separated by streptavidin (GLNDIFEAQKIEWHE, SEQ ID NO: 4); calmodulin tag, a peptide bound by the protein calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL, SEQ ID NO: 5); polyglutamic acid tag, a peptide that efficiently binds to anion exchange resins such as Mono-Q (EEEEEE, SEQ ID NO: 6); E tag, a peptide recognized by an antibody (GAPVPYPDPLEPR, SEQ ID NO: 7); FLAG tag, a peptide recognized by an antibody (DYKDDDDK, SEQ ID NO: 8); HA tag, a peptide derived from hemagglutinin recognized by an antibody (YPYDVPDYA, SEQ ID NO: 9); His tag, 5-10 histidines bound to a nickel or cobalt chelate (HHHHHH, SEQ ID NO: 10); Myc-tag, a peptide derived from c-myc recognized by an antibody. a peptide derived from ribonuclease A (EQKLISEEDL, SEQ ID NO: 11); NE-tag, a novel 18 amino acid synthetic peptide recognized by a monoclonal IgG1 antibody (TKENPRSNQEESYDDNES, SEQ ID NO: 12), which is useful for a wide range of applications including Western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation and affinity purification of recombinant proteins; S-tag, a peptide derived from ribonuclease A (KETAAAKFERQHMDS, SEQ ID NO: 13); SBP-tag, a peptide that binds to streptavidin (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP, SEQ ID NO: 14); Softag1, for mammalian expression (SLAELLNAGLGGS, SEQ ID NO: 15); Softag3, for prokaryotic expression (TQDPSRVG, SEQ ID NO: 16); Strep-tag, a peptide that binds to streptavidin or a modified streptavidin called streptactin (Strep-tag II: WSHPQFEK, SEQ ID NO: 17); TC tag, a tetracysteine ​​tag recognized by FlAsH and ReAsH biarsenical compounds (CCPGCC, SEQ ID NO: 18); V5 tag, a peptide recognized by an antibody (GKPIPNPLLGLDST, SEQ ID NO: 19);VSV tag, a peptide recognized by an antibody (YTDIEMNRLGK, SEQ ID NO: 20); Xpress tag (DLYDDDDK, SEQ ID NO: 21); covalent peptide tag, isopeptag, a peptide covalently attached to the pyrin-C protein (TDKDMTITFTNKKDAE, SEQ ID NO: 22); SpyTag, a peptide covalently attached to the SpyCatcher protein (AHIVMVDAYKPTK, SEQ ID NO: 23); SnoopTag, a peptide covalently attached to the SnoopCatcher protein (KLGDIEFIKVNK, SEQ ID NO: 24); BCCP (biotin carboxyl carrier protein), a peptide covalently attached to streptavidin These include: a protein domain biotinylated by BirA, allowing recognition by IgG; glutathione-S-transferase-tag, a protein that binds immobilized glutathione; green fluorescent protein tag, a protein that is spontaneously fluorescent and can bind to nanobodies; HaloTag, a mutant bacterial haloalkane dehalogenase that covalently binds reactive haloalkane substrates, allowing binding to a wide variety of substrates; maltose-binding protein tag, a protein that binds amylose-agarose; Nus-tag, a thioredoxin tag; Fc-tag, derived from the immunoglobulin Fc domain, allowing dimerization and solubilization; engineered intrinsic disorder tags, including disorders that facilitate amino acids (P, E, S, T, A, Q, G, etc.), and Ty-tag, which can be used for purification on Protein A Sepharose;

[0127] C. Transcription factors In some embodiments, the receptor gene encodes a fusion protein comprising a receptor protein and an auxiliary polypeptide. In some embodiments, the auxiliary polypeptide is a transcription factor. In related embodiments, the inducible reporter comprises a receptor responsive element, where the receptor responsive element is bound by a transcription factor. Such transcription factors and response elements are known in the art and include, for example, reverse tetracycline-controlled transactivator (rtTA), which can induce transcription via a tetracycline responsive element (TRE), Gal4p, which induces transcription via the GAL1 promoter, and estrogen receptor, which induces expression via an estrogen response element when bound to a ligand. Thus, related embodiments include administering a ligand to activate transcription of the auxiliary polypeptide transcription factor.

[0128] IV. Vectors and Nucleic Acids The present disclosure includes embodiments of nucleic acids including one or more of heterologous receptor genes and inducible reporters. The terms "oligonucleotide", "polynucleotide", and "nucleic acid" are used interchangeably and include linear oligomers of natural or modified monomers or bonds (including deoxyribonucleosides, ribonucleosides, their α-anomeric forms, peptide nucleic acids (PNAs), etc.) that can specifically bind to a target polynucleotide by regular patterns of monomer-monomer interactions such as Watson-Crick base pairing, base stacking, Hoogsteen or reverse Hoogsteen base pairing, etc. Typically, the monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., 3-4, to tens of monomeric units. Whenever an oligonucleotide is represented by a series of letters such as "ATGCCTG", it will be understood that unless otherwise specified, the nucleotides are in 5'→3' order from left to right, "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine. Phosphodiester bond analogs include phosphorothioates, phosphorodithioates, phosphoranilidates, phosphoramidates, and the like. It will be apparent to one of skill in the art when oligonucleotides with natural or non-natural nucleotides can be used (e.g., when enzymatic processing is required), but usually when oligonucleotides consisting of natural nucleotides are required.

[0129] A nucleic acid may be an "unmodified oligonucleotide" or "unmodified nucleic acid," which generally refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the nucleic acid molecule is an unmodified oligonucleotide. The term includes oligonucleotides composed of naturally occurring nucleobases, sugars, and covalent internucleoside linkages. The term "oligonucleotide analog" refers to an oligonucleotide having one or more non-naturally occurring moieties that function in a manner similar to an oligonucleotide. Such non-naturally occurring oligonucleotides are often selected over naturally occurring forms for desirable properties (e.g., enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets, and increased stability in the presence of nucleases). The term "oligonucleotide" can be used to refer to an unmodified oligonucleotide or an oligonucleotide analog.

[0130] Specific examples of nucleic acid molecules include nucleic acid molecules that contain modified, i.e., non-naturally occurring, internucleoside linkages. Such non-natural internucleoside linkages are often selected over naturally occurring forms for desirable properties, such as, for example, enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets, and increased stability in the presence of nucleases. In specific embodiments, the modification comprises a methyl group.

[0131] Nucleic acid molecule can have one or more modified internucleoside bonds.As defined herein, the oligonucleotide with modified internucleoside bonds includes the internucleoside bonds that hold phosphorus atom and the internucleoside bonds that do not have phosphorus atom.For the purpose of this specification, and as sometimes referred to in the art, the modified oligonucleotide that does not have phosphorus atom in internucleoside backbone can also be considered as oligonucleoside.

[0132] Modifications to the nucleic acid molecule can include those in which one or both terminal nucleotides are modified.

[0133] One suitable phosphorus-containing modified internucleoside linkage is the phosphorothioate internucleoside linkage. Many other modified oligonucleotide backbones (internucleoside linkages) are known in the art and may be useful in the context of this embodiment.

[0134] Representative United States patents which teach the preparation of phosphorus-containing internucleoside linkages include U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5, These include, but are not limited to, Nos. 466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361, 5,194,599, 5,565,555, 5,527,899, 5,721,218, 5,672,697, 5,625,050, 5,489,677, and 5,602,240.

[0135] Modified oligonucleoside backbones (internucleoside linkages) that do not contain phosphorus atoms have short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or internucleoside linkages formed by one or more short chain heteroatom or heterocyclic internucleoside linkages. These include those with amide backbones; and others, including those with mixed N, O, S and CH2 component moieties.

[0136] Representative U.S. patents which teach the preparation of the above non-phosphorus containing oligonucleosides include U.S. Pat. Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541, 307, 5,561,225, 5,596,086, 5,602,240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, 5,792,608, 5,646,269, and 5,677,439.

[0137] Oligomeric compounds can also include oligonucleotide mimetics.The term mimetics as applied to oligonucleotides is intended to include oligomeric compounds in which only the furanose ring or both the furanose ring and internucleotide bond are replaced with novel groups, and only the furanose ring is replaced with, for example, morpholino ring, also known in the art as sugar substitutes.Heterocyclic base moiety or modified heterocyclic base moiety is maintained for hybridization with appropriate target nucleic acid.

[0138] Oligonucleotide mimetics can include oligomeric compounds such as peptide nucleic acid (PNA) and cyclohexenyl nucleic acid (known as CeNA, see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). Representative United States patents that teach the preparation of oligonucleotide mimetics include, but are not limited to, United States Patents 5,539,082, 5,714,331, and 5,719,262, each of which is incorporated herein by reference. Another class of oligonucleotide mimetics is called phosphonomonoester nucleic acid, which incorporates phosphorus groups into the backbone. This class of oligonucleotide mimetics has been reported to have useful physical and biological and pharmacological properties in the area of ​​inhibiting gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex-forming oligonucleotides), as probes for the detection of nucleic acids, and as aids for use in molecular biology. Another oligonucleotide mimetic has been reported in which the furanosyl ring is replaced by a cyclobutyl moiety.

[0139] The nucleic acid molecule may also contain one or more modified or substituted sugar moieties. The base moiety is appropriately maintained for hybridization with the nucleic acid target compound. The sugar modification may confer nuclease stability, binding affinity, or some other advantageous biological property to the oligomeric compound.

[0140] Representative modified sugars include carbocyclic or acyclic sugars, sugars with a substituent at one or more of their 2', 3' or 4' positions, sugars with a substituent in place of one or more hydrogen atoms of the sugar, and sugars with a bond between any two other atoms in the sugar. Numerous sugar modifications are known in the art, and sugars modified at the 2' position and sugars with a bridge between any two atoms of the sugar (such that the sugar is bicyclic) are particularly useful in this embodiment. Examples of sugar modifications useful in this embodiment include, but are not limited to, compounds containing sugar substituents selected from OH; F; O-, S-, or N-alkyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Particularly preferred are 2-methoxyethoxy (also known as 2'-O-methoxyethyl, 2'-MOE, or 2'-OCH2CHOCH3), 2'-O-methyl (2'-O--CH3), 2'-fluoro (2'-F), or bicyclic sugar modified nucleosides having a bridging group connecting the 4' carbon atom to the 2' carbon atom, exemplary bridging groups include --CH2--O--, --(CH2)2--O-- or --CH2--N(R3)--O, where R3 is H or C1-C12 alkyl.

[0141] One modification that confers increased nuclease resistance and very high binding affinity to nucleotides is the 2'-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). One immediate advantage of the 2'-MOE substitution is the increased binding affinity, which is greater than that of many similar 2' modifications such as O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides with 2'-MOE substituents have also been shown to be antisense inhibitors of gene expression with promising characteristics for in vivo use (Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

[0142] The 2'-sugar substituent may be at the arabino (up) or ribo (down) position. One 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3' position of the sugar on the 3' terminal nucleoside, or the 5' position of 2'-5' linked oligonucleotides and 5' terminal nucleotides. Oligomeric compounds may also have sugar mimetics, such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include U.S. Pat. Nos. 4,981,957, 5,118,800, 5,319,0800, 5,359,044, 5,393,878, 5,446,137, 5,446,786, 5,514,785, 5,519,134, and the like, which are incorporated by reference herein in their entireties. Nos. 5,567,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, 5,792,747, and 5,700,920.

