Multiple assay

Abstract

The present invention provides a multiplex assay method for assays to analyze two or more polypeptides in living cells. In some embodiments, the invention provides a polycistronic reporter vector, a recipient cell for accepting the polycistronic reporter vector, and a multi-reporter cell. Methods of making a polycistron reporter vector, recipient cells for accepting a polycistron reporter vector, and a multi-reporter cell are provided. Libraries and kits comprising a polycistron reporter vector, recipient cells for accepting the polycistron reporter vector, and multiple reporter cells are provided. Methods of analyzing / determining multi-reporter cells and libraries of multi-reporter cells are provided.

Description

[0001] This application is a divisional application of the invention application with the application date of May 15, 2018, the Chinese application number of 201880046483.5, and the invention name of “Multiple Determination”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 507,169, filed May 16, 2017, the disclosure of which is hereby incorporated by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under Grant Nos. 1448764 and 1632576 awarded by the National Science Foundation and Contracts HHSN271201600006C, HHSN271201500028C, and HHSN271201600021C with the National Institutes of Health. The government has certain rights in this invention. Background Art

[0006] Determining the cellular response to internal or external stimuli requires visualization and monitoring of key players and pathways. Current methods for visualizing such responses are inefficient, costly, and only provide snapshots. For example, in vivo preclinical animal models are traditionally used for drug discovery and / or toxicological evaluation of candidate drugs, which are low-throughput, costly, insufficiently predictive of human toxicity, and provide little understanding of the mechanisms of compound toxicity. The throughput of these traditional methods is far less than the speed at which new chemicals are produced, whether they are compounds developed for pharmaceutical, agricultural, or other purposes (such as nutrition, cosmetics, personal care, etc.). The need for assessing the efficacy of these new chemicals or the risks that may be posed to human health requires the development of new screening tools that can provide a meaningful understanding of the mechanisms associated with such compounds at a throughput compatible with evaluating hundreds of thousands of compounds per week. Ideally, new screening tools will be able to pinpoint which aspects of cell physiology a specific chemical interferes with, and measure the resulting cellular responses. These needs can be addressed by appropriately configured living cell methods, which are the focus of the present invention.

[0007] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. Summary of the invention

[0008] The present invention provides compositions and methods for performing multiplex live cell screening (e.g., live cell high content screening (LC-HCS)) in a variety of cells (including immortalized cells, primary cells, human stem cells, human iPSC cells, and human iPSC-derived cells) as models for drug discovery and toxicology screening requiring high-throughput technology.

[0009] In some aspects, the present invention provides a multicistronic reporter vector comprising: a promoter operably linked to an open reading frame, wherein the open reading frame comprises two or more cistrons, and wherein the expression of the open reading frame in a cell produces a separate component polypeptide product from each cistron; wherein each cistron comprises a multiple cloning site (MCS) and a nucleic acid encoding a reporter vector, and wherein each cistron encodes a different reporter polypeptide; and wherein the expression of two or more nucleic acids inserted into the two or more multiple cloning sites and encoding a polypeptide fused to the reporter polypeptide is essentially stoichiometric. In some embodiments, the cistrons are separated from each other by nucleic acids encoding one or more self-cleaving peptides and / or one or more internal ribosome entry sites (IRES). In some embodiments, the one or more self-cleaving peptides are viral self-cleaving peptides. In some embodiments, the one or more viral self-cleaving peptides are one or more 2A peptides. In some embodiments, one or more 2A peptides are T2A peptides, P2A peptides, E2A peptides, or F2A peptides. In some embodiments, the reporter polypeptide further comprises one or more nucleic acids encoding a peptide linker between one or more of the reporter polypeptides and one or more of the self-cleaving peptides. In some embodiments, the peptide linker comprises the sequence Gly-Ser-Gly. In some embodiments, the reporter polypeptide is a fluorescent reporter polypeptide. In some embodiments, the reporter polypeptide of each cistron is selected from GFP, EGFP, Emerald, Citrine, Venus, mOrange, mCherry, TagBFP, mTurquoise, Cerulean, UnaG, dsRed, eqFP611, Dronpa, RFP, TagRFP, TdTomato, KFP, EosFP, Dendra, IrisFP, iRFP and smURFP.

[0010] In some embodiments, the polycistronic reporter vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron and a second cistron, wherein each cistron comprises a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide from 5' to 3'; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a viral cleavage peptide. In some embodiments, the polycistronic reporter vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, and a third cistron, wherein each cistron comprises a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide from 5' to 3'; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, and the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide.

[0011] In some embodiments, the multicistronic reporter vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, a third cistron, and a fourth cistron, wherein each cistron comprises, from 5' to 3', a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide, and the third cistron and the fourth cistron are separated by a nucleic acid encoding a third viral cleavage peptide. In some embodiments, the multicistronic reporter vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, a third cistron, and a fourth cistron, wherein each cistron comprises, from 5' to 3', a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide, and the third cistron and the fourth cistron are separated by a nucleic acid encoding an IRES.

[0012] In some embodiments, the polycistronic reporter vector further comprises one or more inducible elements located between the promoter and the open reading frame. In some embodiments, the polycistronic reporter vector comprises two inducible elements. In some embodiments, the inducible element is a Tet operator 2 (TetO2) inducible element.

[0013] In some embodiments, the polycistronic reporter vector further comprises a constitutive promoter. In some embodiments, the constitutive promoter is cytomegalovirus alpha (CMV), thymidine kinase (TK), eF1-α, ubiquitin C (UbC), phosphoglycerate kinase (PGK), CAG promoter, SV40 promoter or human β-actin promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is a tetracycline responsive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the tissue-specific promoter is specific to cells of the heart, blood, muscle, lung, liver, kidney, pancreas, brain or skin.

[0014] In some embodiments, the polycistronic reporter vector further comprises a site-specific recombinase sequence located 3' of the open reading frame. In some embodiments, the vector further comprises a nucleic acid encoding a selectable marker, wherein when the site-specific recombinase sequence is not recombined, the nucleic acid encoding the selectable marker is not operably connected to the promoter, and when the site-specific recombinase sequence is recombined with its target site-specific recombinase sequence, the nucleic acid encoding the selectable marker is operably connected to the promoter. In some embodiments, the site-specific recombinase sequence is a FRT nucleic acid sequence and / or an attP nucleic acid and / or a loxP nucleic acid sequence.

[0015] In some embodiments, the selectable marker of the vector confers resistance to hygromycin, Zeocin TM , Puromycin, Blasticidin, Neomycin or Hygromycin, Zeocin TM , puromycin, blasticidin or neomycin analogs.

[0016] In some embodiments, a polycistronic reporter vector comprising a nucleic acid encoding one or more polypeptides is inserted in frame into the one or more MCSs. In some embodiments, the one or more polypeptides comprise polypeptides that can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, synthetic lethality, cellular homeostasis, organelle homeostasis, or toxic responses.

[0017] In some embodiments, the multicistronic reporter vector further comprises one, two or three transcription units comprising a promoter located 5' of an open reading frame comprising two or more cistrons and a nucleic acid encoding a transgene, wherein the reporter vector further comprises a core insulator sequence and a polyA sequence located 3' of the transcription unit and 5' of the open reading frame comprising two or more cistrons.

[0018] In some aspects, the present invention provides a recipient cell for receiving a multicistronic reporter vector, wherein the recipient cell comprises a recombinant nucleic acid integrated into a specific site in the host cell genome, wherein the recombinant nucleic acid comprises a first promoter operably linked to a nucleic acid encoding a fusion polypeptide, wherein the fusion polypeptide comprises a reporter domain and a selectable marker domain, and wherein the nucleic acid comprises a site-specific recombinase nucleic acid sequence located at the 5' end of the nucleic acid encoding the fusion polypeptide.

[0019] In some embodiments, the promoter of the recipient cell is a constitutive promoter. In some embodiments, the constitutive promoter is a CMV promoter, a TK promoter, an eF1-α promoter, an UbC promoter, a PGK promoter, a CAG promoter, an SV40 promoter, or a human β-actin promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is a tetracycline responsive promoter.

[0020] In some embodiments, the site-specific recombinase sequence of the recipient cell is a FRT nucleic acid sequence and / or an attP nucleic acid sequence and / or a loxP nucleic acid sequence.

[0021] In some embodiments, the reporter domain of the fusion polypeptide is a fluorescent reporter domain. In some embodiments, the fluorescent reporter domain is selected from GFP, EGFP, Emerald, Citrine, Venus, mOrange, mCherry, TagBFP, mTurquoise, Cerulean, UnaG, dsRed, eqFP611, Dronpa, RFP, TagRFP, TdTomato, KFP, EosFP, Dendra, IrisFP, iRFP and smURFP. In some embodiments, the reporter domain of the fusion polypeptide is an mCherry reporter domain.

[0022] In some embodiments, the selectable marker domain of the fusion polypeptide confers resistance to hygromycin, Zeocin TM , Puromycin, Blasticidin, Neomycin or Hygromycin, Zeocin TM , puromycin, blasticidin, and neomycin analogs.

[0023] In some embodiments, the integrated recombinant nucleic acid of the recipient cell further comprises a nucleic acid encoding a tetracycline repressor polypeptide operably linked to a promoter. In some embodiments, the promoter is a human β-actin promoter or a CAG promoter.

[0024] In some embodiments, the recombinant nucleic acid is integrated into the adeno-associated virus S1 (AAVS1) locus, the chemokine (CC motif) receptor 5 (CCR5) locus, the human homolog of the mouse ROSA26 locus, the hipp11 (H11) locus, or the citrate lyase beta-like gene locus (CLYBL) of the recipient cell.

[0025] In some embodiments, the recipient cell is an immortal cell. In some embodiments, the immortal cell is HEK293T cell, A549 cell, U2OS cell, RPE cell, NPC1 cell, MCF7 cell, HepG2 cell, HaCat cell, TK6 cell, A375 cell or HeLa cell. In some embodiments, the recipient cell is a pluripotent cell, an induced pluripotent stem cell or a multipotent cell. In some embodiments, the induced pluripotent stem cell is a WTC-11 cell or a NCRM5 cell. In some embodiments, the recipient cell is a primary cell.

[0026] In some aspects, the present invention provides a method for producing a recipient cell for receiving a multicistronic reporter vector, the method comprising introducing a recombinant nucleic acid into a cell, wherein the recombinant nucleic acid comprises, from 5' to 3', a first nucleic acid for targeting homologous recombination to a specific site in the cell, a first promoter, a site-specific recombinase nucleic acid, a nucleic acid encoding a first reporter polypeptide and a selectable marker, a second nucleic acid for targeting homologous recombination to a specific site in the cell, a second promoter, and a nucleic acid encoding a second reporter polypeptide, wherein expression of the first reporter polypeptide without expression of the second reporter polypeptide indicates targeting integration of the recombinant nucleic acid to a specific site in the cell genome, and expression of the first and second reporter polypeptides indicates random integration in the cell genome. In some embodiments, the recombinant nucleic acid further comprises a nucleic acid encoding a tetracycline repressor, which is operably linked to the promoter 5' of the second nucleic acid for targeting homologous recombination.

[0027] In some embodiments, the recombinant nucleic acid is integrated into the genome of the recipient cell using: a) an RNA-guided recombination system comprising a nuclease and a guide RNA, b) a TALEN endonuclease, or c) a ZFN endonuclease.

[0028] In some embodiments, cells are selected that express the first reporter polypeptide but not the second reporter polypeptide.

[0029] In some embodiments, the site-specific recombinase nucleic acid introduced into the recipient cell is a FRT nucleic acid sequence and / or an attP nucleic acid sequence and / or a loxP nucleic acid sequence.

[0030] In some embodiments, the first reporter polypeptide introduced into the recipient cell is a fluorescent polypeptide, and the second reporter polypeptide introduced into the recipient cell is a different fluorescent polypeptide. In some embodiments, the fluorescent polypeptide is selected from GFP, EGFP, Emerald, Citrine, Venus, mOrange, mCherry, TagBFP, mTurquoise, Cerulean, UnaG, dsRed, eqFP611, Dronpa, RFP, TagRFP, TdTomato, KFP, EosFP, Dendra, IrisFP, iRFP and smURFP. In some embodiments, the first reporter polypeptide is an mCherry reporter and the second reporter polypeptide is GFP.

[0031] In some embodiments, the selectable marker introduced into the recipient cell confers resistance to hygromycin, Zeocin TM , Puromycin, Blasticidin, Neomycin or Hygromycin, Zeocin TM , puromycin, blasticidin, and neomycin analogs.

[0032] In some embodiments, the first promoter is a CMV promoter, a TK promoter, an eF1-α promoter, an UbC promoter, a PGK promoter, a CAG promoter, an SV40 promoter, or a human β-actin promoter, and the second promoter is a CMV promoter, a TK promoter, an eF1-α promoter, an UbC promoter, a PGK promoter, a CAG promoter, an SV40 promoter, or a human β-actin promoter. In some embodiments, the first nucleic acid for targeting homologous recombination and the second nucleic acid for targeting homologous recombination target the recombination to the AAVS1 locus, the CCR5 locus, the human ortholog of the mouse ROSA26 locus, the H11 locus, or the CLYBL locus.

[0033] In some embodiments, the receptor site is introduced into immortal cells. In some embodiments, the immortal cells are HEK293T cells, A549 cells, U2OS cells, RPE cells, NPC1 cells, MCF7 cells, HepG2 cells, HaCat cells, TK6 cells, A375 cells or HeLa cells. In some embodiments, the cells introduced into the receptor site are pluripotent cells, induced pluripotent stem cells or pluripotent cells. In some embodiments, the receptor site is introduced into primary cells.

[0034] In some embodiments, the invention provides a recipient cell line produced using the methods described herein.

[0035] In some aspects, the invention provides a multi-reporter cell comprising any recipient cell described herein, wherein any multicistronic reporter vector described herein has been integrated into the genome of the recipient cell. In some embodiments, the multicistronic reporter vector has been integrated into the AAVS1 locus of the recipient cell.