[0143] Nucleic acid molecules can also contain one or more nucleobase (often simply referred to in the art as "base") modifications or substitutions that are structurally distinct from naturally occurring or synthetic unmodified nucleobases, but functionally interchangeable. Such nucleobase modifications can impart nuclease stability, binding affinity, or some other advantageous biological property to the oligomeric compound. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases, also referred to herein as heterocyclic base moieties, include other synthetic and natural nucleobases, many examples of which include, among others, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, 7-deazaguanine and 7-deazadenine.

[0144] Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Some nucleobases include those disclosed in U.S. Pat. No. 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, Angewandte Chemie, International Edition, 1991, 30, 613, published by Englisch et al., and Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993, published by Sanghvi, YS. Certain of these nucleobases are particularly effective in increasing the binding affinity of oligomeric compounds. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.

[0145] Additional modifications of the nucleic acid molecules are disclosed in US Patent Publication 2009 / 0221685, which is incorporated herein by reference. Additional suitable conjugates for the nucleic acid molecules are also disclosed herein.

[0146] The heterologous receptor gene and the inducible reporter can be encoded by a nucleic acid molecule, such as a vector. In some embodiments, they are encoded on the same nucleic acid molecule. In some embodiments, they are encoded on separate nucleic acid molecules. In certain embodiments, the nucleic acid molecule can be in the form of a nucleic acid vector. The term "vector" is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell, where it can be replicated, expressed, and / or integrated into the genome of the host cell. The nucleic acid sequence can be "heterologous", meaning that it is foreign to the cell into which it is being introduced or to the nucleic acid into which it is being integrated, including sequences that are homologous to sequences in the cell or nucleic acid, but are at a location within the host cell or nucleic acid where they are not normally found. Vectors include DNA, RNA, plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will be well versed in constructing vectors through standard recombinant techniques (e.g., Sambrook et al., 2001; Ausbel et al., 1996, both incorporated by reference). The vectors can be used in host cells to produce antibodies.

[0147] The term "expression vector" refers to a vector that contains a nucleic acid sequence that is transcribed or stably integrated into the genome of a host cell and that encodes at least a portion of a gene product that can be subsequently transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide or peptide. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors can also contain nucleic acid sequences that perform other functions and are described herein.

[0148] The vector disclosed herein can be any nucleic acid vector known in the art. Exemplary vectors include plasmids, cosmids, bacterial artificial chromosomes (BAC) and viral vectors.

[0149] Any expression vector for animal cells can be used. Examples of suitable vectors include pAGE107 (Miyaji et al., 1990), pAGE103 (Mizukami and Itoh, 1987), pHSG274 (Brady et al., 1984), pKCR (O'Hare et al., 1981), pSG1βd2-4 (Miyaji et al., 1990), etc.

[0150] Other examples of plasmids include replicative plasmids that contain an origin of replication, or integrative plasmids (eg, pUC, pcDNA, pBR, etc.).

[0151] Other examples of viral vectors include adenovirus, lentivirus, retrovirus, herpes virus and AAV vector. Such recombinant viruses can be produced by techniques known in the art (e.g., by transfecting packaging cells or by transient transfection with helper plasmid or virus). Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed procedures for producing such replication-defective recombinant viruses can be found in WO95 / 14785, WO96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056 and WO94 / 19478.

[0152] A "promoter" is a regulatory sequence. A promoter is typically a region of a nucleic acid sequence where the initiation and rate of transcription are controlled. It may contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation relative to a nucleic acid sequence to control the transcription initiation and expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0153] Examples of promoters and enhancers used in expression vectors for animal cells include the SV40 early promoter and enhancer (Mizukami and Itoh, 1987), the Moloney murine leukemia virus LTR promoter and enhancer (Kuwana et al., 1987), and the immunoglobulin H chain promoter (Mason et al., 1985) and enhancer (Gillies et al., 1983).

[0154] Specific initiation signals may also be required for efficient translation of the coding sequence. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would be able to readily determine this and provide the necessary signals.

[0155] A vector can contain a multiple cloning site (MCS), a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant techniques to digest the vector. (See Carbonelli et al., 1999; Levenson et al., 1998; and Cocea, 1997, incorporated herein by reference.)

[0156] Most transcribed eukaryotic RNA molecules undergo RNA splicing to remove introns from the primary transcript. Vectors containing genomic eukaryotic sequences may require donor and / or acceptor splice sites to ensure proper processing of the transcript for protein expression. (See Chandler et al., 1997, incorporated herein by reference.)

[0157] A vector or construct generally includes at least one termination signal. A "termination signal" or "terminator" consists of a DNA sequence involved in the specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments, a termination signal that terminates the production of an RNA transcript is intended. A terminator may be essential in vivo to achieve a desired message level. In eukaryotic systems, the terminator region may also include a specific DNA sequence that allows for region-specific cleavage of the new transcript to expose a polyadenylation region. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail are more stable and appear to be translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that the terminator includes a signal for cleavage of the RNA, and more preferably that the terminator signal promotes polyadenylation of the message.

[0158] In expression, particularly eukaryotic expression, a polyadenylation signal is typically included to provide for proper polyadenylation of the transcript.

[0159] To propagate a vector in a host cell, the vector may contain one or more origin of replication sites (often called "ori"), which are specific nucleic acid sequences at which replication is initiated. Alternatively, when the host cell is yeast, an autonomously replicating sequence (ARS) can be used.

[0160] Some vectors may use control sequences that allow them to be replicated and / or expressed in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions under which all of the above host cells are incubated to maintain them and allow the replication of the vector. Also understood and known are the techniques and conditions that allow large-scale production of vectors and the production of nucleic acids encoded by the vectors and their cognate polypeptides, proteins, or peptides.

[0161] Further aspects of the present disclosure relate to a cell or cells comprising a receptor gene and an inducible reporter as described herein. In some embodiments, a prokaryotic or eukaryotic cell is genetically transformed or transfected with at least one nucleic acid molecule or vector according to the present disclosure. In some embodiments, the cell is infected with a viral particle of the present disclosure.

[0162] The terms "transformation" or "transfection" refer to the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA or RNA sequence into a host cell, such that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA has been "transformed" or "transfected." Construction of expression vectors and transformation or transfection of host cells according to the present disclosure can be carried out using conventional molecular biology techniques.

[0163] Methods suitable for nucleic acid delivery for transformation / transfection of cells, tissues or organisms for use in the present invention are intended to include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into a cell, tissue or organism, as described herein or known to those of skill in the art. (See, e.g., Stadtfeld and Hochedlinger, Nature Methods 6(5):329-330 (2009); Yusa et al., Nat. Methods 6:363-369 (2009); Woltjen et al., Nature 458, 766-770 (9 Apr. 2009)). Such methods include ex vivo transfection (Wilson et al., Science, 244:1344-1346, 1989, Nabel and Baltimore, Nature 326:711-713, 1987), optionally with Fugene6 (Roche) or Lipofectamine (Invitrogen), by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,469, and 5,580,859), by microinjection (Harland and Weintraub, J. Cell Biol., 101:1094-1099, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter et al., Proc. Nat'l Acad. Sci. USA, 81:7161-7165, 1984), by calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10:689-695, 1990), DEAE-dextran followed by polyethylene glycol (Gopal, Mol. Cell Biol., 5:1188-1190, 1985); direct sonication (Fechheimer et al., Proc. Nat'l Acad. Sci. USA, 84:8463-8467, 1987), liposome-mediated transfection (Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982; Fraley et al., Proc. Nat'l Acad. Sci. USA, 76:3348-3352, 1979; Nicolau et al., Methods Enzymol., 149:157-176, 1987; Wong et al., Gene, 10:87-94, 1980; Kaneda et al., Science, 243:375-378, 1989; Kato et al., J. Biol. Chem., 266:3361-3364, 1991), and by receptor-mediated transfection (Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987); as well as any combination, each of which is incorporated herein by reference.

[0164] V. Cell As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include both freshly isolated cells and cells cultured or grown in vitro. All of these terms also include their progeny, which are any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing heterologous nucleic acid sequences, "host cell" or simply "cell" refers to a prokaryotic or eukaryotic cell, including any transformable organism capable of replicating a vector or expressing a heterologous gene encoded by a vector or incorporated nucleic acid. Host cells can be and have been used as recipients of vectors, viruses, and nucleic acids. Host cells can be "transfected" or "transformed," which refers to the process by which exogenous nucleic acid, such as a sequence encoding a recombinant protein, is transferred or introduced into a host cell. Transformed cells include the primary subject cell and its progeny.

[0165] In certain embodiments, nucleic acid transfer can be performed on any prokaryotic or eukaryotic cell.In some embodiments, the cell of the present disclosure is a human cell.In other embodiments, the cell of the present disclosure is an animal cell.In some embodiments, one or more cells are cancer cells, tumor cells, or immortalized cells.In further embodiments, the cell represents a disease model cell. In certain embodiments, the cells are selected from the group consisting of A549, B cells, B16, BHK-21, C2C12, C6, CaCo-2, CAP / , CAP-T, CHO, CH02, CHO-DG44, CHO-K1, COS-1, Cos-7, CV-1, dendritic cells, DLD-1, embryonic stem (ES) cells or derivatives, H1299, HEK, 293, 293T, 293FT, Hep G2, hematopoietic stem cells, HOS, Huh-7, induced pluripotent stem (iPS) cells or derivatives, Jurkat, K562, L5278Y, LNCaP, MCF7, MDA-MB-231, MDCK, mesenchymal cells, Min-6, monocytic cells, Neuro2a, NIH The cells may be 3T3, NIH3T3L1, K562, NK cells, NS0, Panc-1, PC12, PC-3, peripheral blood cells, plasma cells, primary fibroblasts, RBL, Renca, RLE, SF21, SF9, SH-SY5Y, SK-MES-1, SK-N-SH, SL3, SW403, Stimulus-induced Acquisition of Pluripotency (STAP) cells or derivatives SW403, T cells, THP-1, tumor cells, U2OS, U937, peripheral blood lymphocytes, expanded T cells, hematopoietic stem cells, or Vero cells. In some embodiments, the cells are HEK293T cells.

[0166] The term "passage" as used herein is intended to refer to the process of dividing cells to generate a large number of cells from existing cells. Cells may be passaged multiple times before or after any of the steps described herein. Passage involves splitting cells and transferring a small number to each new container. For solid cultures, the cells must first be detached, typically by mixing trypsin-EDTA. A small number of detached cells can then be used to seed a new culture, while the rest are discarded. Also, the amount of cultured cells can be easily increased by distributing all the cells into a fresh flask. Cells can be kept in culture and incubated under conditions that allow cell replication. In some embodiments, cells are maintained in culture conditions that allow the cells to divide 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.

[0167] In some embodiments, cells can be subjected to limiting dilution to allow expansion of a clonal population of cells. Methods of limiting dilution cloning are well known to those skilled in the art. Such methods have been described, for example, for hybridomas, but can be applied to any cell. Such methods are incorporated herein by reference (Cloning hybridoma cells by limiting dilution, Journal of tissue culture methods, 1985, Volume 9, Issue 3, pp 175-177, by Joan C. Rener, Bruce L. Brown, and Roland M. Nardone).

[0168] The disclosed method includes culturing cells. Methods for culturing suspension and adherent cells are well known to those skilled in the art. In some embodiments, cells are cultured in suspension using commercially available cell culture vessels and cell culture media. Examples of commercially available culture vessels that may be used in some embodiments include ADME / TOX plates, cell chamber slides and cover slips, cell counting devices, cell culture surfaces, Corning HYPERFlask cell culture vessels, coated culture vessels, Nalgene Cryoware, culture chambers, culture dishes, glass culture flasks, plastic culture flasks, 3D culture formats, culture multi-well plates, culture plate inserts, glass culture tubes, plastic culture tubes, stackable cell culture vessels, hypoxic culture chambers, Petri dishes and flask carriers, QuickFit culture vessels, scale-up cell culture using roll bottles, spinner flasks, 3D cell culture, or cell culture bags.

[0169] In other embodiments, the medium may be formulated using components well known to those of skill in the art. Preparations and methods for culturing cells are described in detail in the following references: Short Protocols in Cell Biology J. Bonifacino, et al., ed., John Wiley & Sons, 2003, 826 pp; Live Cell Imaging: A Laboratory Manual D. Spector & R. Goldman, ed., Cold Spring Harbor Laboratory Press, 2004, 450 pp.; Stem Cells Handbook S. Sell, ed., Humana Press, 2003, 528 pp.; Animal Cell Culture: Essential Methods, John M. Davis, John Wiley & Sons, Mar 16, 2011; Basic Cell Culture Protocols, Cheryl D. Helgason, Cindy Miller, Humana Press, 2005; Human Cell Culture Protocols, Series: Methods in Molecular Biology, Vol. 806, Mitry, Ragai R.; Hughes, Robin D. (Eds.), 3rd ed. 2012, XIV, 435 p. 89, Humana Press; Cancer Cell Culture: Method and Protocols, Cheryl D. Helgason, Cindy Miller, Humana Press, 2005; Human Cell Culture Protocols, Series: Methods in Molecular Biology, Vol. 806, Mitry, Ragai R.; Hughes, Robin D. (Eds.), 3rd ed. 2012, XIV, 435 p. 89, Humana Press; Cancer Cell Culture: Methods and Protocols, Simon P.Langdon, Springer, 2004; Molecular Cell Biology. 4th edition., Lodish H, Berk A, Zipursky SL, et al., New York: WH Freeman; 2000., Section 6.2 Growth of Animal Cells in Culture, all of which are incorporated herein by reference.

[0170] VI. Genomic integration of nucleic acids A. Targeted Integration The present disclosure provides a method for targeting the integration of nucleic acid. This is also referred to herein and in the art as "gene editing". In some embodiments, targeted integration is achieved by using DNA digestion agents / polynucleotide modifying enzymes, such as site-specific recombinases and / or targeting endonucleases. The term "DNA digestion agent" refers to an agent that can break the bond (i.e., phosphodiester bond) between nucleotide subunits of nucleic acid.

[0171] In one embodiment, the present disclosure includes targeted integration. One way to achieve this is by using an exogenous nucleic acid sequence (i.e., a landing pad) that contains at least one recognition sequence for at least one polynucleotide-modifying enzyme, such as a site-specific recombinase and / or a targeting endonuclease. Site-specific recombinases are well known in the art and may generally be referred to as invertases, resolvases, or integrases. Non-limiting examples of site-specific recombinases may include lambda integrase, Cre recombinase, FLP recombinase, gamma delta resolvase, Tn3 resolvase, ΦC31 integrase, Bxb1 integrase, and R4 integrase. Site-specific recombinases recognize specific recognition sequences (or recognition sites) or variants thereof, all of which are well known in the art. For example, Cre recombinase recognizes LoxP sites, and FLP recombinase recognizes FRT sites.

[0172] Contemplated targeting endonucleases include zinc finger nucleases (ZFN), meganucleases, transcription activator-like effector nucleases (TALEN), CRIPSR / Cas-like endonucleases, I-Tevl nucleases or related monomer hybrids, or artificial targeting DNA double strand break inducers.Exemplary targeting endonucleases are described further below.For example, typically, zinc finger nucleases include a DNA binding domain (i.e., zinc finger) and a cleavage domain (i.e., nuclease), both of which are described below.The definition of polynucleotide modifying enzyme also includes any other useful fusion protein known to those skilled in the art, which may include a DNA binding domain and a nuclease.

[0173] A landing pad sequence is a nucleotide sequence that contains at least one recognition sequence that is selectively bound and modified by a specific polynucleotide modifying enzyme, such as a site-specific recombinase and / or a targeting endonuclease. In general, the recognition sequence in the landing pad sequence is not endogenously present in the genome of the cell to be modified. For example, if the cell to be modified is a CHO cell, the recognition sequence in the landing pad sequence is not endogenously present in the endogenous CHO genome. The rate of targeted integration can be improved by selecting a recognition sequence of a highly efficient nucleotide modifying enzyme that is not endogenously present in the genome of the target cell. The selection of a recognition sequence that is not endogenously present also reduces potential off-target integration. In other embodiments, it may be desirable to use a recognition sequence that is native in the cell to be modified. For example, when multiple recognition sequences are used in a landing pad sequence, one or more can be exogenous and one or more can be natural.

[0174] One of skill in the art can readily determine the sequences bound and cleaved by a site-specific recombinase and / or a targeting endonuclease.

[0175] Multiple recognition sequences may be present in a single landing pad, allowing the landing pad to be targeted sequentially by two or more polynucleotide modifying enzymes so that two or more unique nucleic acids (including, among others, receptor genes and / or inducible reporters) can be inserted. Alternatively, the presence of multiple recognition sequences in a landing pad allows multiple copies of the same nucleic acid to be inserted into the landing pad. When two nucleic acids are targeted to a single landing pad, the landing pad contains a first recognition sequence for a first polynucleotide modifying enzyme (e.g., a first ZFN pair) and a second recognition sequence for a second polynucleotide modifying enzyme (e.g., a second ZFN pair). Alternatively, or in addition, individual landing pads containing one or more recognition sequences may be integrated at multiple locations. Increased protein expression may be observed in cells transformed with multiple copies of a payload. Alternatively, it is also possible to simultaneously express multiple gene products in cells transformed with multiple copies when multiple unique nucleic acid sequences constituting different expression cassettes are loaded into the same or different landing pads. Regardless of the number and type of nucleic acids, when the targeting endonuclease is a ZFN, an exemplary ZFN pair includes hSIRT, hRSK4, and hAAVS1, along with their recognition sequences.

[0176] Generally speaking, the landing pad used to facilitate targeted integration can comprise at least one recognition sequence.For example, the landing pad can comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more recognition sequences.In the embodiment that comprises two or more recognition sequences, the recognition sequences can be unique to each other (i.e., recognized by different polynucleotide modifying enzymes), can be the same repeat sequence, or a combination of repeat sequence and unique sequence.

[0177] Those skilled in the art will easily understand that the exogenous nucleic acid used as landing pad can also contain other sequences in addition to recognition sequences.For example, it may be advantageous to include one or more sequences that code for selectable or screenable genes as described herein, such as antibiotic resistance genes, metabolic selection markers, or fluorescent proteins.There may also be the use of other supplementary sequences, such as transcriptional regulatory elements and control elements (i.e., promoters, partial promoters, promoter traps, start codons, enhancers, introns, insulators and other expression elements).

[0178] In addition to selecting an appropriate recognition sequence, selecting a targeting endonuclease with high cleavage efficiency also improves the rate of targeted integration of the landing pad. The cleavage efficiency of a targeting endonuclease can be determined using methods well known in the art, including, for example, using an assay such as the CEL-1 assay or direct sequencing of insertions / deletions in PCR amplicons.

[0179] The type of targeting endonuclease used in the methods and cells disclosed herein can vary or vary.The targeting endonuclease can be a naturally occurring protein or an engineered protein.One example of a targeting endonuclease is a zinc finger nuclease, which is discussed in more detail below.

[0180] Another example of a targeting endonuclease that can be used is an RNA-guided endonuclease that contains at least one nuclear localization signal, which allows the endonuclease to enter the nucleus of eukaryotic cells. The RNA-guided endonuclease also contains at least one nuclease domain and at least one domain that interacts with the guided RNA. The RNA-guided endonuclease is directed to a specific chromosomal sequence by the guided RNA, so that the RNA-guided endonuclease cleaves the specific chromosomal sequence. Since the guided RNA provides specificity for target cleavage, the endonuclease of the RNA-guided endonuclease is universal and can be used with different guided RNAs to cleave different target chromosomal sequences. Exemplary RNA-guided endonuclease proteins are discussed in more detail below. For example, the RNA-guided endonuclease can be a CRISPR / Cas protein or a CRISPR / Cas-like fusion protein, an RNA-guided endonuclease derived from clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system.

[0181] The targeting endonuclease can also be a meganuclease. Meganucleases are endodeoxyribonucleases characterized by large recognition sites, i.e., the recognition sites generally range from about 12 base pairs to about 40 base pairs. As a result of this requirement, the recognition sites generally occur only once in any given genome. Among the meganucleases, the endonucleases family, named LAGLIDADG, have become valuable tools for the study of genomes and genome engineering. Meganucleases can be targeted to specific chromosomal sequences by modifying their recognition sequences using techniques well known to those skilled in the art. See, for example, Epinat et al., 2003, Nuc Acid Res., 31(11):2952-62 and Stoddard, 2005, Quarterly Review of Biophysics, pp.1-47.

[0182] Another example of a targeting endonuclease that can be used is a transcription activator-like effector (TALE) nuclease. TALE is a transcription factor from the plant pathogen Xanthomonas, which can be easily engineered to bind to new DNA targets. TALE or its truncated form can be linked to the catalytic domain of an endonuclease such as FokI to create a targeting endonuclease called a TALE nuclease or TALEN. See, for example, Sanjana et al., 2012, Nature Protocols 7(1):171-192; Bogdanove AJ, Voytas D F., 2011, Science, 333(6051):1843-6; Bradley P, Bogdanove AJ, Stoddard B L., 2013, Curr Opin Struct Biol., 23(1):93-9.

[0183] Another exemplary targeting endonuclease is a site-specific nuclease.In particular, the site-specific nuclease may be a "rare cutter" endonuclease, whose recognition sequence occurs rarely in genome.Preferably, the recognition sequence of the site-specific nuclease occurs only once in genome.Alternatively, the targeting nuclease may be an artificial targeting DNA double strand break inducer.

[0184] In some embodiments, targeted integration can be achieved by the use of integrase. For example, phiC31 integrase is a sequence-specific recombinase encoded within the genome of the bacteriophage phiC31. phiC31 integrase mediates recombination between two 34-base pair sequences, called attachment sites (att), one found in the phage and the other in the bacterial host. This serine integrase has been shown to function efficiently in many different cell types, including mammalian cells. In the presence of phiC31 integrase, an attB-containing donor plasmid can be directionally integrated into the target genome via recombination at a site with sequence similarity to the natural attP site (called a pseudo attP site). phiC31 integrase can integrate plasmids of any size as a single copy and does not require cofactors. The integrated transgene is stably expressed and heritable.

[0185] In one embodiment, genomic integration of the polynucleotides of the present disclosure is achieved by the use of a transposase. For example, synthetic DNA transposons (e.g., the "Sleeping Beauty" transposon system) designed to introduce precisely defined DNA sequences into vertebrate chromosomes can be used. The Sleeping Beauty transposon system is composed of the Sleeping Beauty (SB) transposase and a transposon designed to insert a specific sequence of DNA into the genome of a vertebrate. DNA transposons move from one DNA site to another in a simple cut-and-paste manner. Transposition is the precise process by which a defined DNA segment is excised from one DNA molecule and moved to another site in the same or a different DNA molecule or genome.