[0036] In some aspects, the present invention provides a multi-reporter cell, wherein the reporter cell comprises a multicistronic reporter construct, wherein the multicistronic reporter construct comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises two or more cistrons; wherein each cistron comprises a nucleic acid encoding a different transgenic product fused to a different reporter polypeptide, wherein the expression of the open reading frame in the cell produces a separate component polypeptide product from each cistron; and wherein the expression of the transgenic product is essentially stoichiometric. In some embodiments, the cistrons are separated from each other by nucleic acids encoding one or more self-cleaving peptides and / or one or more internal ribosome entry sites (IRES). In some embodiments, each of the reporter polypeptides is a fluorescent reporter polypeptide. In some embodiments, the one or more self-cleaving peptides are one or more 2A peptides. In some embodiments, the nucleic acid encoding the transgenic product fused to the reporter polypeptide further comprises one or more nucleic acids encoding a peptide linker between the reporter polypeptide and the viral self-cleaving peptide. In some embodiments, the peptide linker comprises the sequence Gly-Ser-Gly.

[0037] In some embodiments, the multi-reporter cell comprises a multicistronic reporter vector comprising a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, a third cistron, and a fourth cistron, wherein each cistron comprises a nucleic acid encoding a transgenic product fused to a fluorescent reporter polypeptide and a nucleic acid encoding a linker peptide from 5' to 3'; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide, and the third cistron and the fourth cistron are separated by a nucleic acid encoding an IRES. In some embodiments, the multi-reporter cell comprising a multicistronic reporter vector further comprises one or more inducible elements located between the promoter and the open reading frame.

[0038] In some embodiments, the inducible element of the multi-reporter cell is a Tet operator 2 (TetO2) inducible element.

[0039] In some embodiments, the promoter of the multi-reporter cells is a constitutive promoter. In some embodiments, the constitutive promoter is a CMV promoter, a TK promoter, an eF1-α promoter, an UbC promoter, a PGK promoter, a CAG promoter, an SV40 promoter, or a human β-actin promoter. In some embodiments, the promoter of the multi-reporter cells is a tissue-specific promoter. In some embodiments, the tissue-specific promoter is specific to cells of the heart, blood, muscle, lung, liver, kidney, pancreas, brain, or skin. In some embodiments, the promoter of the multi-reporter cells is an inducible promoter. In some embodiments, the inducible promoter is a TRE promoter.

[0040] In some embodiments, the multi-reporter cells encode one or more transgenic products, wherein the one or more transgenic products comprise polypeptides that can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, cellular homeostasis, organelle homeostasis, toxic responses, or phenotypic characteristics. In some embodiments, the analysis is performed on a single cell. In some embodiments, the reporter polypeptides can be visualized by microscopy, high-throughput microscopy, fluorescence activated cell sorting (FACS), luminescence, or using a plate reader.

[0041] In some embodiments, the multi-reporter cell further comprises one, two or three transcription units comprising a promoter located 5' of an open reading frame comprising two or more cistrons and a nucleic acid encoding a transgene, wherein the reporter vector further comprises a core insulator sequence located 3' to the transcription unit and 5' of the open reading frame comprising two or more cistrons.

[0042] In some embodiments, the present invention provides a method for producing a multi-reporter cell, comprising introducing any polycistronic reporter vector described herein into any recipient cell described herein. In some embodiments, the recombinase-related nucleic acid sequence is a FRT nucleic acid sequence, and the recipient cell comprises a flp recombinase. In some embodiments, the recombinase-related nucleic acid is attP, and the recipient cell comprises a Bxb1 recombinase, a PhiC31 recombinase, or an R4 recombinase. In some embodiments, the recombinase-related nucleic acid sequence is a loxP nucleic acid sequence, and the recipient cell comprises a CRE recombinase.

[0043] In some embodiments, the present invention provides a multi-reporter vector library, wherein the library comprises a multicistronic reporter vector or a plurality of reporter cells as described herein, wherein the multicistronic reporter vector comprises different transgenes encoding polypeptides fused to a reporter polypeptide, wherein when introduced into a cell, two or more different transgenes on each vector are expressed essentially stoichiometrically. In some embodiments, the library comprises a reporter vector encoding one or more transgenes, and the one or more polypeptides comprise polypeptides that can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, cell homeostasis, organelle homeostasis, toxic responses, or phenotypic characteristics. In some embodiments, the biological pathway is a pathway associated with a disease. In some embodiments, the disease is cancer, cardiovascular disease, neurodegenerative disease, or autoimmune disease. In some embodiments, the biological pathway is a pathway associated with a toxic response mechanism within the cell. In some embodiments, the biological pathway is a pathway associated with cell proliferation, cell differentiation, cell death, apoptosis, autophagy, DNA damage and repair, oxidative stress, chromatin / epigenetics, MAPK signaling, PI3K / Akt signaling, translation control, cell cycle and checkpoint control, cell metabolism, development and differentiation signaling, immunology and inflammation signaling, tyrosine kinase signaling, vesicle trafficking, cytoskeletal regulation, or ubiquitin pathways. In some embodiments, the library comprises isogenic reporter cells; that is, the reporter cells are prepared from a single receptor cell line by introducing a plurality of different reporter vectors, thereby generating a plurality of different reporter cells based on the single receptor cell line.

[0044] In some embodiments, each polycistronic vector of the library comprises a transgene for analyzing a specific single biological pathway, a specific crosstalk between two or more biological pathways, synthetic lethality, a specific cellular homeostasis, a specific organelle homeostasis, a specific toxic response, a specific tissue or phenotypic characteristic, and for each polycistronic reporter vector comprises a common transgene encoding a polypeptide fused to a different reporter polypeptide. In some embodiments, each polycistronic vector of the library comprising a transgene for analyzing a specific single biological pathway, a specific crosstalk between two or more biological pathways, a specific cellular homeostasis, a specific organelle homeostasis, a specific toxic response, or a phenotypic characteristic comprises a common transgene encoding a polypeptide fused to a reporter polypeptide.

[0045] In some embodiments, the invention provides a library of recipient cells for receiving a polycistronic reporter vector, wherein the library comprises any of the recipient cells described herein. In some embodiments, each cell in the library comprises a polycistronic reporter vector comprising a different transgene encoding a polypeptide fused to a reporter polypeptide, wherein when introduced into the cell, the different transgenes on each vector are expressed essentially stoichiometrically.

[0046] In some embodiments, the library comprises different immortal cells. In some embodiments, the library includes one or more of HEK293T cells, A549 cells, U2OS cells, RPE cells, NPC1 cells, MCF7 cells, HepG2 cells, HaCat cells, TK6 cells, A375 cells or HeLa cells. In some embodiments, the library comprises different pluripotent, multipotent and / or progenitor cells. In some embodiments, the different pluripotent or multipotent cells include one or more of induced pluripotent stem cells, multipotent cells, hematopoietic cells, endothelial progenitor receptor cells, mesenchymal progenitor cells, neural progenitor cells, osteochondroblast progenitor cells, lymphoid progenitor cells or pancreatic progenitor cells. In some embodiments, after introducing the multicistronic reporter vector, the multipotent or multipotent cell multi-reporter cell library is differentiated. In some embodiments, the library comprises different primary cells. In some embodiments, the primary cells include one or more of cardiomyocytes, muscle cells, pneumonocytes, hepatocytes, kidney cells, pancreatic cells, neurons or tumor cells. In some embodiments, each cell in the library comprises the same polycistronic reporter vector. In some embodiments, the cells in the library comprise different polycistronic reporter vectors. In some embodiments, the different polycistronic reporter vectors are introduced into isogenic recipient cells.

[0047] In some embodiments, the present invention provides a cell library as described herein, wherein the reporter vector encodes one or more transgenes, and the one or more polypeptides include polypeptides that can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, cell homeostasis, or phenotypic characteristics. In some embodiments, the biological pathway is a pathway associated with a disease. In some embodiments, the disease is cancer, cardiovascular disease, neurodegenerative disease, or autoimmune disease. In some embodiments, the biological pathway is a pathway associated with a toxic response mechanism within the cell. In some embodiments, the biological pathway is a pathway associated with cell proliferation, cell differentiation, cell death, apoptosis, autophagy, DNA damage and repair, oxidative stress, chromatin / epigenetics, MAPK signaling, PI3K / Akt signaling, translation control, cell cycle and checkpoint control, cell metabolism, development and differentiation signaling, immunology and inflammatory signaling, tyrosine kinase signaling, vesicle transport, cytoskeletal regulation, or ubiquitin pathways.

[0048] In some embodiments, each polycistronic vector of the cells of the library comprising a transgene for analyzing a specific single biological pathway, a specific crosstalk between two or more biological pathways, synthetic lethality, a specific cellular homeostasis, a specific organelle homeostasis, a specific toxic response, a specific tissue and / or a phenotypic characteristic comprises a common transgene encoding a polypeptide fused to a different reporter polypeptide. In some embodiments, each polycistronic vector comprising a transgene for analyzing a specific single biological pathway, a specific crosstalk between two or more biological pathways, a specific cellular homeostasis, a specific organelle homeostasis, a specific toxic response and / or a phenotypic characteristic comprises a common transgene encoding a polypeptide fused to a reporter polypeptide.

[0049] In some aspects, the invention provides a kit comprising one or more polycistronic reporter vectors as described herein. In some embodiments, the invention provides a kit comprising one or more receptor cells as described herein. In some embodiments, the kit comprises one or more polycistronic reporter vectors as described herein and one or more receptor cells as described herein.

[0050] In some aspects, the invention provides a kit comprising one or more of the multi-reporter cells described herein. In some embodiments, the invention provides a kit comprising a library of receptor cells and / or reporter cells arranged in a multi-well plate. In some embodiments, the cells in the multi-well plate are cryopreserved.

[0051] In some aspects, the present invention provides a method for analyzing two or more polypeptides in living cells, the method comprising determining the expression of two or more transgenics and / or the position of two or more transgenic products of any multi-reporter cells described herein. In some embodiments, the method is used to analyze a single biological pathway, crosstalk between two or more biological pathways, synthetic lethality, cell homeostasis, organelle homeostasis, toxic reactions and / or phenotypic characteristics. In some embodiments, the expression of the two or more transgenics and / or the position of the two or more transgenic products are determined at one or more time points. In some embodiments, the expression of the two or more transgenics and / or the position of the two or more transgenic products are determined at one or more time points in 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 4 days, 7 days, 14 days, 21 days, 30 days, 1 month, 3 months, 6 months, 9 months, 1 year or any time between or more than 1 year after the start of the analysis.

[0052] In some embodiments, the invention provides a method of measuring the effect of an agent on the spectrum of two or more polypeptides in a living cell, the method comprising subjecting a multi-reporter cell as described herein to (i.e., contacting) the agent and determining the expression of the two or more transgenes and / or the location of the two or more transgene products in the cell in response to the agent. In some embodiments, the agent is a drug or a drug candidate. In some embodiments, the agent is a cancer drug or a cancer pharmaceutical agent. In some embodiments, the method is a toxicology screen.

[0053] In some embodiments, the invention provides methods for determining the expression of the two or more transgenes and / or the location of the two or more transgene products in a multiple reporter cell library. In some embodiments, the profile is obtained using a single cell.

[0054] In some embodiments of the above methods, the expression of the two or more transgenes and / or the location of the two or more transgene products, the expression and / or location of the two or more transgenes are measured by microscopy, high throughput microscopy, fluorescence activated cell sorting (FACS), luminescence, use of a plate reader, mass spectrometry, or deep sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1A-1F : Figures 1A-1C The generation of recipient cell lines is depicted. Figure 1ADepicted is the AAVS1 locus with the acceptor site integration site (IS) and the associated acceptor site design. The scheme shows the location of the junction PCR primers (bold half arrows) and Southern blot probes. The internal probe detects random integration, while the 5' probe allows for the distinction of mono- or biallelic integration. Figure 1B Depicted are PCR primers (half arrows) designed to amplify homologous recombination junctions and used to verify specific and successful integration. Example of junction PCR of a single clone. * indicates a positive clone. Figure 1C Southern blot analysis of candidate clones is depicted. Genomic DNA digested with EcoRI was probed with a 5' probe (* indicates clones with monoallelic integration) ( Figure 1A Arrows indicate the positions of two AAVS1 hybridizing fragments derived from one targeted allele integration (TI). Figure 1D Depicts further use of internal probes ( Figure 1A ) was probed to verify a single integration in the genome (arrow) of a clone with monoallelic integration (* indicates a nonspecific band). Figure 1E The first and second receptor site designs are shown. Cell lines were initially engineered with single copy receptor sites for reorganization, and the receptor sites contained: SV40 constitutive promoter, flip enzyme recombination target (FRT) site, mCherry fluorescent marker and bleomycin resistance gene. The second receptor site design contains: SV40 constitutive promoter, flip enzyme recombination target (FRT) site, attP site (to be reorganized by serine recombinase), mCherry fluorescent marker, bleomycin resistance gene (bleomycin) or blasticidin resistance gene (blasticidin-S deaminase, Bsd) and CMV-GFP after the right homology arm (AAVS1-R) of AAVS1. The CMV-GFP in the redesigned receptor site plasmid allows to distinguish between random and targeted integration. Cells with random integration emit green fluorescence due to the expression of GFP, and cells with targeted integration do not emit green fluorescence due to the loss of CMV-GFP, because it is located outside the receptor site region integrated into the cell (between AAVS1-L and AAVS1-R). Figure 1F Demonstration of the novel receptor site enables FACS-based single cell sorting. Examples of FACS data for wild-type Hek293 cells (left) and cells after receptor site integration (right). Dual color (mCherry and GFP) allows selection of cells with at least one integration at the AAVS1 locus and no random integrations (mCherry+ / GFP-).

[0056] Figure 2Shown is the design of a polycistronic platform contained in a comparable pFIT vector backbone. The custom design allows expression of four polypeptides from the same ORF under the same promoter and with recombination (FRT sites) capability. Recombination into recipient cell lines results in similar protein expression levels at balanced levels in all cells.

[0057] Figure 3 is a diagram showing that Ras / MAPK signaling proteins are localized in different cellular compartments depending on the activation state of the pathway.

[0058] Figure 4 The following scheme is shown, which describes 1) incorporation of a receptor site into a safe harbor locus of a target cell line; 2) a multicistronic MAPK reporter vector; and 3) recombination of the MAPK reporter vector into the receptor site of a recipient cell line to generate a reporter cell line.

[0059] Figure 5 Depicted is the use of HEK293TA+4-MAPK reporter cell line to track MAPK expression. The engineered multi-reporter cell line was induced to express MAPK reporters, and the cells were treated with GDC-0879 (Raf inhibitor), FR 180204 (Erk inhibitor), lovastatin (KRas inhibitor) and PD0325901 (Mek inhibitor) for 4 or 9 h. The cells were imaged in a wide-field microscope (scale bar 10 μm).

[0060] Figure 6A-6B : Fig. 6A A summary of a library of 24 MAPK polycistronic vectors carrying 4 proteins from the MAPK pathway is shown. mCherry-Erk, Venus-Ras, and mCerulean-Raf are fluorescently labeled, while Mek is unlabeled. mCherry-Erk and unlabeled Mek are fixed for all vectors, while Ras and Raf have different isoforms and mutants that are commonly found in cancer cells. MAPK constructs are denoted "MAPK R" and are numbered from "1-24". Figure 6B Twenty-three MAPK reporter cell lines generated by engineering U2OSA cells with multi-reporter vectors and Bxb1 are shown. All reporter cell lines were under tetracycline induction. TM Epi-fluorescence Nikon for incubator systems TM Cells were imaged using a microscope and Plan Fluor TM 40x objective lens and Software (Nikon TM ) to collect images.

[0061] Figures 7A-7C : Figures 7A-7CDisplay U2OS A-Tet MAPK R19, R20, and R21 reporter cell lines were sensitive to trametinib and PD0325901 inhibition. Fig. 7A Shown are representative examples of cells expressing K-Ras(wt), K-Ras(G12C), and K-Ras(G12D) exposed to trametinib and PD 0325901 for 1 hour showing an increase in mCherry-Erk in the cytoplasm compared to DMSO-treated cells. Figure 7B Results for inhibitors tested in duplicate as single agents are shown for four ten-fold serial dilutions starting at 10 μM (trametinib (black), PD 0325901 (grey)). The percentage of cells with activated MAPK signaling was calculated for each sample and plotted as the mean with standard deviation. The dashed lines represent the minimum and maximum values ​​in the experiment. The subcellular localization of mCherry-labeled ERK was used as a measure of MAPK pathway activation; cells with activated MAPK signaling showed nuclear localization of ERK, while cells with inactivated or inhibited MAPK signaling showed nuclear exclusion of ERK. The ability of MAPK pathway inhibitors to inhibit nuclear localization of mCherry-labeled ERK in three different cell lines expressing K-Ras wild type, K-Ras G12C or K-Ras G12D was tested. To quantify these images, we calculated the ratio of nuclear to cytoplasmic ERK in single cells for four images of each sample and plotted the resulting values ​​as histograms. Figure 7C Shown are the mean ICs from replicate experiments calculated for the two inhibitors in each of the three cell lines. 50 value.

[0062] Figures 8A-8C : Figures 8A-8C Describes the U2OS A-Tet Example of QC steps performed on recipient cell line clone 23. Fig. 8A Shown are the results of genomic DNA sequencing of the acceptor site integration in the AAVS1 locus, which switches the 5' and 3' flanking regions of the integration, indicating that no mutations / deletions or insertions occurred. Figure 8B U2OS (wt) cells and U2OS A-Tet 2D plots and histograms of mCherry fluorescence levels in recipient cells, indicating that U2OS A-Tet More than 99% of the cells were mCherry(+). Figure 8C Shown in U2OS A-Tet2D fluorescence density plot of FACS analysis performed on MTS-Venus:H2B-TagBFP reporter cells. Uninduced reporter cells are mCherry negative, which demonstrates the specificity of integration events into receptor sites. The same reporter cells induced with doxycycline show significant consistency in the reporter expression level in the polyclonal population of cells after the integration of the polycistronic construct. The correlation coefficient (r) is calculated by Venus and TagBFP fluorescence levels, and indicates that there is a linear relationship between different marker protein expressions.

[0063] Fig. 9 Describe the exemplary polycistronic and universal platform that can be used.The polycistronic and universal platform constructs are designed to include exemplary constitutive promoters, including cytomegalovirus immediate early promoter (CMV), human thymidine kinase promoter (TK) and chicken beta-actin promoter coupled to CMV early enhancer (CAG).The reporter vector is also designed to contain genes encoding exemplary resistance markers, including hygromycin resistance gene (Hygro), bleomycin resistance gene (Zeo) and puromycin resistance gene (Puro).

[0064] Figures 10A-10C : Figures 10A-10C Depicting U2OS A 3-Tox ORG Characterization of reporter cell lines. Fig. 10A Engineered multi-reporter cell lines imaged in wide-field microscopy are shown (scale bar 10 μm). Fig. 10B and 10C Shown are engineered multi-reporter cell lines analyzed using flow cytometric analysis (FACS). Fig. 10B shows a histogram of three fluorescent reporters expressed by U2OSAToxORG cells, which shows similar coefficients of variation (CV) for all three fluorescently labeled proteins; and Fig. 10C A 2D density plot is shown, which shows that the expression of the MTS-Venus and H2B-TagBFP reporters is highly correlated (r=87%).

[0065] Figures 11A-11B : Fig.11A and 11B New U2OS for DNA damage inducers is shown A Tox DUPR Reporter cell line report. Fig.11A U2OS Tox expressing 53BP1-mCerulean, XBP1-Venus, and H2B-mCherry (not shown) exposed to vehicle, etoposide, neocarcinostatin (NCS), aphidicolin, thapsigargin, and tunicamycin for 8 hDUPR Representative examples of stable cell lines. Only cells exposed to etoposide and NCS showed an increase in the number of foci per nucleus. All other compounds showed similar numbers of foci to the DMSO control. Fig. 11B Results are shown for compounds tested in quadruplicate in four-point ten-fold serial dilutions. The number of foci / nuclei was scored for each sample and plotted as mean versus standard deviation. The mean EC is indicated in the legend. 50 value.

[0066] Figures 12A-12E Depicts the DUPR Measurement of 53BP1 reporter in cells. DUPR Reporter cells are plated in 384-well plates treated with vehicle (DMSO) or 1 μM etoposide and 500 ng / μl NCS-DNA damage inducer (which increases the number of 53BP1 foci). For each condition, there are 48 holes on the whole plate to determine spatial consistency. Images are processed to count the number of foci / nuclei using CellProfiler (open source software for image analysis, Kamensty, L et al. Bioinformatics (2011)). Data are further calculated using python programming language (Python Software Foundation, World Wide Web website python.org), and graphs are generated using GraphPad Prism 7.0 software (La Jolla, California, USA). Fig. 12A and 12B No drift or edge effects were observed. The scatter plots of the responses (number of 53BP1 foci / nucleus) were plotted against the well number, where the wells were sorted first by row and then by column ( Fig. 12A ), or sort by columns first and then by rows ( Fig. 12B ). Figures 12C-12E It is shown that the use of etoposide ( Fig. 12C ) or NCS( Fig.12D ) and compared with vehicle D-UPR Scatter plot of the average number of foci / nucleus in cells. The bold line represents the mean and the dashed line represents 3 times the standard deviation. Fig.12E A summary of the mean, StDev and Z' for each compound is provided. Z' was calculated using the following formula: Z' = 1 - ((3*StDev(Cpd) + 3*StDev(DMSO))) / ((Average(Cpd) - Average(DMSO)).

[0067] Fig.13 Shows the use of U2OS A Tox ORGand Tox DUPR Reporter cell lines were used as single reporters or combined in the same well to assess toxicity. A Tox ORG or Tox DUPR Representative images of , and representative images after mixing the two cell lines at a 1:1 ratio. Cells were plated 24h before image acquisition. Below is a table depicting the fluorescently labeled reporters contained in each Tox reporter. Note that the DNA marker (H2B) is common to both reporters, however it is labeled with two different fluorophores - this allows the two reporter cell lines to be separated during image analysis.

[0068] Fig.14 Shown is the use of U2OSA Tox ORG and Tox DUPR Reporter cell lines were combined in the same well to assess toxicity. The top image shows U2OS Tox ORG and U2OS Tox DUPR Representative example of . Cells were exposed to vehicle and neocarcinomastatin (NCS) for 8 hours. Only cells expressing U2OSA Tox DUPR And cells exposed to NCS showed an increase in the number of foci per nucleus. Scale bar 10 μm. The lower panel shows the results of testing NCS in duplicate at four ten-fold serial dilutions and in quadruplicate for the highest concentration. The number of foci / nucleus was scored for each sample and plotted as mean with standard deviation. The mean EC is indicated in the legend. 50 value.

[0069] Fig.15 Depicted is the expression of a 4-color multi-reporter transiently expressed in U2OS cells. U2OS cells were transiently transfected with a polycistronic reporter containing H2B-TagBFP, mCherry-LC3, MTS-Venus, and palm-miRFP. 48 h after transfection, cells were plated in glass-bottom plates and imaged 24 h later.

[0070] Fig.16A and 16B The successful generation of iPS recipient cell lines for reporter recombination is shown. Fig.16A The receptor site design is shown. Fig. 16B NCRM5 iPSC colonies with correct integration of the receptor site (mCherry positive and GFP negative) are shown.

[0071] Figures 17A-17C The process of engineering iPS recipient cell lines, designing 4-TOX reporter constructs, and cloning the constructs into recipient lines is described. Specifically, Fig.17A The design of a targeting vector is shown to engineer a recipient cell line with a "landing pad" in the AAVS1 locus using Cripsr-Cas9-guided sgRNA. Note: mCherry is expressed under the SV40 promoter and ATG-Frt start site. Fig. 17B Shown is the design of the 4-TOX reporter: a tetracistronic expression construct incorporating four multiple cloning sites (MCS) separated by three unique tandem viral 2A cleavage peptide inserts under the αMHC promoter (or CAG promoter) and an IRES element followed by a Frt. Fig. 17C It is shown how once recombination has occurred and the 4-color reporter is inserted into the genome at the FRT site, the SV40 promoter and ATG start codon are brought into proximity and in frame with the hygromycin resistance gene and the mCherry gene is inactivated.

[0072] Fig.18 Depicted is a scheme for generating alternative reporter cell lines. A multi-reporter system was developed to incorporate plug-and-play components (indicated by black boxes) and testable elements (indicated by gray boxes). The plasmid includes several previously validated modular elements (indicated by dashed boxes). Fig.18 As shown, the reporter constructs were designed to include exemplary constitutive promoters, including the cytomegalovirus immediate early promoter (CMV), the human elongation factor 1α promoter (EF1α), the human ubiquitin C promoter (Ubc), and the chicken β-actin promoter coupled to the CMV early enhancer (CAG). The reporter vectors were also designed to contain genes encoding exemplary resistance markers, including the hygromycin resistance gene (Hygro), the Zeocin TM resistance gene (Zeo), neomycin resistance gene (Neo) and puromycin resistance gene (Puro). DETAILED DESCRIPTION

[0073] The present invention provides multiple high-content assays that can analyze multiple polypeptides in living cells (e.g., single living cells). In some aspects, the present invention provides a multicistronic reporter vector, comprising: a promoter operably connected to an open reading frame, wherein the open reading frame comprises two or more cistrons, and wherein the expression of the open reading frame in the cell produces a component polypeptide product separated from each cistron; wherein each cistron comprises a multiple cloning site (MCS) and a nucleic acid encoding a reporter vector, wherein each cistron encodes a different reporter polypeptide; and wherein the expression of two or more nucleic acids inserted into the two or more multiple cloning sites and encoding a polypeptide fused to the reporter polypeptide is essentially stoichiometric. In some aspects, the present invention provides a receptor cell for receiving a multicistronic reporter vector. In yet other aspects, the present invention provides a multi-reporter cell for multiple high-content assays, wherein the multi-reporter cell comprises any multicistronic reporter vector described herein. The multi-reporter cells described herein can be used in live cell assays as a means to analyze aspects of cell behavior and perturbations of these behaviors that may be induced by candidate therapeutic agents or other chemical compounds or other stimuli or combinations thereof, to analyze the expression and activity of multiple polypeptides in living cells (e.g., single living cells), including but not limited to biological pathways, crosstalk between biological pathways, cellular homeostasis, organelle homeostasis, and toxicity.

[0074] Live cell screening (e.g., LC-HCS) provides the opportunity to screen compounds in a cell system that recapitulates the dynamic properties of signal transduction and cell phenotypes that cannot be captured by endpoint assays. Immortal cell lines can be used to monitor the toxicity of various compounds and determine the effects that molecules (such as target therapeutic candidate compounds) may have on specific pathways. The ability to easily maintain and use these cells allows immortal cell lines to become powerful tools in cell and molecular biology. Stem cells or human induced pluripotent stem cells (iPSCs) have great potential as cell models used in live cell screening because they provide physiological relevance and high fecundity.

[0075] This article describes novel methods, cells and multiple high-throughput assays that provide mechanical and phenotypic readouts of cell stress, homeostasis and related events in immortalized, primary and human iPS cells (which have the potential to differentiate into a variety of cell types in vitro). A wide variety of chemicals are known to perturb homeostasis and cause cell stress, and therefore are important aspects of cell physiology monitoring in the context of understanding the activity or toxicity mechanisms of candidate therapeutic agents. The methods described herein can be used to interrogate any potential incidental cytotoxicity of therapeutic agents, particularly anticancer drugs.

[0076] In some embodiments, the methods, cells and multiple high-throughput assays are used to analyze cardiotoxicity. Cardiotoxicity represents a harmful side effect of cancer therapy, resulting in a large amount of morbidity and mortality. Cytotoxic agents and targeted therapies (including classical chemotherapeutic agents, antibodies and small molecule tyrosine kinase inhibitors and chemopreventive agents) used to treat cancer all affect the cardiovascular system and may cause serious effects such as heart failure, ventricular dysfunction and myocardial ischemia. The rise of cancer therapy-induced cardiomyopathy shows that the risk of cardiotoxicity must be carefully weighed during the evaluation and development of any anticancer drug.

[0077] The molecular mechanisms linking cancer therapy to cardiomyopathy, including the specific contribution of stress-induced transcription factors to cell survival or death, are not fully understood due to the lack of systems to monitor this aspect of toxicology in real time in biologically relevant cardiac cells. The assay described herein meets this need by providing a multiplexed fluorescent reporter system that provides a readout of cellular stress and organelle homeostasis using human iPSC-derived cardiomyocytes. Molecules and potential therapeutics can be assessed for the effects of neurotoxicity, hepatotoxicity, or any other type of tissue toxicity.