[0186] Like all other Tc1 / mariner type transposases, SB transposase inserts the transposon into the TA dinucleotide base pair of the recipient DNA sequence. The insertion site can be anywhere on the same DNA molecule or anywhere on another DNA molecule (or chromosome). There are approximately 200 million TA sites in mammalian genomes, including humans. The TA insertion site is duplicated during the process of transposon integration. This duplication of the TA sequence is a hallmark of transposition and is used in some experiments to confirm the mechanism. The transposase can either be encoded within the transposon or the transposase can be provided by another source, in which case the transposon becomes a non-autonomous element. Non-autonomous transposons are most useful as genetic tools because they cannot continue to excise and reinsert independently after insertion. All of the DNA transposons identified in the human genome and other mammalian genomes are non-autonomous because, even though they contain a transposase gene, the gene is non-functional and cannot generate a transposase that can mobilize the transposon.

[0187] VII. How to use The assays described herein make large-scale screening both time- and cost-effective.Furthermore, the assays described herein are useful for screening ligands for on- and off-target effects, determining the activity of one or more receptor variants against a particular ligand or set of ligands, mapping the critical residues in a receptor that are required for ligand binding, and determining which residues in a receptor are not important for ligand binding.

[0188] In some aspects, the assay method relates to an assay in which the receptor is a variant of one receptor. In some embodiments, each variant comprises or consists of one substitution relative to the wild-type protein sequence. In some embodiments, each variant comprises or consists of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions (or any range derivable therein) compared to the wild-type amino acid sequence. In some aspects, the method comprises determining the activity of a population of receptors to a ligand, wherein the population of receptors comprises at least two variants of the same receptor, and wherein the activity is determined in response to the ligand. In some embodiments, the receptor population comprises and is screened at least, at most, or about 2, 10, 100, 200, 300, 400, 500, 1000, 1500, 2000, 3000, 4000, or 5000 receptors (or any derivable range therein). In some embodiments, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ligands (or any derivable range therein) are screened. In some embodiments, at least, at most, or about 2, 10, 100, 200, 300, 400, 500, 1000, 1500, 2000, 3000, 4000, or 5000 receptors (or any range of inducible therein) are screened in response to at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ligands (or any range of inducible therein). In some embodiments, the assay can be used to predict the response of a patient to a ligand based on the determined activity of the variant receptor to the ligand. For example, the assay described herein can be used to predict the therapeutic response of a variant receptor to a ligand. This information can then be used in a treatment method for treating a patient with a variant receptor.In some embodiments, the method includes treating a patient with a ligand, where the patient has been determined to have a variant receptor. In some embodiments, the activity of the variant receptor for the ligand has been determined by the techniques described herein.

[0189] In some embodiments, the assay is for determining the activity of a class of receptors towards one or more ligands.

[0190] In some embodiments, the receptor class is an olfactory, GPCR, nuclear hormone, hormone, or catalytic receptor. In some embodiments, the receptor is an alpha or beta adrenergic receptor, or an adrenergic receptor such as an alpha-1, alpha-2, beta-1, beta-2, or beta-3 adrenergic receptor, or an alpha-1A, alpha-1B, alpha-1D, alpha-2A, alpha-2B, or alpha-2C adrenergic receptor. In some embodiments, the receptor or receptor class is as described herein.

[0191] VIII. Kit Certain aspects of the present disclosure also relate to kits that include the nucleic acid, vector, or cell of the present disclosure. The kits can be used to carry out the methods of the present disclosure. In some embodiments, the kits can be used to evaluate the activation of a receptor gene or a group of receptor genes. In some embodiments, the kits can be used to evaluate the variants of a single gene. In certain embodiments, the kit comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more, or at least or at most 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more nucleic acid probes, primers, or synthetic RNA molecules, or any values ​​or ranges and combinations derivable therein. In some embodiments, there is a kit for evaluating the activation or engagement of a receptor by a ligand. In some embodiments, a universal probe or primer for amplifying, identifying, or sequencing a barcode or receptor is included. Such reagents can also be used to generate or test host cells that can be used in screening.

[0192] In certain embodiments, the kits may include materials for analyzing cell morphology and / or phenotype, such as histological slides and reagents, histological stains, alcohol, buffers, tissue embedding media, paraffin, formaldehyde, and tissue dehydrating agents.

[0193] The kits may contain components that may be individually packaged or disposed in containers, such as tubes, bottles, vials, syringes, or other suitable packaging means.

[0194] Individual components may also be provided in the kit in concentrated amounts, and in some embodiments, components are provided individually at the same concentration as they are in solution with the other components. The concentrations of components can be 1x, 2x, 5x, 10x, or 20x or more.

[0195] Kits for using the probe, polypeptide or polynucleotide detection agents of the present disclosure for drug discovery are contemplated.

[0196] In certain aspects, negative and / or positive control agents are included in some kit embodiments. Control molecules can be used to verify transfection efficiency and / or control of transfection-induced changes in cells.

[0197] An embodiment of the present invention includes a kit for analyzing a pathological sample by evaluating a nucleic acid or polypeptide profile for the sample, comprising in a suitable container two or more RNA probes or primers for detecting expressed polynucleotides. Additionally, the probes or primers may be labeled. Labels are known in the art and are described herein. In some embodiments, the kit may further comprise a reagent for labeling the probe, nucleic acid, and / or detection agent. The kit may also comprise a labeling reagent comprising at least one of an amine-modified nucleotide, poly(A) polymerase, and poly(A) polymerase buffer. The labeling reagent may comprise an amine-reactive dye. The kit may comprise any one or more of the following materials: enzymes, reaction tubes, buffers, detergents, primers, probes, antibodies. In some embodiments, these kits include the equipment necessary to perform RNA extraction, RT-PCR, and gel electrophoresis. Instructions for performing the assay may also be included in the kit.

[0198] The kit may further include instructions for using the kit to assess expression, a means for converting expression data into expression values, and / or a means for analyzing the expression values ​​to generate ligand / receptor interaction data.

[0199] The kit may include a container having a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials, such as glass or plastic. The containers may hold compositions that include probes useful in the disclosed methods. The kit may include the above-mentioned containers and one or more other containers that contain materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. EXAMPLES

[0200] IX. Working Examples The following examples are included to demonstrate preferred embodiments of the present disclosure. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present disclosure, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should understand in light of the present disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure.

[0201] Example 1 - Multiplexed odorant-receptor screening system. Mammalian olfaction is a highly complex process and perhaps the least understood sense. Olfactory receptors (ORs) are the first layer of odor perception. Human ORs are a set of 400 G protein-coupled receptors (GPCRs) that are monoallelically expressed in neurons located in the nasal epithelium. Odorants bind to receptors in a many-to-many manner, a pattern that is transmitted to the olfactory bulb and translated into perception in the cortex. High affinity ligands have been identified for only about 5% of human ORs, and the large number of orphan receptors has hindered the possibility of investigating the downstream neurobiology that governs olfaction. Previous dealphablation attempts utilized heterogeneous cell-based assays that screened each odorant-receptor pair individually. The large number of potential receptor-odorant combinations, and the difficulty of achieving heterogeneous OR expression, limited the throughput of the "one at a time" approach. Instead, we engineered stable OR-expressing cell lines that allow for multiplexed odorant-receptor screening.

[0202] To measure receptor-odorant interactions, we adapted a genetic reporter for cAMP signaling in HEK293T cells. Upon odorant binding, G protein signaling stimulates the production of cAMP, which leads to phosphorylation of the transcription factor CREB. CREB binds to the short tandem repeat sequence CRE and turns on transcription of a downstream reporter gene, usually luciferase. The assay was modified to contain a DNA barcode in the 3'UTR of the reporter gene that uniquely associates with one OR in the library expressed on the same plasmid (Figure 1). To ensure that cAMP signaling does not trigger expression of a barcode corresponding to a receptor not bound by the odorant but present in the same cell, we incorporate a single library member in each cell. We seeded the cell lines in 96-well plates, induced each well with a different odor, and sequenced the barcoded transcripts. We converted the relative abundance of each barcode into a heatmap that displays the affinity of the odor for each receptor.

[0203] A typical genetic reporter assay for GPCR activation co-transfects the receptor and reporter individually. To map each barcode to its corresponding OR, it is necessary to express all components for the assay on a single plasmid that allows for the association of the barcode and OR via sequencing. We constructed a plasmid to express all the necessary components (Figure 3). We tentatively screened a range of concentrations for two ORs, MOR42-3 and MOR9-1, with known high affinity ligands for both structures and observed comparable reporter activation.

[0204] The multiplexing strategy requires stable clonal integration of the OR library. Initially, we decided to use Bxb1 recombination because it allowed each library member to be integrated at a single copy per cell in a single pot reaction. We engineered a "landing pad" containing a Bxb1 attp recombinase site into the H11 safe harbor locus of HEK293T cells (Figure 4). The engineered cell line is called Mukku1a (Table 1). Bxb1 recombination irreversibly integrates plasmid DNA containing a complementary attb recognition site, disrupting the genomic attp sequence that limits one single recombination per cell. We were unable to observe reporter activation when inducing MOR42-3 in the landing pad. However, the beta-2 adrenergic receptor (a canonical GPCR that also activates adenylate cyclase) strongly activated the reporter upon induction when expressed from the landing pad. TIFF0007671316000098.tif46152

[0205] ORs are known to be difficult to express heterologously, and stable heterologous expression has not been reported. We hypothesized that stable constitutive expression of ORs would lead to many possible avenues of downregulation and resolved to attempt inducible expression. We engineered Mukku1a cells to express the inverse Tet transactivator and replaced the promoter driving OR expression with a Tet-On inducible promoter (Figure 5). Although the inducible system transiently achieved reporter activation comparable to the previous system, we still could not observe reporter expression when in the landing pad. The next hypothesis was that a single OR gene was insufficient to achieve the expression required to activate the genetic reporter. We flanked the intermediate terminal repeats to the gene construct and integrated the plasmid using a transposase (Figure 6). Under constitutive OR expression, the reporter still did not respond to odorants. Unexpectedly, the combination of reporter transposition and control of OR expression inducibly restored the odorant response of the reporter. QPCR confirmed that the transposon was integrated at an average of 4–6 copies per cell.

[0206] Many ORs, when transiently expressed in heterologous systems, require the co-expression of accessory factors for cell membrane trafficking and proper signaling (Figure 7). This is achieved by stable expression as well as by the co-expression of four genomically integrated accessory factor transgenes: RTP1S and RTP2 (chaperones that increase surface expression), G αolf (G protein alpha subunit that naturally interacts with ORs), and Ric8b (G αolf The inventors predicted that the problem was for the OR-associated guanine nucleotide exchange factor (Olfr62 and OR7D4) in Mukku2a cells. We pooled these four factors under Tet-inducible regulation and transferred them into Mukku2a cells. To generate cell lines with strong OR expression capacity, we isolated single clones and transiently screened them for gene reporter activation for two ORs, Olfr62 and OR7D4, previously known to require accessory factors for heterologous function expression.