[0078] The main limitation of using primary cardiomyocytes, primary neurons or other primary cell types is the technical difficulties associated with obtaining and maintaining these cells. For example, although immortalized cardiac cells are convenient because they can easily proliferate, beat, and in some cases can stably maintain cardiac phenotypes, their metabolism and morphology may be different from cardiomyocytes, thus limiting their use in toxicological studies. Cardiomyocytes derived from stem cells or iPSC overcome these shortcomings and provide tools that not only assess the effects of molecules on terminally differentiated cells, but also study the development or effects of various molecules through different differentiation stages. This is particularly important because reliable tests for progenitor cells and differentiated cells are valuable but rare. In addition, iPSCs can be generated from human subjects to examine various disease phenotypes and normal phenotypes.

[0079] In some embodiments, the methods, cells and multiple high-throughput assays are used for drug discovery. For example, the methods, cells and multiple high-throughput assays are used for drug discovery for the treatment of neurodegeneration. In some embodiments, the invention provides the use of iPSC-derived cells in drug development for the treatment of neurodegenerative diseases.

[0080] definition

[0081] As used herein, "vector" refers to a recombinant plasmid or virus comprising a nucleic acid to be delivered into a host cell in vitro or in vivo.

[0082] As used herein, term " polynucleotide " or " nucleic acid " refers to the polymeric form of nucleotide (ribonucleotide or deoxyribonucleotide) of any length.Therefore, this term includes but is not limited to single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrid, or the polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.The backbone of nucleic acid can include sugar and phosphate group (as can usually be found in RNA or DNA) or modified or substituted sugar or phosphate group.Alternately, the backbone of nucleic acid can include the polymer of synthetic subunit (such as phosphoramidate), and therefore can be oligodeoxynucleoside phosphoramidate (P-NH2) or mixed phosphoramidate-phosphodiester oligomer.In addition, double-stranded nucleic acid can be obtained from chemically synthesized single-stranded polynucleotide product by synthesizing complementary chain and making chain annealing under appropriate conditions or by using DNA polymerase to synthesize complementary chain from the beginning with appropriate primer.

[0083] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers and polymers of amino acid residues. The definition encompasses both full-length proteins and fragments thereof. The term also includes post-translational modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In addition, for the purposes of the present invention, "polypeptide" refers to a protein (usually conservative in nature) including modifications (such as deletions, additions and substitutions) relative to the native sequence, as long as the protein maintains the desired activity. These modifications may be intentional (such as by site-directed mutagenesis), or may be accidental (such as by mutations of the host producing the protein or due to errors caused by PCR amplification).

[0084] As used herein, "biosensor" refers to a reporter compound attached to another protein sequence to make it sensitive to small biomolecules or other physiological intracellular processes. In a non-limiting example, the biosensor is a fluorescent biosensor comprising a genetically encoded fluorescent polypeptide. The biosensor is introduced into a cell, tissue or organism to allow detection (e.g., by fluorescence microscopy) of the regulation of the reporter properties as a FRET efficiency difference, translocation of a fluorescent protein or a single reporter protein. Many biosensors allow long-term imaging and can be designed to specifically target cell compartments or organelles. Another advantage of biosensors is that they allow the study of signal transduction pathways or the measurement of biomolecules while retaining spatial and temporal cellular processes to a large extent.

[0085] A "recipient cell" is a cell that has been engineered to contain a receptor construct in its genome.

[0086] A "receptor construct" is a nucleotide sequence that comprises a nucleic acid sequence that may comprise a reporter nucleic acid.

[0087] The term "transgene" refers to a nucleic acid that is introduced into a cell and is capable of being transcribed into RNA and optionally translated and / or expressed under appropriate conditions.

[0088] As used herein, unless otherwise defined, "stem cell" refers to any non-somatic cell. Any cell that is not a terminally differentiated or terminally committed cell can be referred to as a stem cell. This includes embryonic stem cells, induced pluripotent stem cells, progenitor cells, and partially differentiated progenitor cells. For the purposes of this application, any cell with the potential to differentiate into two different types of cells is considered a stem cell.

[0089] As used herein, "iPS" cells refer to any pluripotent cells obtained by reprogramming non-pluripotent cells. Reprogrammed cells may be generated by reprogramming progenitor cells, partially differentiated cells, or fully differentiated cells of any embryonic or extraembryonic tissue lineage.

[0090] As used herein, "reprogramming" refers to the process of dedifferentiating cells that have been at least partially differentiated into a pluripotent state.

[0091] As used herein, "immune privileged cells" refer to cells that elicit a diminished immune response when introduced into a foreign host organism.

[0092] As used herein, "cistron" refers to a nucleic acid segment that is equivalent to a gene and encodes a single functional unit (e.g., a single polypeptide or a fusion polypeptide comprising a transgenic product and a reporter domain). As used herein, a polycistronic vector is a nucleic acid comprising two or more cistrons. In some embodiments, a polycistronic vector comprises two or more cistrons in a single open reading frame. In some embodiments, a single open reading frame produces two or more polypeptides separated from each other when translated.

[0093] As used herein, the term "essentially stoichiometric" with respect to the expression of two or more reporter polypeptides refers to the expression of two or more reporter polypeptides, wherein the expression levels of the two or more reporter polypeptides are equal or vary by no more than about 5%, 10%, 15%, 20% or 25% of each other.

[0094] As used herein, "site-specific recombinase sequence" refers to the target sequence of a site-specific recombination system. Site-specific recombination systems include, but are not limited to, Tyr recombinase, Ser integrase, Cre recombinase with a loxP target sequence, and FLP recombinase with a FRT target sequence. The site-specific recombination nucleic acid sequences of Tyr recombinase and Ser integrase (e.g., PhiC31) integrase include, but are not limited to, attB and attP. The site-specific recombination nucleic acid sequences of CRE recombinase include, but are not limited to, loxP. The site-specific recombination nucleic acid sequences of FLP recombinase include, but are not limited to, FRT.

[0095] References herein to "about" a value or parameter include (and describe) embodiments involving the value or parameter itself. For example, description referring to "about X" includes description of "X".

[0096] As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated.

[0097] It should be understood that aspects and embodiments of the present invention described herein include "comprising," "consisting," and / or "consisting essentially of" aspects and embodiments.

[0098] Receptor cells

[0099] The present disclosure provides multi-reporter cells and methods for producing multi-reporter cells, which can be used to analyze two or more polypeptides in living cells. Multi-reporter cells are developed by cloning a multicistronic reporter vector into an insertion site of a recipient cell. Receptor cells are developed by incorporating a recombinant nucleic acid encoding a receptor sequence into the genome of a cell. The receptor sequence comprises an insertion site that allows the multicistronic reporter vector to be site-specifically integrated into the genome of the recipient cell. As described herein, the multicistronic reporter vector comprises a nucleic acid encoding two or more polypeptides, wherein the polypeptide is fused to a reporter domain. The two or more nucleic acid sequences encoding the target polypeptide are located in the same open reading frame, thereby allowing the recombinant peptide to be expressed essentially stoichiometrically. In some embodiments, essentially stoichiometric expression is the expression of two or more reporter polypeptides, wherein the expression levels of the two or more reporter polypeptides are essentially the same; that is, with a stoichiometry of 1:1. In some embodiments, the expression levels of the two or more reporter polypeptides are equal or vary by no more than about 5%, 10%, 15%, 20%, or 25% of each other. In some embodiments, stoichiometric expression is essentially the stoichiometric expression of two, three, four or more reporter polypeptides. The expression levels of the two or more reporter polypeptides can be measured by any means known in the art; for example, by fluorescence detection, by immunoassay, by enzyme assay, by measuring RNA levels (e.g., qPCR), etc.

[0100] In some aspects, the present invention provides a recipient cell for receiving a multicistronic reporter vector, wherein the recipient cell comprises a recombinant nucleic acid integrated into a specific site in the host cell genome, wherein the recombinant nucleic acid comprises a first promoter operably linked to a nucleic acid encoding a fusion polypeptide, wherein the fusion polypeptide comprises a reporter domain and a selectable marker domain, and wherein the nucleic acid comprises a site-specific recombinase nucleic acid sequence located at the 5' end of the nucleic acid encoding the fusion polypeptide. Figure 1E Non-limiting examples of receptor sites are provided in .

[0101] In some embodiments, the promoter (e.g., the first promoter) is a constitutive promoter. Examples of constitutive promoters include, but are not limited to, cytomegalovirus immediate early (CMV) promoter, thymidine kinase (TK) promoter, eF1-α, ubiquitin C (UbC), phosphoglycerate kinase (PGK), CAG promoter, SV40 promoter, or human β-actin promoter. In some embodiments, the promoter is an inducible promoter. Examples of inducible promoters include, but are not limited to, tetracycline responsive promoters, rapamycin regulated promoters, and sterol inducible promoters. In some embodiments, the inducible promoter is a tetracycline responsive promoter.

[0102] In some embodiments of the present invention, the acceptor site comprises a site-specific recombinase sequence. Examples of site-specific recombinase sequences include, but are not limited to, FRT nucleic acid sequences, attP nucleic acid sequences, and loxP nucleic acid sequences. In some embodiments, the site-specific recombinase sequence is a FRT nucleic acid. In some embodiments, the site-specific recombinase sequence is an attP nucleic acid. In some embodiments, the site-specific recombinase sequence is an attB nucleic acid. In some embodiments, the site-specific recombinase sequence is a loxP nucleic acid. In some embodiments, the site-specific recombinase sequence is a FRT nucleic acid and an attP nucleic acid. In some embodiments, the site-specific recombinase sequence is a FRT nucleic acid and an attB nucleic acid.

[0103] In some embodiments, the receptor site comprises a nucleic acid encoding a reporter polypeptide. In some embodiments, the receptor site comprises a nucleic acid encoding a selection polypeptide. In some embodiments, the receptor site comprises a nucleic acid encoding a reporter polypeptide (e.g., a reporter domain) fused to a selection polypeptide (e.g., a selection domain). In some embodiments, the reporter polypeptide is at the N-terminus of the fusion polypeptide, and the selection polypeptide is at the C-terminus of the fusion polypeptide. In other embodiments, the selection polypeptide is at the N-terminus of the fusion polypeptide, and the reporter polypeptide is at the C-terminus of the fusion polypeptide.

[0104] Reporter peptides are peptides that can be easily identified; for example, via microscopy, plate reader, FACS, chemically, mass spectrometry or deep sequencing. For example, the reporter domain can be a fluorescent or luminescent polypeptide. In some embodiments, the reporter domain can be green fluorescent protein (GFP) or any derivative thereof. In some embodiments, the reporter domain is a non-GFP derived fluorescent peptide. In some embodiments, the reporter domain encodes GFP, EGFP, Emerald, Citrine, Venus, mOrange, mCherry, TagBFP, mTurquoise, Cerulean, UnaG, dsRed, eqFP611, Dronpa, RFP, TagRFP, TdTomato, KFP, EosFP, Dendra, IrisFP, iRFP or smURFP. The reporter domain can be a luciferase. The reporter domain can be an enzyme that allows visualization of the product of a chemical reaction when expressed. In some embodiments, the reporter is a firefly luciferase or a Renilla luciferase. In some embodiments, the reporter domain is β-glucuronidase or β-galactosidase.

[0105] The selectable marker domain can be a polypeptide that confers resistance to a molecule to which the cell is not normally resistant or at a dose to which the cell is not normally resistant. For example, the selectable marker domain can be a polypeptide that confers resistance to an antibiotic. In some embodiments, the selectable marker is a polypeptide that confers resistance to blasticidin, geneticin, hygromycin, puromycin, neomycin, zeocin, pyrimidine ... TM , kanamycin, carbenicillin, ampicillin, antinomycin, apramycin, mycophenolic acid, histidinol, methotrexate, or any salt or derivative thereof.

[0106] In some embodiments, the recipient cell comprises a receptor site comprising a nucleic acid encoding a fusion polypeptide comprising a reporter domain and a selection domain. In some embodiments, the reporter domain of the fusion polypeptide is an mCherry reporter domain. In some embodiments, the selectable marker domain of the fusion polypeptide confers resistance to hygromycin, Zeocin, TM , Puromycin, Blasticidin, Neomycin or Hygromycin, Zeocin TM , puromycin, blasticidin, and neomycin analogs.

[0107] In some embodiments, the receptor site further comprises a nucleic acid encoding a gene expression repressor polypeptide. In some embodiments, the receptor site comprises a nucleic acid encoding a tetracycline repressor polypeptide operably connected to a promoter. In some embodiments, the constitutive promoter is a CMV promoter, a TK promoter, an eF1-α, an UbC promoter, a PGK promoter, a CAG promoter, a SV40 promoter, or a human β-actin promoter. In some embodiments, the promoter is a human β-actin promoter or a CAG promoter.

[0108] The receptor site is integrated at a specific site in the genome of the receptor cell. In some embodiments, the specific site is a harmless site in the genome of the receptor cell. For example, inserting the nucleic acid into the specific site has little effect on the function of the receptor cell. In some embodiments, the recombinant nucleic acid is integrated into adeno-associated virus S1 (AAVS1) locus, chemokine (CC motif) receptor 5 (CCR5) locus, human orthologs of the mouse ROSA26 locus, hipp11 (H11) locus or citrate lyase β-like gene locus (CLYBL). In some embodiments, the receptor site comprises a heterologous nucleic acid sequence, which is used to target the recombinant nucleic acid encoding the site-specific recombinase nucleic acid sequence to a specific target locus in the genome of the receptor cell. In some embodiments, the receptor cell comprises a nucleic acid for targeting the AAVS1 locus, the CCR5 locus, the mouse ROSA26 locus or the human orthologs of the mouse ROSA26 locus, the hipp11 (H11) locus or the CLYBL locus.

[0109] The present disclosure provides a method for producing a receptor cell line. The method includes engineering cells so that the cells can contain a reporter nucleic acid. Any cell can be a receptor cell. In some embodiments, the cells used are prokaryotic cells. In some embodiments, the cells used are eukaryotic cells. In some embodiments, the cells used are plant cells. In some embodiments, the cells used are fungal cells. In some embodiments, the cells used are mammalian cells. In some embodiments, the cells used are human cells. In some embodiments, the receptor cells are produced by engineering immortal cells. In some embodiments, the immortal cells are HEK293T cells, A549 cells, U2OS cells, RPE cells, NPC1 cells, MCF7 cells, HepG2 cells, HaCat cells, TK6 cells, A375 cells or HeLa cells. In some embodiments, the receptor cell line is produced by engineering primary cells. The primary cells can be harvested from plants or animals. In some embodiments, the primary cells are harvested from mammals. In some embodiments, the primary cells are harvested from humans. In some embodiments, primary cells of rodents are harvested. In some embodiments, the cells used are patient-specific cells.