[0207] Forty-two mouse ORs were cloned into a transposon vector containing random barcodes in the 3'UTR of a reporter gene, and the barcodes were mapped to each receptor using sequenced clones. Each construct was then transposed individually into Mukku3a cells, and the cells were then pooled together after transposition. Finally, the integrated Mukku3a cells inducibly express both accessory factors and ORs under the control of the Tet-On system (data not shown). To confirm that the stable cell lines recapitulate and work reliably with previous receptor-odorant associations for a large receptor cohort, we tested a small number of receptors with known ligands at both the protein and transcript levels (Figures 2A-B).

[0208] To make this assay amenable to high-throughput screening, we developed a 96-well plate-compatible in-lysate protocol for library preparation (Figure 8). Each well of the plate, as well as the plate itself, was barcoded with a custom index. We screened 96 odorants at four separate concentrations against our 42-receptor library, yielding 16,128 unique receptor-ligand interactions. A heat map was constructed to show the relative activation of each receptor under each condition (Figure 2C).

[0209] The odorant-receptor interaction space is complex and difficult to traverse. We have developed a platform that overcomes the challenges of heterologous OR expression and compresses the interaction space through multiplexing. This platform makes large-scale dealphaylation of mammalian ORs economically and technically feasible.

[0210] Example 2 - Olfactory-Seq: A multiplexed GPCR activity assay to decode olfactory receptor-ligand interactions We developed a platform for multiplexed receptor-ligand profiling by constructing a library of stable human cell line reporters that can be multiplexed read by next-generation sequencing in a high-throughput format. This technology can be generalized to many other classes of receptors and enables high-throughput screening for drug discovery for pharma- ceutical relevant GPCRs.

[0211] Interactions between small molecules and receptors underpin the ability of living organisms to sense and respond to internal conditions and environments. For many drugs and natural products, the ability to modulate the function of many biological targets at once is critical to their efficacy. Such multidrug resistance is difficult to study because it is often not known which chemical interacts with which target. This many-to-many challenge makes studying one interaction at a time arduous, which is particularly evident in mammalian olfaction.

[0212] Olfaction is mediated by a class of G protein-coupled receptors (GPCRs) known as olfactory receptors (ORs). GPCRs are central players in small molecule signaling in mammals and are targeted by over 30% of U.S. Food and Drug Administration-approved drugs. ORs are a large family of class A GPCRs specialized in many different evolutionary contexts with approximately 396, 1130, and 1948 intact receptors in humans, mice, and elephants, respectively. Each OR can potentially interact with a near-infinite number of odorants, and each odorant can interact with many ORs. The vast majority of ORs remain orphans because of this complexity and the difficulty of recapitulating mammalian GPCR function in vitro. Furthermore, no crystal structures exist for any OR, which hampers computational efforts to predict which odorants activate each OR.

[0213] Here, we report a new HTS-compatible system for characterizing small molecule libraries against mammalian OR libraries in a multiplexed fashion (Figure 9A). To do this, we developed both stable cell lines capable of functional OR expression (Figure 11) and multiplexed reporters for OR activity (Figure 12). The final platform contains inducibly expressed multicopy ORs located inside engineered cell lines with inducibly expressed proteins required for OR trafficking and signaling (Figure 13). Activation of each OR leads to expression of a reporter transcript with a unique 15-nucleotide barcode sequence. Each barcode identifies the OR and allows multiple readouts with barcode amplicon RNA sequences (Figure 9A, Figure 13). Using this platform, we screened at least 42 different receptors, and we adapted the platform for high-throughput screening that enabled the discovery of novel odorant pairs. We found that multicopy integration and inducible expression allowed reporter activation. Individually, these features did not produce a response; however, their combination produced a functional OR reporter cell line, demonstrating a synergistic response not found when either multicopy integration or inducible expression was used alone. G_alpha_olf, Ric8b, RTP1S, RTP2 were then inducibly expressed (Figure 9B, Figure 11). To engineer the reporter construct, we used protein trafficking tags to increase surface expression, added DNA insulator sequences to reduce background reporter activation, modified the cAMP response element (CRE) enhancer to improve reporter signal, and combined these elements into a single transferable vector to facilitate cell line development (Figure 12). We validated our system against three mouse ORs with known ligands and observed induction and dose-dependent activation, including Olfr62, which had previously been difficult to express (Figure 9C).

[0214] After modification, we generated a library of 42 mouse OR-expressing cell lines and tested multiple readouts of activation. First, we cloned the ORs and mapped them to their corresponding barcodes by Sanger sequencing, transposed the plasmids individually into HEK-293T cells, and pooled the cell lines together after selection (Figure 10A). To derive the multiplexed assay, we seeded the cell library in 6-well culture dishes and added odorants known to activate specific ORs (Figure 14); all but three ORs were present in enough cells to obtain a robust estimate of activation. Analysis of the sequencing readouts reproduced previously identified odorant-receptor pairs, and the chemical mixtures appropriately activated multiple ORs. Interestingly, we found that this assay was robust against chemicals such as the direct adenylate cyclase stimulator forskolin, which nonspecifically stimulate cells independent of the ORs they express. Such nuisance chemicals can be easily eliminated, as they activate all barcodes equally. We then adapted the platform for high-throughput screening in a 96-well format. To reduce reagent costs and analysis time, we developed an in-lysate reverse transcription protocol and used double indexing to uniquely identify each well (see Methods). Using these improvements, we were able to reproduce dose-response curves of known odorant-receptor pairs (Figure 10B, Figure 14). We observed reproducible results between identically treated but biologically independent wells (Figures 15-16).

[0215] We then screened 182 odorants in triplicate at three concentrations against the OR cell library. This corresponds to approximately 85,000 separate luciferase assays including controls (Figure 10A, Table 2). Each 96-well plate in the assay contained a positive control odorant and a solvent DMSO well for normalization (Figure 16). The EdgeR software package was used to determine differential response ORs based on a negative binomial model of barcode counts. We found 114 OR-odorant interactions (out of a possible 7,200), of which 81 were novel, and 24 interactions were with 15 orphan receptors (Figure 10C, Figure 17, and Supplementary Table 4) (FDR=1%; Benjamini-Hochberg correction). Twenty-eight of the 39 total receptors were activated by at least one odorant, and 68 of the 182 odorants activated at least one OR (Table 4). We selected 37 interactions with at least 1.2-fold induction and tested them individually using a previously developed transient OR assay with some important differences (Figure 18). Of the 28 interactions at 1% FDR called hits, 21 were reproduced in this orthogonal system (Figure 17). Some of the seven that were not reproduced are also likely to be true. For example, our assay recorded two hits (methyl salicylate and benzyl salicylate) with high chemical similarity to MOR19-1, suggesting that they may not be false positives (Figure 18). Furthermore, three of the nine interactions that did not exceed the 1% FDR threshold showed activation in the orthogonal assay, indicating a modest threshold. Although the previous large-scale OR deodorization study used some of the same receptors and chemicals, and we did not identify most of the previous low affinity interactions, we found that 9 / 12 of the reported interactions with EC50s below 100 μM were also detected in our platform (Figure 19). Conversely, we also detected 14 interactions that were tested and negative in this previous study.Finally, our assay reproduced most of the odorant and OR combinations that did not interact (493 / 507).

[0216] We found that chemicals with similar features activate similar sets of ORs, such as the receptors we deorphanized in this study. For example, the previously orphaned MOR13-1 is activated by four chemicals, with polar groups attached to a rigid, non-rotatable scaffold in three cases. Another example is MOR19-1, which has a distinct affinity for salicylic acid functional groups. To better understand how chemical similarity relates to receptor activation without relying on incomplete, sometimes arbitrary, chemical descriptors, we used a previously validated computational autoencoder to represent each chemical in a roughly 292-dimensional latent space, which allowed for nearly lossless compression of chemical structures (data not shown). We found that chemicals that activate the same ORs tend to cluster distinctly (Figure 10D, Figure 20). For example, MOR5-1 ligands cluster in latent space, showing that 10 / 13 odorants that are long-chain (>5 carbons) aldehydes and carboxylic acids activate the receptor. Furthermore, MOR170-1 shows a broad activation pattern, i.e., binding of about 50% of all odorants containing a benzene ring and either a carbonyl or ether group, a pattern that is also reflected in the latent space; many, but not all, receptors. The activation landscape of the entire set of interactions suggests that some ORs are activated by truncated chemical subspaces (Figure 20). Understanding the space of chemicals that activate each OR establishes a basis for predicting novel odorant-OR interactions.

[0217] Because a given chemical can interact with a large number of targets, our incomplete understanding of how chemicals interact with potential targets, whether endogenous ligands, pharmaceuticals, natural products, or odors, limits our ability to rationally develop new ones with a large number of possible targets and functional pathways. This is becoming increasingly evident in both natural and therapeutic contexts. We predicted that Smell-seq could be scaled up to the 396-member human OR repertoire and comprehensively define the response of ORs to odors. The approximate cost per well of Smell-seq is comparable to extant assays, but multiplexing dramatically reduces the cost and effort per interaction investigated. Efforts to hit more selectively to specific targets or activate a set of receptors more broadly utilize machine learning methods that rely on huge data sets. Multiplexing methods like Smell-seq provide a scalable solution to generate quality data at this scale.

[0218] table (Table 2) Olfactory receptors screened in this study TIFF0007671316000099.tif93128

[0219] (Table 3) Odorants screened in this study TIFF0007671316000100.tif63165TIFF0007671316000101.tif252165TIFF0007671316000102.tif97165

[0220] Table 4. Odorant-receptor pairs called hits TIFF0007671316000103.tif116161TIFF0007671316000104.tif243161TIFF0007671316000105.tif241161TIFF0007671316000106.tif149161

[0221] Table 5 Primers and sequences used in this study TIFF0007671316000107.tif73163TIFF0007671316000108.tif229163TIFF0007671316000109.tif243163TIFF0007671316000110.tif242163 TIFF0007671316000111.tif241163TIFF0007671316000112.tif241163TIFF0007671316000113.tif241163TIFF0007671316000114.tif95163

[0222] method 1. Odor Receptor Activation Luciferase Assay (Transient) The Dual-Glo Luciferase Assay System (Promega) was used to measure OR-odorant responses as previously described (Zhuang and Matsunami 2008). HEK293T cells (ATCC#11268) were plated in poly-D-lysine-coated white 96-well plates (Corning) at a density of 7,333 cells per well in 100ul of DMEM (Thermo Fisher Scientific). After 24 hours, cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) with 5ng / well of a plasmid encoding an OR and 10ng / well of luciferase driven by a cyclic AMP response element or with 10ng / well of a plasmid encoding both an OR and a luciferase gene, in both cases with 5ng / well of a plasmid encoding Renilla luciferase. Experiments performed with accessory factors included 5ng / well of plasmids encoding RTP1S (Gene ID: 132112) and RTP2 (Gene ID: 344892). Inducibly expressed ORs were transfected by adding 1ug / ml doxycycline (Sigma-Aldrich) to the transfection medium. 10-100mM bromine stocks were established in DMSO or ethyl alcohol. 24 hours after transfection, the transfection medium was removed and replaced with 25ul / well of the appropriate concentration of odorant diluted from stock in CD293 (Thermo Fisher Scientific). Four hours after odorant stimulation, the Dual-Glo Luciferase Assay Kit was administered according to the manufacturer's instructions. Luminescence was measured using an M1000 plate reader (Tecan). All luminescence values ​​were normalized to Renilla luciferase activity to control for transfection efficiency in a given well. Data were analyzed in Microsoft Excel and R.