[0110] In some embodiments, the recipient cell is a stem cell. The stem cell can be a totipotent, multipotent or pluripotent stem cell. Any totipotent, multipotent, multipotent or progenitor stem cell can be used to produce a recipient cell line. The stem cell can be an animal cell. In some embodiments, the stem cell is from a mammal. In some embodiments, the stem cell is from a human. In some embodiments, the stem cell is a patient-specific stem cell. In some embodiments, the stem cell is an autologous stem cell. In some embodiments, the stem cell is an allogeneic stem cell. In some cases, the stem cell is from a non-human primate, a dog or a rodent. The stem cell can be derived from the trophectoderm, the inner cell mass of the blastocyst or a specific tissue. The stem cell can be an embryonic stem cell, an induced pluripotent stem cell or a progenitor stem cell. Any progenitor cell can be used to produce a recipient cell line. For example, the progenitor cell used can be a hematopoietic cell, an endothelial progenitor cell, a mesenchymal progenitor cell, a neural progenitor cell, an osteochondroprogenitor cell, a lymphoid progenitor cell or a pancreatic progenitor cell.

[0111] In some embodiments, the recipient cell is a bacterial cell, a plant cell, a fungal cell or an animal cell. In some embodiments, the animal is an invertebrate. In some embodiments, the recipient cell is a cell from a member of the species Drosophila melanogaster. In some embodiments, the recipient cell is a cell from a member of the species Caenorhabditis elegans. In some embodiments, the recipient cell is a vertebrate animal cell. In some embodiments, the recipient cell is a mammalian cell. In some embodiments, the recipient cell is a human cell, a primate cell, a rodent cell, a cat cell, a dog cell, a cattle cell, a pig cell or a sheep cell.

[0112] In some embodiments, the present invention provides a method for producing a recipient cell for receiving a multicistronic reporter vector, the method comprising introducing a recombinant nucleic acid into a cell, wherein the recombinant nucleic acid comprises, from 5' to 3', a first nucleic acid for targeting homologous recombination to a specific site in the cell, a first promoter, a site-specific recombinase nucleic acid, a nucleic acid encoding a first reporter polypeptide and a selectable marker, and a second nucleic acid for targeting homologous recombination to a specific site in the cell. In some embodiments, the recombinant nucleic acid comprises any of the above-mentioned recipient sites to produce any of the above-mentioned recipient cells.

[0113] In some embodiments, the present invention provides a method for producing a recipient cell for receiving a multicistronic reporter vector, the method comprising introducing a recombinant nucleic acid into a cell, wherein the recombinant nucleic acid comprises, from 5' to 3', a first nucleic acid for targeting homologous recombination to a specific site in the cell, a first promoter, a site-specific recombinase nucleic acid, a nucleic acid encoding a first reporter polypeptide and a selectable marker, a second nucleic acid for targeting homologous recombination to a specific site in the cell, a second promoter, and a nucleic acid encoding a second reporter polypeptide, wherein expression of the first reporter polypeptide without expression of the second reporter polypeptide indicates targeting integration of the recombinant nucleic acid to a specific site in the cell genome, and expression of the first and second reporter polypeptides indicates random integration in the cell genome. In some embodiments, the recombinant nucleic acid comprises any of the above-mentioned recipient sites to produce any of the above-mentioned recipient cells.

[0114] In some embodiments, the receptor site further comprises a nucleic acid encoding a gene expression repressor polypeptide. In some embodiments, the receptor site comprises a nucleic acid encoding a tetracycline repressor polypeptide operably connected to a promoter. In some embodiments, the constitutive promoter is a CMV promoter, a TK promoter, an eF1-α, an UbC promoter, a PGK promoter, a CAG promoter, a SV40 promoter, or a human β-actin promoter. In some embodiments, the promoter is a human β-actin promoter or a CAG promoter.

[0115] In some embodiments, the recipient cell is generated by introducing a receptor site into the recipient cell. In some embodiments, the receptor site comprises 1) two AAVS1 sequences that direct the receptor site to integrate into the AAVS1 locus; 2) a fluorescent marker (mCherry) visible by microscopy; 3) an antibiotic (bleomycin-ThermoFisher) resistance selection marker; 4) an FRT site for site-specific recombination of a reporter construct by flippase (Flp); 5) a constitutive promoter SV40 that drives bleomycin (Thermo Scientific TM ) resistance gene and mCherry expression, enabling antibiotic selection and fluorescence screening to identify positive receptor cell clones, respectively; 6) human b-actin promoter driving tetracycline repressor protein (TetR).

[0116] The receptor cell is produced by engineering the genome of the cell to include a receptor construct. There are several techniques known in the art that can be used to engineer the cell to include an exogenous nucleic acid sequence. For example, the receptor cell can be produced by inserting the receptor construct into a cell via a viral transfection system. In some embodiments, the retrovirus used is a lentivirus or an adenovirus. In some embodiments, the receptor construct can be a vector. The vector can be a viral vector. In some embodiments, the vector is a viral vector, such as a lentiviral vector, a baculovirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector. In some embodiments, the receptor construct is delivered to the receptor cell using an AAV transfection system. The AAV used can be modified and optimized according to the cell type or locus used. For example, AAV1, AAV2, AAV5, or any combination thereof can be used.

[0117] The receptor construct can be delivered by other methods known in the art. Many delivery means are known (such as yeast systems, microbubbles, gene guns / means of attaching the carrier to gold nanoparticles). In some embodiments, the receptor construct can be delivered via liposomes, nanoparticles, exosomes, microbubbles, or gene guns.

[0118] In some embodiments, the receptor construct is inserted into the genome of the cell by using an RNA-guided endonuclease system. In some embodiments, a CRISPR system is used. In some embodiments, the receptor construct is inserted into the genome of the cell using RNA-guided genome engineering via Cas9. However, any nuclease that works in the RNA-guided genome engineering system works. Available nucleases include Cas3, Cas8a, Cas5, Cas8b, Cas8C, Cas10d, Cse, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, Cas9, Cas4, Csn2, Cpf1, C2c1, C2c3, C2c2. The type of endonuclease used may depend on the cell to be engineered and the target locus for insertion.

[0119] In some embodiments, the receptor construct is inserted into the genome of the cell by using TALENs or zinc finger endonucleases (ZFNs).

[0120] Genome engineering via RNA guidance of Cas9 provides an improvement over TALEN and ZFN methods for cell line engineering. For example, there are some limitations using ZFN. First, ZFN technology requires the synthesis of new vectors and RNA for specific DNA binding sites in each new genome integration locus to be modified. These generally require expensive optimization, so cost and complexity limit the flexibility of applying these technologies to one or more loci. In contrast, the RNA-guided system uses a single protein (Cas9), which only requires short RNA molecules to program it for site-specific DNA recognition. Therefore, Cas9-RNA complexes are easier to prepare than similar ZFN targeting proteins, and therefore the system is more flexible. Cas9-RNA complexes also have lower toxicity in mammalian cells compared to TALEN and ZFN. In addition to Cas9, other nucleases related to RNA-guided genome editing can also be used. RNA-guided genome engineering is known in the art.

[0121] The nucleic acid encoding the receptor site can be inserted into any part of the cell genome, into which an exogenous sequence of DNA can be inserted without disrupting the transcription of endogenous genes. In some embodiments, the receptor site targets the AAVS1 locus, the CCR5 locus, the mouse ROSA26 locus or the human straight homolog of the mouse ROSA26 locus, the H11 locus or the CLYBL locus. In some embodiments, the construct is inserted into a position in the genome without epigenetic silencing. In some embodiments, the receptor construct is inserted into the AAVS1 genomic locus of the host cell. In some embodiments, a single copy of the nucleic acid encoding the receptor site is incorporated into the recipient cell genome (e.g., on a single allele of the recipient cell genome).

[0122] The nucleic acid encoding the receptor site may include two nucleic acid sequences that allow homologous recombination into the cell genome. In some embodiments, in the case where the receptor construct is integrated into the AAVS1 genomic locus of the cell, the receptor construct includes two AAVS1 sequences that allow the receptor construct to be directly integrated into the cell. The reporter construct may include one or more sequences that allow the construct to be integrated into different genomic loci of the cell. The receptor construct may be inserted into the locus in the cell genome via homologous recombination or any other genome engineering method known in the art. In some embodiments, the receptor construct includes two AAVS1 sequences that allow the receptor construct to be directly integrated into the AAVS1 locus of the cell. In some embodiments, the receptor construct includes two CCR5 sequences that allow the receptor construct to be directly integrated into the CCR5 locus of the cell. In some embodiments, the receptor construct includes two ROSA26 sequences that allow the receptor construct to be directly integrated into the ROSA26 locus of mouse cells. In some embodiments, the receptor construct comprises two human orthologs of the mouse ROSA26 sequence, which allows the receptor construct to be directly integrated into the human ortholog of the mouse ROSA26 locus of human cells. In some embodiments, the receptor construct comprises two CLYBL sequences, which allows the receptor construct to be directly integrated into the CLYBL locus of cells. In some embodiments, the receptor construct comprises two H11 sequences, which allows the receptor construct to be directly integrated into the H11 locus of cells.

[0123] In some embodiments, the library enables pooled screening of different reporter cell lines. For example, two or more reporter cell lines can be developed from a single isogenic recipient cell line, wherein the two or more reporter cell lines can be distinguished; for example, by differentially labeling nuclei. The two or more reporter cell lines can be mixed together in a single assay to provide results for multiple polypeptides in one or more pathways.

[0124] In some embodiments, up to at least five quality control steps can be performed to select a recipient cell line with complete integration sites and full functionality at optimal performance. In some embodiments, the genomic DNA at the 5' and 3' insertion sites in the AAVS1 genomic locus of the recipient cell line is sequenced to confirm that the recombination process does not trigger any small insertions, deletions or mutations flanking the recombination site. In some embodiments, the homogeneity of the recipient cells is assessed to ensure that they come from a single cell-flow cytometry analysis is performed for this purpose and the coefficient of variation (CV) of the fluorophore is calculated. In some embodiments, it is determined that the recipient cells contain functional tetracycline regulation; for example, under tetracycline regulation, the cells are co-transfected with BxB1 and a dual-color multicistronic construct containing MTS-Venus and H2B-TagBFP. In some embodiments, reporter cells are selected by antibiotic resistance. For example, the number of colonies formed is scored. After selection, the cells are treated with 1 μg / ml doxycycline for 12-15h. The following verification and QC steps are performed using flow cytometry analysis. In some embodiments, induced cells are compared with uninduced cells to verify the loss of mCherry and the acquisition of Venus and TagBFP fluorescence. In some embodiments, the variability of protein expression in the polyclonal population after reporter integration is determined. In some embodiments, protein expression is similar to protein expression in the recipient cell line population. In some embodiments, the CV of the fluorescence intensity of each fluorophore present in the polycistronic vector is determined and compared with the parent CV. In some embodiments, the expression of different proteins placed on the receptor site is homogeneous and the payload is verified; for example, the correlation coefficient (r) is determined by the flow cytometry value based on the fluorescence intensity of the expressed protein.

[0125] Polycistronic reporter vector

[0126] In some aspects, the present invention provides a multicistronic reporter vector comprising: a promoter operably linked to an open reading frame, wherein the open reading frame comprises two or more cistrons, and wherein expression of the open reading frame in a cell produces separate component polypeptide products from each cistron; wherein each cistron comprises a multiple cloning site (MCS) and a nucleic acid encoding a reporter vector, wherein each cistron encodes a different reporter polypeptide; and wherein expression of two or more nucleic acids inserted into the two or more multiple cloning sites and encoding a polypeptide fused to the reporter polypeptide is stoichiometric in nature. The vector is designed for a "plug and play" mode, wherein different promoters can be exchanged to drive expression of the open reading frame, different target polypeptides can be exchanged, different reporter polypeptides can be exchanged, and different selection polypeptides can be exchanged, depending on the specific use of the multicistronic reporter vector. Similarly, the multicistronic reporter vector is designed by inserting nucleic acids encoding any target polypeptide using various MCS sequences so that a transgenic product labeled as a reporter polypeptide is expressed by the multicistronic reporter vector. In some embodiments, the polycistronic vector comprises a "backbone" vector in which the transgene of interest has not yet been inserted into the MCS sequence. In other embodiments, the polycistronic vector comprises a vector in which the transgene of interest has been inserted into the MCS sequence, such that expression of the open reading frame produces a different reporter-labeled polypeptide. Figure 2 and Fig.18 Non-limiting examples of multicistronic reporter vectors are provided in .

[0127] In some embodiments, essentially stoichiometric expression is the expression of two or more polypeptides fused to a reporter polypeptide, wherein the expression levels of the two or more polypeptides fused to the reporter polypeptide are essentially the same; that is, with a 1:1 stoichiometry. In some embodiments, the expression levels of the two or more reporter polypeptides are equal or vary by no more than about 5%, 10%, 15%, 20%, or 25% of each other. In some embodiments, essentially stoichiometric expression is the stoichiometric expression of two, three, four, or more than four, two or more polypeptides fused to the reporter polypeptide. The expression levels of the two or more reporter polypeptides can be measured by any means known in the art; for example, by fluorescence detection, by immunoassay, by enzyme assay, by measuring RNA levels (e.g., qPCR), etc.

[0128] In some embodiments, the cistrons of the polycistronic reporter vector are separated from each other by nucleic acids encoding one or more self-cleaving peptides and / or one or more internal ribosome entry sites (IRES). In some embodiments, the one or more self-cleaving peptides are viral self-cleaving peptides. In some embodiments, the one or more viral self-cleaving peptides are one or more 2A peptides. In some embodiments, the one or more 2A peptides are T2A peptides, P2A peptides, E2A peptides or F2A peptides. In some embodiments, one or more cistrons of the open reading frame are separated from other cistrons in the open reading frame by an IRES sequence. In some embodiments, the IRES is encephalomyocarditis virus (EMCV) IRES, hepatitis C virus (HCV) IRES or enterovirus 71 (EV71) IRES.

[0129] In some embodiments, the polycistronic reporter vector comprises two cistrons, wherein the two cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide or an IRES. In some embodiments, the polycistronic reporter vector comprises three cistrons, wherein the three cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide. In some embodiments, the polycistronic reporter vector comprises three cistrons, wherein the three cistrons are separated by an IRES sequence. In some embodiments, the polycistronic reporter vector comprises three cistrons, wherein the first and second cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, and the second and third cistrons are separated by an IRES sequence. In some embodiments, the polycistronic reporter vector comprises three cistrons, wherein the first and second cistrons are separated by an IRES sequence, and the second and third cistrons are separated by a nucleic acid encoding a self-cleaving peptide. In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the cistrons are separated from each other by a nucleic acid encoding a viral self-cleaving peptide. In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the cistrons are separated from each other by an IRES sequence. In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the first and second cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, the second and third cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, and the third and fourth cistrons are separated by an IRES sequence. In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the first and second cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, the second and third cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, and the third and fourth cistrons are separated by an IRES sequence. In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the first and second cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, the second and third cistrons are separated by an IRES sequence, and the third and fourth cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide. In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the first and second cistrons are separated by an IRES sequence, the second and third cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, and the third and fourth cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide. In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the first and second cistrons are separated by an IRES sequence, the second and third cistrons are separated by an IRES sequence, and the third and fourth cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide.In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the first and second cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, the second and third cistrons are separated by an IRES sequence, and the third and fourth cistrons are separated by an IRES sequence. In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the first and second cistrons are separated by an IRES sequence, the second and third cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, and the third and fourth cistrons are separated by an IRES sequence. In some embodiments, the polycistronic reporter vector comprises five or more cistrons, wherein the cistrons are separated from each other by any combination of nucleic acids encoding viral self-cleaving peptides and IRES sequences.

[0130] In some embodiments, the polycistronic reporter vector of the present invention comprises one or more nucleic acids encoding a peptide linker between one or more of the reporter polypeptides and one or more of the self-cleaving peptides. In some embodiments, the peptide linker comprises the sequence Gly-Ser-Gly. In some embodiments, the polycistronic reporter vector comprises a peptide linker between one or more reporter polypeptides. In some embodiments, the polycistronic reporter vector comprises a peptide linker between two reporter polypeptides. In some embodiments, the polycistronic reporter vector comprises three reporter polypeptides, wherein the first peptide linker is between the first reporter polypeptide and the second reporter polypeptide, and the second peptide linker is between the second reporter polypeptide and the third reporter polypeptide. In some embodiments, the polycistronic reporter vector does not have the first peptide linker and / or the second peptide linker. In some embodiments, the polycistronic reporter vector comprises four reporter polypeptides, wherein the first peptide linker is between the first reporter polypeptide and the second reporter polypeptide, the second peptide linker is between the second reporter polypeptide and the third reporter polypeptide, and the third peptide linker is between the third reporter polypeptide and the fourth reporter polypeptide. In some embodiments, the polycistronic reporter vector does not have the first peptide linker and / or the second peptide linker and / or the third peptide linker. In some embodiments, the peptide linkers are identical peptide linkers (eg, Gly-Ser-Gly). In other embodiments, at least two peptide linkers in the multicistronic reporter vector are different.

[0131] In some embodiments, the present invention provides a multicistronic reporter vector comprising an open reading frame, the open reading frame is operably connected to a promoter, and wherein the open reading frame includes two or more MCS sequences, which are connected to a nucleic acid encoding a reporter polypeptide, so that when the nucleic acid encoding the target transgene is inserted into the MCS, the resulting polypeptide encoded by the multicistronic reporter vector includes the product of the target transgene marked with the reporter polypeptide. Each cistron of the open reading frame encodes a different reporter polypeptide, so that each labeled transgenic product can be analyzed in living cells. In some embodiments, the reporter polypeptide is a fluorescent reporter polypeptide. In some embodiments, the reporter polypeptide can be green fluorescent protein (GFP) or any derivative thereof. In some embodiments, the reporter polypeptide is a non-GFP derived fluorescent peptide. In some embodiments, the reporter polypeptide is GFP, EGFP, Emerald, Citrine, Venus, mOrange, mCherry, TagBFP, mTurquoise, Cerulean, UnaG, dsRed, eqFP611, Dronpa, RFP, TagRFP, TdTomato, KFP, EosFP, Dendra, IrisFP, iRFP or smURFP. In some embodiments, the reporter polypeptide is a luciferase. In some embodiments, the reporter polypeptide is an enzyme that allows visualization of the product of a chemical reaction when expressed. In some embodiments, the reporter domain is a firefly luciferase or a Renilla luciferase. In some embodiments, the reporter domain is a β-glucuronidase or a β-galactosidase.

[0132] In some embodiments, the present invention provides a multicistronic reporter vector, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron and a second cistron, wherein each cistron comprises a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide from 5' to 3'; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a viral cleavage peptide. In some embodiments, the present invention provides a multicistronic reporter vector, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, and a third cistron, wherein each cistron comprises a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide from 5' to 3'; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, and the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide. In some embodiments, the present invention provides a multicistronic reporter vector, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, a third cistron and a fourth cistron, wherein each cistron comprises, from 5' to 3', a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide, and the third cistron and the fourth cistron are separated by a nucleic acid encoding a third viral cleavage peptide. In some embodiments, the present invention provides a multi-cistronic reporter vector, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, a third cistron and a fourth cistron, wherein each cistron comprises, from 5' to 3', a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide, and the third cistron and the fourth cistron are separated by a nucleic acid encoding an IRES.

[0133] In some embodiments, the polycistronic reporter vector further comprises one or more inducible elements located between the promoter and the open reading frame. In some embodiments, the polycistronic reporter vector comprises two inducible elements. In some embodiments, the inducible element is a Tet operator 2 (TetO2) inducible element.

[0134] In some embodiments, the present invention provides a multicistronic reporter vector, wherein the vector comprises an open reading frame containing two or more cistrons, wherein the open reading frame is operably connected to a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is cytomegalovirus alpha (CMV), thymidine kinase (TK), eF1-α, ubiquitin C (UbC), phosphoglycerate kinase (PGK), CAG promoter, SV40 promoter or human β-actin promoter.

[0135] In some embodiments, the present invention provides a multicistronic reporter vector, wherein the vector comprises an open reading frame containing two or more cistrons, wherein the open reading frame is operably linked to an inducible promoter. In some embodiments, the inducible promoter is a tetracycline responsive promoter. In some embodiments, the inducible promoter is a rapamycin regulated promoter or a sterol inducible promoter.

[0136] In some embodiments, the invention provides a multicistronic reporter vector, wherein the vector comprises an open reading frame comprising two or more cistrons, wherein the open reading frame is operably linked to a tissue-specific promoter. In some embodiments, the tissue-specific promoter is specific for cells of the heart, blood, muscle, lung, liver, kidney, pancreas, brain, or skin.

[0137] In some embodiments, the present invention provides a multicistronic reporter vector, wherein the vector comprises an open reading frame comprising two or more cistrons, wherein the vector further comprises a site-specific recombinase sequence located 3' to the open reading frame.

[0138] Examples of site-specific recombinase sequences include, but are not limited to, FRT nucleic acid sequences, attP nucleic acid sequences, and loxP nucleic acid sequences. In some embodiments, the site-specific recombinase sequence is a FRT nucleic acid. In some embodiments, the site-specific recombinase sequence is an attP nucleic acid. In some embodiments, the site-specific recombinase sequence is an attB nucleic acid. In some embodiments, the site-specific recombinase sequence is a loxP nucleic acid. In some embodiments, the site-specific recombinase sequence is a FRT nucleic acid and an attP nucleic acid. In some embodiments, the site-specific recombinase sequence is a FRT nucleic acid and an attB nucleic acid.

[0139] In some embodiments, the present invention provides a multicistronic reporter vector, wherein the vector comprises an open reading frame comprising two or more cistrons, wherein the vector further comprises a nucleic acid encoding a selectable marker, wherein when the site-specific recombinase sequence is not recombined, the nucleic acid encoding the selectable marker is not operably linked to the promoter, and when the site-specific recombinase sequence is recombined with its target site-specific recombinase sequence, the nucleic acid encoding the selectable marker is operably linked to the promoter. In some embodiments, the selectable marker confers resistance to hygromycin, Zeocin TM , puromycin, neomycin or hygromycin, Zeocin TM , puromycin, blasticidin or neomycin analogs.

[0140] In some embodiments, the present invention provides a multicistronic reporter vector, wherein the vector comprises an open reading frame containing two or more cistrons, wherein a nucleic acid encoding one or more polypeptides is inserted into the one or more MCSs. In some embodiments, the one or more polypeptides comprise polypeptides that can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, cell homeostasis, organelle homeostasis, or toxicity. In some embodiments, the one or more polypeptides include ATF4, ATF6, XBP1ΔDBBD, and H2B; α-tubulin, mitochondrial targeting sequence (MTS), LC3, and H2B; or 53BP1, Nrf2, p53RE, and H2B; Mek, Erk, Raf, and Ras; H2B, palmitoylation signal, and MTS; or H2B, MTS, and α-actinin 2. In some embodiments, the present invention provides a plurality of polycistronic reporter vectors, wherein the plurality of polycistronic reporter vectors are used to analyze a specific target selected from a single biological pathway, crosstalk between two or more biological pathways, cellular homeostasis, organelle homeostasis, and toxicity response; wherein each vector encodes at least one common polypeptide (e.g., H2B) that can be used to identify cells that have received one or more of the polycistronic vectors encoding polypeptides targeting a specific target. In some aspects, the common polypeptide can be considered a barcode for the specific target.

[0141] In some embodiments, the present invention provides a multicistronic reporter vector as described above, wherein the vector comprises one, two or three transcription units comprising a promoter located 5' of an open reading frame comprising two or more cistrons and a nucleic acid encoding a transgene, wherein the reporter vector further comprises a core insulator sequence and a polyA sequence located 3' of the transcription unit and 5' of an open reading frame comprising two or more cistrons. In some embodiments, the one, two or three transcription units encode transcription factors, or other factors that may contribute to the assays described herein.

[0142] In some embodiments, the present invention provides a polycistronic reporter vector as described above, wherein the vector comprises a nucleic acid encoding H2B fused to a reporter polypeptide and operably linked to an MLC2v promoter, a SLN promoter, or a SHOX2 promoter, thereby enabling the reporter to be expressed in cardiac subtype cells. In some embodiments, the present invention provides a polycistronic reporter vector as described above, wherein the vector comprises a nucleic acid encoding H2B fused to a reporter polypeptide and operably linked to a vGAT promoter, a TH promoter, a GFAP promoter, or a vGLUT promoter, thereby enabling the reporter to be expressed in neural subtype cells.

[0143] Multiple reporter cells

[0144] In some embodiments, the present invention provides a multi-reporter cell comprising any of the above-mentioned receptor cells, wherein the above-mentioned multicistronic reporter vector has been integrated into the genome of the receptor cell. In some embodiments, the multicistronic reporter vector has been integrated into a specific site in the genome of the receptor cell. In some embodiments, the specific site in the genome of the receptor cell is adeno-associated virus S1 (AAVS1) locus, chemokine (CC motif) receptor 5 (CCR5) locus, human ortholog of the mouse ROSA26 locus, hipp11 (H11) locus or citrate lyase β-like gene locus (CLYBL). In some embodiments, a single copy of the multicistronic reporter vector is integrated into the genome of the receptor cell.

[0145] In some embodiments, any of the multicistronic reporter vectors described above is inserted into a recipient cell to generate a multi-reporter cell of the invention.

[0146] In some embodiments, the present invention provides a multi-reporter cell, wherein the reporter cell comprises a multicistronic reporter construct, wherein the multicistronic reporter construct comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises two or more cistrons; wherein each cistron comprises a nucleic acid encoding a different transgene product fused to a different reporter polypeptide, wherein expression of the open reading frame in the cell produces separate component polypeptide products from each cistron; and wherein expression of the transgene products is stoichiometric in nature. In some embodiments, the cistrons are separated from each other by nucleic acids encoding one or more self-cleaving peptides and / or one or more internal ribosome entry sites (IRES). In Figure 5 Non-limiting examples of reporter constructs in multi-reporter cells are provided in .

[0147] In some embodiments, essentially stoichiometric expression is the expression of two or more transgenic products, wherein the expression levels of the two or more transgenic products are essentially the same; that is, with a 1:1 stoichiometry. In some embodiments, the expression levels of the two or more reporter polypeptides are equal or vary by no more than about 5%, 10%, 15%, 20%, or 25% of each other. In some embodiments, essentially stoichiometric expression is the stoichiometric expression of two, three, four, or more transgenic products. The expression levels of the two or more reporter polypeptides can be measured by any means known in the art; for example, by fluorescence detection, by immunoassay, by enzyme assay, by measuring RNA levels (e.g., qPCR), etc.

[0148] In some embodiments, the cistrons of the multicistronic reporter vector inserted into the multi-reporter cell are separated from each other by nucleic acids encoding one or more self-cleaving peptides and / or one or more internal ribosome entry sites (IRES). In some embodiments, the one or more self-cleaving peptides are viral self-cleaving peptides. In some embodiments, the one or more viral self-cleaving peptides are one or more 2A peptides. In some embodiments, the one or more 2A peptides are T2A peptides, P2A peptides, E2A peptides or F2A peptides. In some embodiments, one or more cistrons of the open reading frame are separated from other cistrons in the open reading frame by an IRES sequence. In some embodiments, the IRES is encephalomyocarditis virus (EMCV) IRES, hepatitis C virus (HCV) IRES or enterovirus 71 (EV71) IRES.