[0223] 2. Olfactory Receptor Activation Luciferase Assay (Integrated) HEK293T and HEK293T-derived cells integrated with the combined receptor / reporter plasmids were seeded at a density of 7333 cells / well in 100uL DMEM in poly-D-lysine coated 96-well plates. After 24 hours, 1ug / ml doxycycline was added to the well medium. Odor stimulation, luciferase reagent addition, and luminescence measurement were performed in the same manner as for the transient assay. Constitutively expressed ORs were assayed in the same manner without the addition of doxycycline. Data were analyzed in Microsoft Excel and R.

[0224] 3. Odor Stimulation and RNA Extraction for Pilot-scale Multiple Odorant Screening HEK293T and HEK293T-derived cells transfected with the combined receptor / reporter plasmids were seeded in 6-well plates at a density of 200k cells / well in 2mL of DMEM. After 24 hours, 1ug / ml of doxycycline was added to the well media. 10-100mM bromide stocks were established in DMSO or ethyl alcohol. 24 hours after doxycycline addition, odorants were diluted in OptiMEM, media was aspirated, and replaced with 1mL of odorant-OptiMEM solution. Three hours after odor stimulation, odor media was aspirated and 600uL of buffer RLT (Qiagen) was added to each well. Cells were lysed with a Qiashredder Tissue and Cell Homogenizer (Qiagen) and RNA was purified with an optional on-column DNAse step using the RNEasy MiniPrep Kit (Qiagen) according to the manufacturer's protocol.

[0225] 4. Pilot-scale Library Preparation and RNA-seq 5ug of total RNA per sample was reverse transcribed using Superscript IV (Thermo-Fisher) with gene-specific primers for the barcoded reporter gene (OL003). Reaction conditions are as follows: Annealing: [65°C for 5 min, 0°C for 1 min] Extension: [52°C for 60 min, 80°C for 10 min] 10% of the cDNA library volume was amplified for 5 cycles (OL004F and R) using HiFi Master Mix (Kapa Biosystems). Reaction and cycle conditions are optimized as follows: 95°C for 3 min, 5 cycles of 98°C for 20 sec, 59°C for 15 sec, and 72°C for 10 sec, followed by extension at 72°C for 1 min. The PCR products were purified to 10 ul using the DNA Clean and Concentrator Kit (Zymo Research) and 1 ul of each sample was amplified (OL005F and R) using SYBR FAST qPCR Master Mix (Kapa Biosystems) with a CFX Connect Thermocycler (Biorad) to determine the number of PCR cycles required for library amplification. The reaction and cycle conditions are optimized as follows: 95°C for 3 min, 40 cycles of 95°C for 3 s and 60°C for 20 s. After qPCR, 5 ul of the pre-amplified cDNA library was amplified a second time with the same primers used for qPCR over 4 cycles greater than the previously determined Cq and with the same cycle conditions as the first amplification. The PCR products were then gel isolated from a 1% agarose gel using the Zymoclean Gel DNA Recovery Kit (Zymo Research). Library concentrations were quantified using a Tape Station 2200 (Agilent) and loaded equimolarly onto a Hi-Seq 3000 with 20% PhiX spike-in and sequenced with custom primers: Read 1 (OL003) and i7 Index (OL006).

[0226] 5. Cloning of the OR Library The backbone plasmid (all genetic elements except OR and barcode) was generated using isothermal assembly with Gibson Assembly Hifi Mastermix (SGI-DNA). Short fragments were amplified with primers containing 15 random nucleotides to generate barcode sequences (OL007F and R) using HiFi Mastermix. Reaction and cycle conditions were optimized as follows: 95°C for 3 min, 35 cycles of 98°C for 20 s, 60°C for 15 s, and 72°C for 20 s, followed by 1 min extension at 72°C. The amplicon and backbone plasmid were digested with restriction enzymes MluI and AgeI (New England Biolabs) and ligated together with T4 DNA ligase (New England Biolabs). DH5α E. coli competent cells (New England Biolabs) were transformed directly into liquid culture with antibiotics to maintain the diversity of the barcode library.

[0227] OR genes were individually amplified with primers (OL008) that add homology to the barcoded backbone plasmid using HiFi Master Mix. Reaction and cycle conditions are optimized as follows: 95°C for 3 min, 35 cycles of 98°C for 20 s, 61°C for 15 s, and 72°C for 30 s, followed by 1 min extension at 72°C. Amplified ORs were purified with DNA Clean and Concentrator and pooled together. Barcoded backbone plasmids were digested with NdeI and SbfI, and OR amplicon pools were cloned into them using isothermal assembly with Gibson Assembly HifiMastermix. DH5α E. coli competent cells were transformed with the assemblies, and antibiotic resistant clones were picked and grown overnight in 96-well plates. Plasmid DNA was prepared with Zyppy-96 Plasmid Miniprep Kit (Zymo Research). Plasmids were Sanger sequenced (OL109-111) to identify barcode-associated reporter gene and error-free ORs.

[0228] 6. OR Library Genome Integration HEK293T cells and HEK293T-derived cells were seeded at a density of 350k cells / well in 6-well plates in 2ml DMEM. 24 hours after seeding, cells were transfected with plasmids encoding the receptor / reporter transposon and Super PiggyBac Transposase (Systems Bioscience) according to the manufacturer's instructions. 1ug of transposon DNA and 200ng of transposase DNA were transfected per well with Lipofectamine 3000. 3 days after transfection, cells were passaged 1:10 into 6-well plates and 1 day after passaging, 8ug / ml blasticidin was added to the cells. Cells were grown for 7-10 days with selection. OR libraries were transposed individually and pooled together in equal cell numbers.

[0229] 7. Generation of Accessory Factor Cell Lines HEK293T-derived cells were transfected with plasmids encoding accessory factor genes RTP1S, RTP2, Gα olf (Gene ID: 2774), and Ric8b (Gene ID: 237422) inducibly driven by equimolar Tet-On promoter pools, following the transposition protocol in the OR library integration section. Cells were selected with 2ug / ml puromycin (Thermo Fisher). After selection, cells were seeded into 96-well plates at a density of 0.5 cells / well. Wells were examined for single colonies after 3 days and expanded to 24-well plates after 7 days. Clones were screened for accessory factor expression by screening for strong activation of Olfr62 and OR7D4 in transient luciferase assays (Figure 11). Clones with the highest fold activation for both receptors and no significant growth defects were established for multiplex screening.

[0230] 8. Transposon Copy Number Validation gDNA was purified from cells transposed with the OR reporter vector and from cells containing single-copy landing pads using the Quick-gDNA Miniprep kit. 50 ng of gDNA was amplified with primers that anneal to regions of exogenous DNA from each sample using SYBR FAST qPCR Master Mix (Kapa Biosystems) on a CFX Connect thermocycler using the manufacturer's protocol. Reaction and cycling conditions were optimized as follows: 95°C for 3 min, 40 cycles of 95°C for 3 s, and 60°C for 20 s. Cq values ​​of transposed ORs were normalized to the single-copy landing pad to determine copy number.

[0231] 9. Lentiviral Transduction Lentiviral vectors were generated by transient transfection of 293T cells with lentiviral transfer plasmids, pCMVΔR8.91 and pCAGGS-VSV-G, using Mirus TransIT-293. HEK293T cells were transduced to express m2rtTA transcription factor (Tet-On) at 50% confluence and seeded 1 day prior to transduction. Clones were isolated by seeding cells in 96-well plates at a density of 0.5 cells / well. Wells were tested for single colonies after 7 days and expanded to 24-well plates. Clones were assessed for m2rtTA expression by screening for strong activation of MOR42-3 (Gene ID: 257926) using a transient luciferase assay.

[0232] 10. High-throughput Odorant Screening OR library cell lines were thawed from liquid nitrogen frozen stocks into T-225 flasks (Corning) for 3 days before seeding into 96-well plates for screening. The library was seeded at 6,666 cells per well in 100ul of DMEM. After 24 hours, a working concentration of 1ug / ml doxycycline in DMEM was added to the wells. After 24 hours of induction, the medium was removed from each plate and replaced with 25ul of odorant diluted in OptiMEM. Each odorant was added in triplicate at three different concentrations (10uM, 100uM, 1mM) with the same amount of final DMSO (1%). Each plate contained two control odorants at three concentrations (10uM, 100uM, 1mM) in triplicate, and three wells containing 1% DMSO dissolved in medium. The library was incubated with odorants for 3 hours in a cell culture incubator with the lid removed.

[0233] After odor incubation, the medium was pipetted out of the plate and cells were lysed by adding 25uL of ice-cold Cells-to-cDNA II Lysis Buffer (Thermo Fisher) and pipetting up and down to homogenize and lyse the cells. The lysates were then heated to 75°C for 15 minutes, flash frozen in liquid nitrogen, and kept at -80°C until further processing. 0.5uL of DNase I (New England Biolabs) was then added to the lysates and incubated at 37°C for 15 minutes. To anneal the RT primers, 5ul of lysate from each well was combined with 2.5ul of 10mM dNTPs (New England Biosciences), 1ul of 2uM gene-specific RT primer (OL003), and 1.5ul of H2O. The reactions were heated to 65°C for 5 minutes and cooled to 0°C. After annealing, 1 ul of M-MuLV reverse transcriptase (Enzymatics), 1 ul of buffer, and 0.25 ul of RNase inhibitor (Enzymatics) were added to each reaction. Reactions were incubated at 42°C for 60 minutes and the RT enzyme was heat inactivated at 85°C for 10 minutes.

[0234] For each batch, qPCR was performed on several wells (OL005F and OL013) using SYBR FAST qPCR Mastermix to determine the number of cycles required for PCR-based library preparation. Reaction and cycle conditions are optimized as follows: 95°C for 3 min, 40 cycles of 95°C for 3 s, and 60°C for 20 s. After qPCR, 5 ul of each RT reaction was combined with 0.4 ul of 10 uM primers containing sequencing adaptors (OL005F and OL013), 10 ul of NEB-Next Q5 Mastermix (New England Biosciences) and 4.2 ul of H2O, and PCR was performed according to the manufacturer's protocol. The forward primer contains the P7 adaptor sequence and an index that identifies the well in the assay, and the reverse primer contains the P5 adaptor sequence and an index that identifies the plate in the assay. PCR products were pooled together by plate and purified with the DNA Clean and Concentrator Kit. Library concentrations were quantified using a Tape Station 2200 and Qubit (Thermo Fisher). Libraries were sequenced on a NextSeq500 in high output mode (Illumina) using two index reads and a single-end 75 bp read.

[0235] 11. Analysis of Next-generation Sequencing Data Samples were identified by indexing the adapters by PCR index, which were unique to each well (5' end) and unique to each plate (3' end). Well barcodes followed the 7bp indexing scheme of (Illumina Sequencing Library Preparation for Highly Multiplexed Target Capture and Sequencing Matthias Meyer, Martin Kircher, Cold Spring Harb Protoc; 2010; doi:10.1101 / pdb.prot5448). Plate indexing scheme followed the Illumina indexing scheme. Sequencing data was demultiplexed and 15bp barcode sequences were counted for exact matches only by custom python and bash scripts.