[0149] In some embodiments, the multi-reporter cell comprises a multicistronic reporter vector, wherein the multicistronic reporter vector comprises two cistrons, wherein the two cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide or an IRES. In some embodiments, the multicistronic reporter vector comprises three cistrons, wherein the three cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide. In some embodiments, the multicistronic reporter vector comprises three cistrons, wherein the three cistrons are separated by an IRES sequence. In some embodiments, the multicistronic reporter vector comprises three cistrons, wherein the first and second cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, and the second and third cistrons are separated by an IRES sequence. In some embodiments, the multicistronic reporter vector comprises three cistrons, wherein the first and second cistrons are separated by an IRES sequence, and the second and third cistrons are separated by a nucleic acid encoding a self-cleaving peptide. In some embodiments, the multicistronic reporter vector comprises four cistrons, wherein the cistrons are separated from each other by nucleic acids encoding viral self-cleaving peptides. In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the cistrons are separated from each other by an IRES sequence. In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the first and second cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, the second and third cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, and the third and fourth cistrons are separated by an IRES sequence. In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the first and second cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, the second and third cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, and the third and fourth cistrons are separated by an IRES sequence. In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the first and second cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, the second and third cistrons are separated by an IRES sequence, and the third and fourth cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide. In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the first and second cistrons are separated by an IRES sequence, the second and third cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, and the third and fourth cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide. In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the first and second cistrons are separated by an IRES sequence, the second and third cistrons are separated by an IRES sequence, and the third and fourth cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide.In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the first and second cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, the second and third cistrons are separated by an IRES sequence, and the third and fourth cistrons are separated by an IRES sequence. In some embodiments, the polycistronic reporter vector comprises four cistrons, wherein the first and second cistrons are separated by an IRES sequence, the second and third cistrons are separated by a nucleic acid encoding a viral self-cleaving peptide, and the third and fourth cistrons are separated by an IRES sequence. In some embodiments, the polycistronic reporter vector comprises five or more cistrons, wherein the cistrons are separated from each other by any combination of nucleic acids encoding viral self-cleaving peptides and IRES sequences.

[0150] In some embodiments, the multi-reporter cell comprises a multicistronic reporter vector comprising one or more nucleic acids encoding a peptide linker between one or more of the reporter polypeptides and one or more of the self-cleaving peptides. In some embodiments, the peptide linker comprises the sequence Gly-Ser-Gly.

[0151] In some embodiments, the present invention provides a multi-reporter cell comprising a multicistronic reporter vector, wherein the multicistronic reporter vector comprises an open reading frame, which is operably linked to a promoter, and wherein the open reading frame includes two or more MCS sequences, which are linked to a nucleic acid encoding a reporter polypeptide, such that when a nucleic acid encoding a transgene of interest is inserted into the MCS, the resulting polypeptide encoded by the multicistronic reporter vector includes a product of the transgene of interest labeled with the reporter polypeptide.

[0152] Each cistron of the open reading frame encodes a different reporter polypeptide, so that each labeled transgenic product can be analyzed in living cells. In some embodiments, the reporter polypeptide is a fluorescent reporter polypeptide. In some embodiments, the reporter polypeptide can be green fluorescent protein (GFP) or any derivative thereof. In some embodiments, the reporter polypeptide is a non-GFP derived fluorescent peptide. In some embodiments, the reporter polypeptide is GFP, EGFP, Emerald, Citrine, Venus, mOrange, mCherry, TagBFP, mTurquoise, Cerulean, UnaG, dsRed, eqFP611, Dronpa, RFP, TagRFP, TdTomato, KFP, EosFP, Dendra, IrisFP, iRFP or smURFP. In some embodiments, the reporter polypeptide is a luciferase. In some embodiments, the reporter polypeptide is an enzyme that allows visualization of the product of a chemical reaction when expressed. In some embodiments, the reporter domain is a firefly luciferase or a Renilla luciferase. In some embodiments, the reporter domain is β-glucuronidase or β-galactosidase.

[0153] In some embodiments, the present invention provides a multi-reporter cell comprising a multicistronic reporter vector, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron and a second cistron, wherein each cistron comprises a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide from 5' to 3'; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a viral cleavage peptide. In some embodiments, the present invention provides a multi-cistronic reporter vector, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, and a third cistron, wherein each cistron comprises a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide from 5' to 3'; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, and the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide. In some embodiments, the present invention provides a multicistronic reporter vector, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, a third cistron and a fourth cistron, wherein each cistron comprises, from 5' to 3', a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide, and the third cistron and the fourth cistron are separated by a nucleic acid encoding a third viral cleavage peptide. In some embodiments, the present invention provides a multi-cistronic reporter vector, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, a third cistron and a fourth cistron, wherein each cistron comprises, from 5' to 3', a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide, and the third cistron and the fourth cistron are separated by a nucleic acid encoding an IRES.

[0154] In some embodiments, the multicistronic reporter vector of the multi-reporter cell further comprises one or more inducible elements located between the promoter and the open reading frame. In some embodiments, the multicistronic reporter vector comprises two inducible elements. In some embodiments, the inducible element is a Tet operator 2 (TetO2) inducible element.

[0155] In some embodiments, the present invention provides a multi-reporter cell comprising a multicistronic reporter vector, wherein the vector comprises an open reading frame containing two or more cistrons, wherein the open reading frame is operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is cytomegalovirus alpha (CMV), thymidine kinase (TK), eF1-α, ubiquitin C (UbC), phosphoglycerate kinase (PGK), CAG promoter, SV40 promoter or human β-actin promoter.

[0156] In some embodiments, the invention provides a multi-reporter cell comprising a multicistronic reporter vector, wherein the vector comprises an open reading frame comprising two or more cistrons, wherein the open reading frame is operably linked to an inducible promoter. In some embodiments, the inducible promoter is a tetracycline responsive promoter. In some embodiments, the inducible promoter is a rapamycin regulated promoter or a sterol inducible promoter.

[0157] In some embodiments, the invention provides a multi-reporter cell comprising a multicistronic reporter vector, wherein the vector comprises an open reading frame comprising two or more cistrons, wherein the open reading frame is operably linked to a tissue-specific promoter. In some embodiments, the tissue-specific promoter is specific for cells of the heart, blood, muscle, lung, liver, kidney, pancreas, brain, or skin.

[0158] In some embodiments, the present invention provides a multi-reporter cell comprising a multicistronic reporter vector, wherein the vector comprises an open reading frame containing two or more cistrons, wherein the vector further comprises a site-specific recombinase sequence located at 3' of the open reading frame, which is used to target the multicistronic reporter vector to a specific site in the cell. Examples of site-specific recombinase sequences include, but are not limited to, FRT nucleic acid sequences, attP nucleic acid sequences, and loxP nucleic acid sequences. In some embodiments, the site-specific recombinase sequence is a FRT nucleic acid. In some embodiments, the site-specific recombinase sequence is an attP nucleic acid. In some embodiments, the site-specific recombinase sequence is an attB nucleic acid. In some embodiments, the site-specific recombinase sequence is a loxP nucleic acid. In some embodiments, the site-specific recombinase sequence is a FRT nucleic acid and an attP nucleic acid. In some embodiments, the site-specific recombinase sequence is a FRT nucleic acid and an attB nucleic acid.

[0159] In some embodiments, the present invention provides a multi-reporter cell comprising a multicistronic reporter vector, wherein the vector comprises an open reading frame comprising two or more cistrons, wherein the vector further comprises a nucleic acid encoding a selectable marker, wherein when the site-specific recombinase sequence is not recombined, the nucleic acid encoding the selectable marker is not operably linked to the promoter, and when the site-specific recombinase sequence is recombined with its target site-specific recombinase sequence, the nucleic acid encoding the selectable marker is operably linked to the promoter. In some embodiments, the selectable marker confers resistance to hygromycin, Zeocin TM , puromycin, neomycin or hygromycin, Zeocin TM , puromycin, blasticidin or neomycin analogs.

[0160] In some embodiments, the present invention provides a multi-reporter cell comprising a multicistronic reporter vector, wherein the vector comprises an open reading frame containing two or more cistrons, wherein each cistron encodes a transgenic product fused to a reporter polypeptide. In some embodiments, the transgenic encoding can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, cellular homeostasis, organelle homeostasis, or a toxic response. In some embodiments, the polypeptides include ATF4, ATF6, XBP1ΔDBBD, and H2B; α-tubulin, mitochondrial targeting sequence (MTS), LC3, and H2B; or 53BP1, Nrf2, p53RE, and H2B; Mek, Erk, Raf, and Ras; H2B, palmitoylation signal, and MTS; or H2B, MTS, and α-actinin 2.

[0161] In some embodiments, the present invention provides a multi-reporter cell comprising a multicistronic reporter vector as described above, wherein the vector comprises one, two or three additional transcription units comprising a promoter located 5' of an open reading frame comprising two or more cistrons and a nucleic acid encoding a transgene, wherein the reporter vector further comprises a core insulator sequence and a polyA sequence located 3' of the transcription unit and 5' of an open reading frame comprising two or more cistrons. In some embodiments, the one, two or three transcription units encode transcription factors, or other factors that may contribute to the assays described herein. In some embodiments, at least the additional transcription unit comprises a nucleic acid encoding a reporter molecule operably linked to a promoter.

[0162] In some embodiments, the multi-reporter cell can be a stem cell that can differentiate into different lineages. For example, the multi-reporter cell can be a totipotent, pluripotent, multipotent or progenitor stem cell. In some embodiments, the multi-reporter cell is a totipotent stem cell that has the ability to differentiate into at least all embryonic and extraembryonic lineages. In some embodiments, the multi-reporter cell is a pluripotent stem cell. In some embodiments, the reporter pluripotent stem cell is an embryonic pluripotent stem cell isolated from an animal. In a specific embodiment, the reporter pluripotent stem cell is a mammalian embryonic stem cell. In some embodiments, the reporter pluripotent stem cell is a human embryonic stem cell. In some embodiments, the reporter pluripotent stem cell is an induced pluripotent stem cell. The iPS cells used to develop reporter iPS cells may be produced by reprogramming via transfection, piggy-Bac, additional or protein reprogramming methods. The iPS used to develop reporter iPS cells may be produced by reprogramming somatic cells, terminally differentiated cells or partially differentiated cells of the ectoderm, endoderm, mesoderm, placenta, chorion or trophectoderm lineage. For example, the reporter iPS cells may be derived from fibroblasts, peripheral blood cells, umbilical cord blood endothelial cells, umbilical cord blood stem cells, adipose-derived stem cells, hepatocytes, keratinocytes, neural stem cells, pancreatic β cells, or amniotic membrane cells. The reporter iPS cells may be derived from established iPS cell lines, or may be derived from patient-specific iPS cells. In some embodiments, the reporter iPS cells are derived from iPS cells generated by reprogramming immune privileged cells.

[0163] In some embodiments, the multi-reporter cell is a multipotent or progenitor cell. The multi-reporter cell can be a hematopoietic cell, an endothelial progenitor cell, a mesenchymal progenitor cell, a neural progenitor cell, an osteochondroma progenitor cell, a lymphoid progenitor cell, or a pancreatic progenitor cell. In some embodiments, the multi-reporter cell is an umbilical cord endothelial cell, an umbilical cord blood stem cell, an adipose-derived stem cell, a hepatocyte, a keratinocyte, a neural stem cell, a pancreatic beta cell, or an amniotic cell.

[0164] The stem cells can be differentiated into any progenitor cell or terminal cell lineage. General or lineage-specific differentiation methods are known in the art. Any method known in the art can be used to differentiate the stem cells. For example, one or more factors or molecules that drive differentiation, one or more cell matrices, embryoid body formation, or a combination of the above can be used to differentiate the stem cells. In some embodiments, the differentiated cells are multi-reporter cells.

[0165] The cells can be differentiated into cardiomyocytes, endothelial cells, neurons, GABAergic neurons, astrocytes, dopaminergic neurons, glutamatergic neurons, hepatocytes, hepatoblasts, skeletal muscle myoblasts, macrophages, cortical neurons, atrial cardiomyocytes, ventricular cardiomyocytes, Purkinje fibers, basal cells, squamous cells, kidney cells, pancreatic beta cells, epithelial cells, mesenchymal cells, adrenal cortex cells, osteoblasts, osteocytes, chondroblasts, chondrocytes, gastrointestinal cells, colorectal cells, ductal cells, lobular cells, lymphocytes, retinal cells, photoreceptor cells or cochlear cells.

[0166] In some embodiments, the reporter iPS cell comprises a multicistronic reporter as described in any one of the embodiments above, wherein the multicistronic reporter is driven by a promoter that is active only in pluripotent cells. For example, the promoter operably linked to an open reading frame comprising two or more cistrons can be Oct-4, Sox2, Nanog, KLF4, TRA-1-60, TRA-2-54, TRA-1-81, SSEA1, SSEA4, or any promoter of pluripotency-related genes.

[0167] Toxicity can be tested by monitoring the expression and / or movement of various peptides associated with toxicity within or between cells. For example, the expression and movement of proteins involved in the unfolded protein response, autophagy, DNA damage, oxidative stress, and p53-dependent stress responses.

[0168] In some embodiments, the multi-reporter cells are immortal cells. For example, the reporter can be HEK293T cells, A549 cells, U2OS cells, RPE cells, NPC1 cells, MCF7 cells, HepG2 cells, HaCat cells, TK6 cells, A375 cells or HeLa cells. In some embodiments, the multi-reporter cells are primary cells.

[0169] Multi-reporter cells can be used to test toxicity, test and monitor the effects of various molecules in cells, test the effects of different therapeutics in cells, or monitor the movement of proteins in cells in response to stimuli. Examples of molecules and therapeutics include chemicals, chemical compositions, small biologics, nanoparticles, peptides, antibodies, vaccines, and combinations thereof.

[0170] In some embodiments, the present invention provides a method for producing a multi-reporter cell, the method comprising introducing a polycistronic reporter vector as described herein into any recipient cell as described herein. In some embodiments, the multi-specific reporter vector is inserted into the receptor site of the recipient cell recombinase system. In some embodiments, the polycistronic reporter vector comprises a recombinase-related nucleic acid, which can be inserted into the recombinase-related nucleic acid of the recipient cell via the recombinase protein in the recipient cell. In some embodiments, the nucleic acid encoding the recombinase protein is stably introduced into the recipient cell. In some embodiments, the nucleic acid encoding the recombinase is transiently introduced into the recipient cell. In some embodiments, before, at the same time, or after the introduction of the polycistronic reporter vector, the nucleic acid encoding the recombinase is transiently introduced into the recipient cell. In some embodiments, the recombinase protein is introduced into the recipient cell. In some embodiments, the recombinase-related nucleic acid sequence is a FRT nucleic acid sequence, and the recipient cell comprises a flp recombinase. In some embodiments, the recombinase-related nucleic acid is attP, and the recipient cell comprises a Bxb1 recombinase, a PhiC31 recombinase, or an R4 recombinase. In some embodiments, the recombinase-associated nucleic acid sequence is a loxP nucleic acid sequence, and the recipient cell comprises a CRE recombinase.