[0236] 12. Statistical methods for calling hits The count data were then analyzed using the differential expression package EdgeR. To filter out under-represented ORs, a cutoff was set where an OR must contain at least 0.5% of the reads from more than 399 of the 1954 test samples. This filtered out 3 of the 42 ORs that were under-represented in the cell library (MOR172-1, MOR176-1 and MOR181-1). Normalization factors were determined using the EdgeR package function calcNormFactors and glmFit was used with variance set to variance per tag since only 40 ORs were present in the library and the trend variance value fitted the data well. To determine whether odorants stimulated specific ORs, fitting a generalized linear model to the count data allowed the determination of both the mean activation and the p-value for each OR-odorant interaction. This p-value was then corrected for multiple hypothesis testing using the built-in p.adjust function with Benjamini&Hochberg correction to obtain the false discovery rate (FDR). We set a conservative cutoff of 1% to determine interacting odorant-OR pairs. For each interaction between an odorant and an OR, we further required that the OR-odorant interaction exceed the cutoff at two different concentrations of odorant or at exactly 1000 uM concentration.

[0237] 13. Molecular Autoencoder We visualized OR-chemical interactions in chemical space using an autoencoder as described in Gomez-Bombarelli et al. Following the authors' advice, we used a reimplementation of the autoencoder since the original implementation requires a non-working Python package. The model was pre-trained to a validation accuracy of 0.99 on the entire ChEMBL23 database, excluding molecules whose SMILES are longer than 120 characters. We used this pre-trained model to generate potential representations for both our 168 chemicals (for which we could find a SMILES representation) and 250,000 randomly sampled chemicals from ChEMBL23. We then performed a principal component analysis using scikit-learn and projected the resulting matrix into two dimensions.

[0238] Example 3 - ADRB2 variant screening Overview of mutant library creation and functional evaluation. We synthesize mutant sequences on oligonucleotide microarrays, with the length limit of each oligo being about 230 nt, whereas ADRB2 is about 1200 nt long. To cover the length of the protein, we had to segment it into 8 parts and synthesize each mutant 8th and clone it into a separate background vector. When amplifying and cloning the variant segments, we attached a 15 nt random barcode to each sequence. Upon cloning, we mapped each barcode to each variant using subsequent gene sequencing. We then cloned the barcode into the remainder of the protein and transposed it into the 3'UTR of a cyclic AMP response element (CRE) reporter gene that is expressed upon Gs signaling. From there, we used serine recombinase technology to integrate the library at a defined genomic locus in ΔADRB2 HEK293T cells at a single copy per cell (essential to prevent crosstalk between mutants in multiplexed assays). We integrated and stimulated the library cell lines with various isoproterenol concentrations and performed RNA-seq with barcode sequencing. The relative abundance of each barcode can be inferred as the relative activity of each B2 variant after normalization for expression. This is shown in Figure 21.

[0239] In Figure 22, we show the distribution activity relative to the median wild-type signal for both frameshifts (a common error mode of oligonucleotide microarray synthesis) and our single mutant library across two biological replicates. To construct our variant distribution, we average the measurements of all barcodes associated with a given variant. To construct the frameshift distribution, we average the measurements of all barcodes associated with insertions or deletions at a particular codon (excluding the C-terminus). As expected, frameshifts have a more deleterious effect than average missense mutations. We also find that at high isoproterenol concentrations, a higher proportion of our missense mutations approach wild-type levels of activity.

[0240] In Figure 23, we show the variant activity landscape of β2 at 0.625 uM isoproterenol. The mutational landscape reveals general trends in β2 structure and function. For example, we find that the transmembrane domain is more sensitive to substitutions of prolines and charged residues than the termini or intracellular loop 3 (mutation tolerance is the average effect of all mutations). We also see that the effect of frameshifting is greatly reduced at the C-terminus. We see that the mutational data correlates with the EV mutation score, and from the GNOMAD data, we can see how rare variants affect function.

[0241] In Figure 24, we show a comparison between missense variants assayed individually using a luciferase reporter compared to a multiplexed sequencing approach. Mutant activity compared to WT is largely recapitulated. The multiplexed assay is able to distinguish between fully dead and partially deleterious variants over a range of isoproterenol stimulation.

[0242] We investigated the mutational tolerance (average of all substitutions) of the ligand-binding pocket of β2, as annotated from Ring et al.'s contact map of hydroxybenzylisoproterenol with the receptor. In our assays, we stimulated with isoproterenol alone and found that mutations in residues that interact with isoproterenol were significantly less tolerant to mutations compared to residues that interact with the hydroxybenzyl tail. This is shown in Figure 25.

[0243] We also found that simple algorithms such as k-means clustering can group our data into distinct classes that map to the structure of β2 in a functionally relevant way. In this particular example, we grouped amino acid mutations together into functional classes and averaged their signals. Importantly, we did not provide any spatial information to the algorithm. Future deep mutation scanning is likely to be a powerful method for investigating protein structure. This is shown in Figure 26.

[0244] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods and steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be understood that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar test results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0245] References The following references and publications referenced throughout the specification, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF0007671316000115.tif202160TIFF0007671316000116.tif179160

[0246] Array information SEQUENCE LISTING <110> THE REGENTS OF THE UNIVERSITY OF CALIFORNIA <120> MULTIPLEXED RECEPTOR-LIGAND INTERACTION SCREENS <150> US 62 / 528,833 <151> 2017-07-05 <160> 189 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> DNA <213> Unknown <220> <223> Description of Unknown: CRE motif sequence <400> 1 tgacgtca 8 <210> 2 <211> 316 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 2 cgtcgtgacg tcagacagac cacgcgatcg ctcgagtccg ccggtcaatc cggtgacgtc 60 acgggcctct tcgctattac gccagctggc gaaagggggt tgacgtcaca ttaaatcggc 120 caacgcgcgg ggagaggcgg tgacgtcaac aggcatcgtg gtgtcacgct cgtcgtgacg 180 tcagtcgctt taactggccc tggctttggc agcctgtagc ctgacgtcag agagcctgac 240 gtcagagagc ggagactcta gagggtatat aatggaagct cgaattccag cttggcattc 300 cggtactgtt ggtaaa 316 <210> 3 <211> 232 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 3 gagggacagc ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg 60 gcagcagcga gccgcccggg gctccgctcc ggtccggcgc tccccccgca tccccgagcc 120 ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa 180 cgcttctcgc tgctctttga gcctgcagac acctgggggg atacggggaa aa 232 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 4 Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 1 5 10 15 <210> 5 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 5 Lys Arg Arg Trp Lys Lys Asn Phe Ile Ala Val Ser Ala Ala Asn Arg 1 5 10 15 Phe Lys Lys Ile Ser Ser Ser Gly Ala Leu 20 25 <210> 6 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 6 Glu Glu Glu Glu Glu Glu 1 5 <210> 7 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 7 Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg 1 5 10 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 8 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 9 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <210> 10 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic 6xHis tag <400> 10 His His His His His His 1 5 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 11 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 12 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 12 Thr Lys Glu Asn Pro Arg Ser Asn Gln Glu Glu Ser Tyr Asp Asp Asn 1 5 10 15 Glu Ser <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 13 Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser 1 5 10 15 <210> 14 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 14 Met Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly 1 5 10 15 Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro 20 25 30 Gln Gly Gln Arg Glu Pro 35 <210> 15 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 15 Ser Leu Ala Glu Leu Leu Asn Ala Gly Leu Gly Gly Ser 1 5 10 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 16 Thr Gln Asp Pro Ser Arg Val Gly 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 17 Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 18 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 18 Cys Cys Pro Gly Cys Cys 1 5 <210> 19 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 19 Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 1 5 10 <210> 20 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 20 Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys 1 5 10 <210> 21 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 21 Asp Leu Tyr Asp Asp Asp Asp Lys 1 5 <210> 22 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 22 Thr Asp Lys Asp Met Thr Ile Thr Phe Thr Asn Lys Lys Asp Ala Glu 1 5 10 15 <210> 23 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 23 Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys 1 5 10 <210> 24 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 24 Lys Leu Gly Asp Ile Glu Phe Ile Lys Val Asn Lys 1 5 10 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 25 ccctttaatc agatgcgtcg 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 26 ctgcctgctt caccaccttc 20 <210> 27 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 27 aagtgccttc ctgcccttta atcagatgcg tcg 33 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 28 cgccgaagtg aaaaccacct a 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 29 aagtgccttc ctgcccttta a 21 <210> 30 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <220> <221> modified_base <222> (25)..(32) <223> a, c, t or g <400> 30 caagcagaag acggcatacg agatnnnnnn nncgaagtga aaaccaccta 50 <210> 31 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 31 aatgatacgg cgaccaccga gatctacaca agtgccttcc tgccctttaa 50 <210> 32 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 32 cgggtttctt ggccttgtag gtggttttca cttcg 35 <210> 33 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <220> <221> modified_base <222> (13)..(27) <223> a, c, t, g, unknown or other <400> 33 ggaataacgc gtnnnnnnnn nnnnnnncga cgcatctgat taaaggg 47 <210> 34 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 34 ggaaggaccg gttctagtca aggcactata cat 33 <210> 35 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 35 tgctcctggc cctgctgacc ctaggcctgg ctcatatgaa tggcacagaa ggccc 55 <210> 36 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 36 agtcggccct gctgaggagt ctttccacct gcaggtctta tcatgtctgc tcgaa 55 <210> 37 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 37 cttctacgtg cccttctc 18 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 38 cctgcaggtc ttatcatgtc 20 <210> 39 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 39 tacaggcgga atggacgag 19 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 40 aagtgaaaac cacctacaag g 21 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 41 ccctttaatc agatgcgtcg 20 <210> 42 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <220> <221> modified_base <222> (30)..(37) <223> a, c, t or g <400> 42 aatgatacgg cgaccaccga gatctacacn nnnnnnnaag tgccttcctg ccctttaa 58 <210> 43 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 43 tgggcagttc caggcttata gtc 23 <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 44 gggcgtactt ggcatatgat acac 24 <210> 45 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 45 atcacg 6 <210> 46 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 46 cgatgt 6 <210> 47 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 47 ttaggc 6 <210> 48 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 48 tgacca 6 <210> 49 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 49 acagtg 6 <210> 50 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 50 gccaat 6 <210> 51 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 51 cagatc 6 <210> 52 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 52 acttga 6 <210> 53 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 53 gatcag 6 <210> 54 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 54 tagctt 6 <210> 55 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 55 ggctac 6 <210> 56 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 56 cttgta 6 <210> 57 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 57 agtcaa 6 <210> 58 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 58 agttcc 6 <210> 59 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 59 atgtca 6 <210> 60 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 60 ccgtcc 6 <210> 61 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 61 gtagag 6 <210> 62 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 62 gtccgc 6 <210> 63 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 63 gtgaaa 6 <210> 64 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 64 gtggcc 6 <210> 65 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 65 gtttcg 6 <210> 66 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 66 cgtacg 6 <210> 67 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 67 gagtgg 6 <210> 68 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 68 ggtagc 6 <210> 69 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 69 actgat 6 <210> 70 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 70 atgagc 6 <210> 71 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 71 attcct 6 <210> 72 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 72 caaaag 6 <210> 73 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 73 caacta 6 <210> 74 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 74 caccgg 6 <210> 75 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 75 cacgat 6 <210> 76 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 76 cactca 6 <210> 77 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 77 caggcg 6 <210> 78 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 78 catggc 6 <210> 79 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 79 cctgcga 7 <210> 80 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 80 ctctctat 8 <210> 81 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 81 tgcagag 7 <210> 82 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 82 tatcctct 8 <210> 83 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 83 acctagg 7 <210> 84 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 84 gtaaggag 8 <210> 85 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 85 ttgatcc 7 <210> 86 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 86 actgcata 8 <210> 87 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 87 atcttgc 7 <210> 88 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 88 aaggagta 8 <210> 89 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 89 tctccat 7 <210> 90 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 90 ctaagcct 8 <210> 91 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 91 catcgag 7 <210> 92 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 92 cgtctaat 8 <210> 93 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 93 ttcgagc 7 <210> 94 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 94 tctctccg 8 <210> 95 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 95 agttggt 7 <210> 96 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 96 ctagtcga 8 <210> 97 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 97 gtaccgg 7 <210> 98 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 98 agctagaa 8 <210> 99 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 99 cggagtt 7 <210> 100 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 100 actctagg 8 <210> 101 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 101 acttcaa 7 <210> 102 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 102 tcttacgc 8 <210> 103 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 103 tgatagt 7 <210> 104 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 104 cttaatag 8 <210> 105 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 105 gatccaa 7 <210> 106 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 106 caggtcg 7 <210> 107 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 107 cgcatta 7 <210> 108 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 108 ggtacct 7 <210> 109 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 109 ggacgca 7 <210> 110 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 110 gagattc 7 <210> 111 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 111 gagcatg 7 <210> 112 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 112 gttgcgt 7 <210> 113 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 113 ccaatgc 7 <210> 114 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 114 cgagatc 7 <210> 115 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 115 catattg 7 <210> 116 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 116 gacgtca 7 <210> 117 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 117 tggcatc 7 <210> 118 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 118 gtaattg 7 <210> 119 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 119 cctatct 7 <210> 120 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 120 caatcgg 7 <210> 121 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 121 gcggcat 7 <210> 122 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 122 agtactg 7 <210> 123 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 123 tactatt 7 <210> 124 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 124 ccggatg 7 <210> 125 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 125 accatga 7 <210> 126 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 126 cggttct 7 <210> 127 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 127 tattcca 7 <210> 128 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 128 cctcctg 7 <210> 129 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 129 aggtatt 7 <210> 130 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 130 gcattcg 7 <210> 131 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 131 ttgcgaa 7 <210> 132 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 132 ttgaatt 7 <210> 133 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 133 ctgcgcg 7 <210> 134 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 134 agacctt 7 <210> 135 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 135 gtccagt 7 <210> 136 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 136 acctgct 7 <210> 137 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 137 ccggtac 7 <210> 138 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 138 cttgacc 7 <210> 139 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 139 catcatt 7 <210> 140 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 140 tctgact 7 <210> 141 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 141 tctagtt 7 <210> 142 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 142 gccatag 7 <210> 143 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 143 accgtcg 7 <210> 144 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 144 cttggtt 7 <210> 145 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 145 tacgccg 7 <210> 146 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 146 ggactgc 7 <210> 147 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 147 gcgcgag 7 <210> 148 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 148 gtcgcag 7 <210> 149 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 149 catacgt 7 <210> 150 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 150 tcagtat 7 <210> 151 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 151 ctaagta 7 <210> 152 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 152 ttagctt 7 <210> 153 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 153 cgccgtc 7 <210> 154 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 154 gtcttct 7 <210> 155 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 155 gccggac 7 <210> 156 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 156 aagctga 7 <210> 157 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 157 gcgctct 7 <210> 158 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 158 cgtaggc 7 <210> 159 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 159 atgatta 7 <210> 160 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 160 gcaggtt 7 <210> 161 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 161 aatcgtc 7 <210> 162 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 162 cggccta 7 <210> 163 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 163 ctatgcc 7 <210> 164 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 164 ggttgaa 7 <210> 165 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 165 gagttaa 7 <210> 166 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 166 tagacta 7 <210> 167 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 167 tcatgca 7 <210> 168 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 168 gcttatt 7 <210> 169 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 169 caaggct 7 <210> 170 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 170 aggttgg 7 <210> 171 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 171 cttctgc 7 <210> 172 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 172 taattct 7 <210> 173 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 173 gatgctg 7 <210> 174 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 174 cctagaa 7 <210> 175 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 175 ctagagg 7 <210> 176 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 176 tatccgg 7 <210> 177 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 177 aggcggc 7 <210> 178 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 178 ggtcgtt 7 <210> 179 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 179 ccgctgg 7 <210> 180 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 180 ggaacta 7 <210> 181 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 181 attgcca 7 <210> 182 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 182 atatacg 7 <210> 183 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 183 gattagc 7 <210> 184 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 184 agaagtc 7 <210> 185 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 185 atagtac 7 <210> 186 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 186 gatctcg 7 <210> 187 <211> 7 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 187 ggctgcg 7 <210> 188 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MISC_FEATURE <222> (1)..(10) <223> This sequence may encompass 5-10 residues <400> 188 His His His His His His His His His His 1 5 10 <210> 189 <211> 413 <212> PRT <213> Unknown <220> <223> Description of Unknown: Beta-2 adrenergic receptor sequence <400> 189 Met Gly Gln Pro Gly Asn Gly Ser Ala Phe Leu Leu Ala Pro Asn Gly 1 5 10 15 Ser His Ala Pro Asp His Asp Val Thr Gln Glu Arg Asp Glu Val Trp 20 25 30 Val Val Gly Met Gly Ile Val Met Ser Leu Ile Val Leu Ala Ile Val 35 40 45 Phe Gly Asn Val Leu Val Ile Thr Ala Ile Ala Lys Phe Glu Arg Leu 50 55 60 Gln Thr Val Thr Asn Tyr Phe Ile Thr Ser Leu Ala Cys Ala Asp Leu 65 70 75 80 Val Met Gly Leu Ala Val Val Pro Phe Gly Ala Ala His Ile Leu Met 85 90 95 Lys Met Trp Thr Phe Gly Asn Phe Trp Cys Glu Phe Trp Thr Ser Ile 100 105 110 AspValLeuCysValThrAlaSerIleGluThrLeuCysValIleAla 115 120 125 Val Asp Arg Tyr Phe Ala Ile Thr Ser Pro Phe Lys Tyr Gln Ser Leu 130 135 140 Leu Thr Lys Asn Lys Ala Arg Val Ile Ile Leu Met Val Trp Ile Val 145 150 155 160 Ser Gly Leu Thr Ser Phe Leu Pro Ile Gln Met His Trp Tyr Arg Ala 165 170 175 Thr His Gln Glu Ala Ile Asn Cys Tyr Ala Asn Glu Thr Cys Cys Asp 180 185 190 Phe Phe Thr Asn Gln Ala Tyr Ala Ile Ala Ser Ser Ile Val Ser Phe 195 200 205 Tyr Val Pro Leu Val Ile Met Val Phe Val Tyr Ser Arg Val Phe Gln 210 215 220 Glu Ala Lys Arg Gln Leu Gln Lys Ile Asp Lys Ser Glu Gly Arg Phe 225 230 235 240 His Val Gln Asn Leu Ser Gln Val Glu Gln Asp Gly Arg Thr Gly His 245 250 255 Gly Leu Arg Arg Ser Ser Lys Phe Cys Leu Lys Glu His Lys Ala Leu 260 265 270 Lys Thr Leu Gly Ile Ile Met Gly Thr Phe Thr Leu Cys Trp Leu Pro 275 280 285 Phe Phe Ile Val Asn Ile Val His Val Ile Gln Asp Asn Leu Ile Arg 290 295 300 Lys Glu Val Tyr Ile Leu Leu Asn Trp Ile Gly Tyr Val Asn Ser Gly 305 310 315 320 Phe Asn Pro Leu Ile Tyr Cys Arg Ser Pro Asp Phe Arg Ile Ala Phe 325 330 335 Gln Glu Leu Leu Cys Leu Arg Arg Ser Ser Leu Lys Ala Tyr Gly Asn 340 345 350 Gly Tyr Ser Ser Asn Gly Asn Thr Gly Glu Gln Ser Gly Tyr His Val 355 360 365 Glu Gln Glu Lys Glu Asn Lys Leu Leu Cys Glu Asp Leu Pro Gly Thr 370 375 380 Glu Asp Phe Val Gly His Gln Gly Thr Val Pro Ser Asp Asn Ile Asp 385 390 395 400 Ser Gln Gly Arg Asn Cys Ser Thr Asn Asp Ser Leu Leu 405 410

Claims

1. A population of mammalian cells, Each mammalian cell of the population of mammalian cells is (i) a heterologous receptor gene operably linked to a doxycycline responsive promoter, the heterologous receptor gene being integrated into the genome of each mammalian cell of the population of mammalian cells by a transposase or a site-specific recombinase; (ii) Inducible reporters containing receptor-responsive elements Including, The inducible reporter is induced upon activation of a heterologous receptor protein encoded by the heterologous receptor gene; and The inducible reporter comprises a barcode that includes an index region that is unique to the heterologous receptor gene. A population of said mammalian cells.

2. 2. The population of mammalian cells of claim 1, wherein the heterologous receptor gene encodes a tyrosine kinase or a G protein-coupled receptor (GPCR).

3. 3. The population of mammalian cells of claim 1 or 2, wherein each individual mammalian cell of the population of mammalian cells further comprises one or more genes encoding one or more accessory proteins, the one or more accessory proteins comprising one or more of Gα subunits, Ric-8B, RTP1L, RTP2, RTP3, RTP4, CHMR3, or RTP1S.

4. The population of mammalian cells according to any one of claims 1 to 3, wherein each individual mammalian cell of said population of mammalian cells lacks endogenous nucleic acid encoding said heterologous receptor protein.

5. A population of mammalian cells described in any one of claims 1 to 4, wherein the inducible reporter is integrated into the genome of individual mammalian cells of the population of mammalian cells.

6. The population of mammalian cells of claim 5 , wherein the integrated inducible reporter is integrated by targeted integration.

7. The population of mammalian cells of claim 6 , wherein the integration is within the H11 safe harbor locus.

8. The population of mammalian cells according to any one of claims 1 to 7, wherein each individual mammalian cell of the population of mammalian cells comprises at least two copies of said heterologous receptor gene and / or said inducible reporter gene.

9. The population of mammalian cells according to any one of claims 1 to 8, wherein the barcode and / or index region is at least 10 nucleic acids.

10. 10. The population of mammalian cells of any one of claims 1 to 9, wherein the inducible reporter comprises or further comprises a gene for a fluorescent protein, said gene comprising a 3' untranslated region (UTR).

11. The population of mammalian cells of claim 10, wherein the barcode is located in the 3'UTR of the gene for the fluorescent protein.

12. 12. The population of mammalian cells of claim 10 or 11, wherein the gene encodes a luciferase protein.

13. 13. The population of mammalian cells of any one of claims 1 to 12, wherein the heterologous receptor gene is flanked by insulator sequences at the 5' and 3' ends and / or the inducible reporter is flanked by insulator sequences at the 5' and 3' ends.

14. The population of mammalian cells according to any one of claims 1 to 13, wherein each individual mammalian cell of said population of mammalian cells expresses a different heterologous receptor gene.

15. 1. A method for screening ligand and receptor binding, comprising: Contacting a population of mammalian cells according to any one of claims 1 to 14 with a ligand; detecting one or more reporters; determining the identity of the one or more reporters; Including, The identity of the reporter indicates the identity of the bound receptor. method.

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