[0171] library

[0172] In some aspects, the invention provides one or more polycistronic reporter vector libraries, wherein the library comprises polycistronic reporter molecules containing different transgenes, the transgene encoding a polypeptide fused to a reporter polypeptide, wherein when introduced into a cell, two or more of the different transgenes on each vector are expressed essentially stoichiometrically. In some embodiments, the library comprises a reporter vector encoding one or more transgenes, the transgene encoding a polypeptide that can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, cell homeostasis, organelle homeostasis, toxic responses, and / or phenotypic characteristics. In some embodiments, the library is used to visualize biological pathways or target readouts using phenotypic readouts. In some embodiments, the library comprises two or more different polycistronic reporter vectors. In some embodiments, the library comprises between about two and about 10, about 10 and about 20, about 20 and about 30, about 30 and about 40, about 40 and about 50, about 50 and about 100, about 100 and about 500, about 500 and about 100, about 1000 and about 10,000 different polycistronic reporter vectors. In some embodiments, the library comprises more than about 10,000 different polycistronic reporter vectors.

[0173] In some aspects, the invention provides one or more polycistronic reporter cell libraries, wherein the library comprises a plurality of polycistronic reporter cells containing different transgenes encoding polypeptides fused to a reporter polypeptide, wherein when introduced into a cell, two or more of the different transgenes on each vector are expressed essentially stoichiometrically. In some embodiments, the library comprises a plurality of reporter cells encoding one or more transgenes encoding polypeptides that can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, cell homeostasis, organelle homeostasis, or toxic responses. In some embodiments, the library comprises different polycistronic reporter cells between about two and about 10, about 10 and about 20, about 20 and about 30, about 30 and about 40, about 40 and about 50, about 50 and about 100, about 100 and about 500, about 500 and about 100, about 1000 and about 10,000. In some embodiments, the library comprises more than about 10,000 different polycistronic reporter cells. In some embodiments, the plurality of polycistronic reporter cells are isogenic; that is, derived from a corecipient cell into which a different polycistronic vector was introduced.

[0174] In some embodiments, essentially stoichiometric expression is the expression of two or more transgenes, wherein the expression levels of the two or more transgenes are essentially the same; that is, having a 1:1 stoichiometry. In some embodiments, the expression levels of the two or more reporter polypeptides are equal or vary by no more than about 5%, 10%, 15%, 20%, or 25% of each other. In some embodiments, essentially stoichiometric expression is the stoichiometric expression of two, three, four, or more transgenes of the multicistronic reporter vector. The expression levels of the two or more transgenes can be measured by any means known in the art; for example, by fluorescence detection, by immunoassay, by enzyme assay, by measuring RNA levels (e.g., qPCR), etc.

[0175] In some embodiments, the library comprises a polycistronic reporter vector to analyze biological pathways associated with a disease. In some embodiments, the disease is cancer, cardiovascular disease, neurodegenerative disease, or autoimmune disease. In some embodiments, the biological pathway is a pathway associated with a toxic response mechanism within the cell. In some embodiments, the library comprises polycistronic reporter vectors to analyze biological pathways related to cell proliferation, cell differentiation, cell death, apoptosis, autophagy, DNA damage and repair, or oxidative stress, chromatin / epigenetics (e.g., chromatin acetylation), MAPK signaling (e.g., MAPK / Erk), PI3K / Akt signaling (e.g., mTor signaling), translation control (e.g., eIF2 regulation), cell cycle and checkpoint control (G1 / S checkpoint), cellular metabolism (e.g., insulin receptor signaling), developmental and differentiation signaling (e.g., Wnt signaling), immunology and inflammatory signaling (e.g., JAK / STAT signaling), tyrosine kinase signaling (e.g., ErbB / HER signaling), vesicular trafficking, cytoskeletal regulation, or protein degradation (e.g., ubiquitin pathway), and any synthetic lethal combination of these pathways. In some embodiments, each polycistronic vector of the library comprising a transgene for analyzing a specific single biological pathway, a specific crosstalk between two or more biological pathways, a specific cellular homeostasis, a specific organelle homeostasis, or a specific toxic response comprises a common transgene encoding a polypeptide fused to a reporter polypeptide. Such a common reporter transgene can be used to identify cells that receive the polycistronic reporter vector for a common spectrum target.

[0176] In some embodiments, the invention provides a receptor cell library for accepting a polycistronic reporter vector. In some embodiments, the library comprises two or more different receptor cells as described herein. In some embodiments, the library comprises about two and about 10, about 10 and about 20, about 20 and about 30, about 30 and about 40, about 40 and about 50, about 50 and about 100, about 100 and about 500 different receptor cells between any one. In some embodiments, the library comprises more than about 500 different receptor cells. In some embodiments, a common polycistronic reporter vector can be introduced into two or more receptor cells to compare the spectrum under different cell backgrounds.

[0177] In some embodiments, the invention provides a library of multiple reporter cells, wherein each cell comprises a multicistronic reporter vector comprising a different transgene encoding a polypeptide fused to a reporter polypeptide, wherein when introduced into the cell, the different transgenes on each vector are expressed essentially stoichiometrically. In some embodiments, the library comprises between about two and about 10, about 10 and about 20, about 20 and about 30, about 30 and about 40, about 40 and about 50, about 50 and about 100, about 100 and about 500 different multi-reporter cells. In some embodiments, different multicistronic reporter vectors targeting a common pathway have a common reporter polypeptide as a means of identifying cells that have received the relevant multicistronic reporter vector.

[0178] In some embodiments, the receptor cell library and / or the multi-reporter cell library comprises different immortalized cells. In some embodiments, the library comprises one or more of HEK293T cells, A549 cells, U2OS cells, RPE cells, NPC1 cells, MCF7 cells, HepG2 cells, HaCat cells, TK6 cells, A375 cells or HeLa cells.

[0179] In some embodiments, the receptor cell library and / or the multi-reporter cell library include different pluripotency, multipotency and / or progenitor cells. In some embodiments, the different pluripotency or multipotency cells include induced pluripotent stem cells, multipotency cells, hematopoietic cells, endothelial progenitor receptor cells, mesenchymal progenitor cells, neural progenitor cells, osteochondrocyte progenitor cells, lymphoid progenitor cells or pancreatic progenitor cells. In some embodiments, after introducing the multicistronic reporter vector, the multipotency or multipotency cell multi-reporter cell library is differentiated. In some embodiments, the library includes one or more of WTC-11iPSC or NCRM5 iPSC.

[0180] In some embodiments, the receptor cell library and / or the multiple reporter cell library comprises different primary cells. In some embodiments, the primary cells comprise one or more of cardiomyocytes, muscle cells, lung cells, liver cells, kidney cells, pancreatic cells, neurons or tumor cells.

[0181] In some embodiments, each cell in the multiple reporter cell library comprises the same polycistronic reporter vector. In other embodiments, the cells in the multiple reporter cell library comprise different polycistronic reporter vectors. In some embodiments, the different polycistronic reporter vectors are introduced into isogenic recipient cells.

[0182] In some embodiments, the present invention provides a multi-reporter cell library, wherein the reporter cell comprises a polycistronic reporter vector encoding one or more polypeptides fused to a reporter polypeptide, and the reporter polypeptide can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, or cellular homeostasis. In some embodiments, the biological pathway is a pathway associated with a disease. In some embodiments, the disease is cancer, cardiovascular disease, neurodegenerative disease, or autoimmune disease. In some embodiments, the biological pathway is a pathway associated with a toxic response mechanism within the cell. In some embodiments, the biological pathway is a pathway related to cell proliferation, cell differentiation, cell death, apoptosis, autophagy, DNA damage and repair or oxidative stress, chromatin / epigenetics (e.g., chromatin acetylation), MAPK signaling (e.g., MAPK / Erk), PI3K / Akt signaling (e.g., mTor signaling), translation control (e.g., eIF2 regulation), cell cycle and checkpoint control (G1 / S checkpoint), cellular metabolism (e.g., insulin receptor signaling), developmental and differentiation signaling (e.g., Wnt), immunology and inflammatory signaling (e.g., JAK / STAT signaling), tyrosine kinase signaling (e.g., ErbB / HER signaling), vesicular trafficking, cytoskeletal regulation, or protein degradation (e.g., ubiquitin pathway), and synthetic lethal combinations of these pathways. In some embodiments, the multiple reporter cell library comprises different polycistronic vectors containing transgenes for analysis of a specific single biological pathway, a specific crosstalk between two or more biological pathways, a specific cellular homeostasis, a specific organelle homeostasis, or a specific toxic response, wherein each different polycistronic reporter vector comprises a common transgene fused to a nucleic acid encoding a reporter polypeptide. In some embodiments, the common transgene product fused to the reporter polypeptide is used as a means of identifying cells that have received the relevant polycistronic reporter vector.

[0183] Determination

[0184] The present invention provides living cell assays using cells and vectors as described herein. In some embodiments, the assay is performed on a single living cell. In some embodiments, the present invention provides a method for analyzing two or more polypeptides in living cells, the method comprising determining the expression of two or more transgenics of multi-reporter cells as described herein and / or the position of the two or more transgenic products. In some embodiments, the method is used to analyze crosstalk between a single biological pathway, two or more biological pathways, cell homeostasis, organelle homeostasis, toxic reactions, and / or phenotypic characteristics. In some embodiments, the assay is used to visualize biological pathways or target readouts using phenotypic readouts. In some embodiments, the expression of the two or more transgenics and / or the position of the two or more transgenic products are determined at one or more time points. In some embodiments, expression of the two or more transgenes and / or the location of the two or more transgene products is determined at one or more time points of 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 4 days, 7 days, 14 days, 21 days, 30 days, 1 month, 3 months, 6 months, 9 months, 1 year, or any time in between or more than 1 year after initiation of the analysis.

[0185] In some embodiments, the location of the transgene product is one or more of the cell membrane, the nucleus, the nuclear membrane, the mitochondria, the mitochondrial membrane, the autophagosome, the lysosome, the endosome, the Golgi apparatus, or the cytosol.

[0186] In some embodiments, the invention provides a method of measuring the effect of an agent on the spectrum of two or more polypeptides in a living cell, the method comprising subjecting a multi-reporter cell as described herein to the agent and determining the expression of the two or more transgenes and / or the location of the two or more transgene products in the cell in response to the agent. In some embodiments, the agent is a drug or a drug candidate. In some embodiments, the agent is a cancer drug or a cancer pharmaceutical agent. In some embodiments, the method is a toxicology screen.

[0187] In some embodiments, the location of the transgene product is one or more of the cell membrane, the nucleus, the nuclear membrane, the mitochondria, the mitochondrial membrane, the autophagosome, the lysosome, the endosome, the Golgi apparatus, or the cytosol.

[0188] In some embodiments of the above assays and methods, the profile is obtained from a single living cell. In some embodiments, the profile is determined for a plurality of living cells. In some embodiments, the cells are cultured on a tissue culture plate, including but not limited to a multi-well tissue culture plate, such as a 96-well or 384-well tissue culture plate. In some embodiments, the cells are cultured in suspension.

[0189] In some embodiments of the above assays and methods, the expression of the two or more transgenes and / or the localization of the two or more transgene products is determined in a multiple reporter cell library.

[0190] In some embodiments, the expression and / or location of the two or more polypeptides is measured by microscopy, high throughput microscopy, fluorescence activated cell sorting (FACS), luminescence, use of a plate reader, mass spectrometry, or deep sequencing.

[0191] In some embodiments, the profiles are configured as a set of three multicolor reporters for a common cellular response. These reporters enable 10 different fluorescence readouts to distinguish aspects of the cellular response. All three reporters have H2B as a DNA / nuclear marker, which we labeled with different fluorescent marker profiles to enable differentiation of their profiles when used in a combined assay. Since the H2B / nuclear marker allows for automated image analysis clustering by reporter, combining isogenic reporter cells in the same well enables highly multiplexed assays with at least 10 readouts.

[0192] In some embodiments, the spectrum is configured as a set of three multicolor "Tox" reporters for unfolded protein response panels, organelle panels, and DNA damage, oxidation, and p53 stress panels. These reporters enable 10 different fluorescence readouts to distinguish early stress responses (oxidative stress, UPR, and p53-dependent cellular stress responses) from late stress responses (DNA double-strand breaks, autophagy, cell cycle, and overall nuclear and mitochondrial homeostasis). All three Tox reporters have H2B as a DNA / nuclear marker, which we label with different fluorescent marker spectra to enable their spectra to be distinguished when used in a combined assay. Since the H2B / nuclear marker allows for automated image analysis clustering by the reporter, merging isogenic reporter cells in the same well enables highly multiplexed assays of at least 10 readouts.

[0193] Kits and Products

[0194] In some embodiments, the present invention provides a kit comprising one or more polycistronic reporter vectors as described herein. In some embodiments, the present invention provides a kit comprising one or more receptor cells as described herein. In some embodiments, the present invention provides a kit comprising one or more multi-reporter cells as described herein. In some embodiments, the present invention provides a kit comprising one or more polycistronic reporter vectors as described herein and one or more receptor cells as described herein. In some embodiments, the kit also includes instructions for using the polycistronic reporter vectors, receptor cells and / or multi-reporter cells as described herein. In some embodiments, the kit includes a mixture of isogenic but differentially labeled multi-reporter cells. Such cells enable direct plating and merging assays.

[0195] In some embodiments, the present invention provides a receptor cell and / or reporter cell library arranged in a multi-well plate (eg, a 96-well plate or a 384-well plate). In some embodiments, the cells in the multi-well plate are cryopreserved.

[0196] The multicistronic reporter vectors, recipient cells and / or multi-reporter cells described herein may be included in an article of manufacture. The article of manufacture may include a container containing the multicistronic reporter vectors, recipient cells and / or multi-reporter cells described herein. In some embodiments, the article of manufacture comprises: (a) a container, in which the multicistronic reporter vectors, recipient cells and / or multi-reporter cells described herein are included; and (b) a package insert having instructions for using the multicistronic reporter vectors, recipient cells and / or multi-reporter cells described herein.

[0197] In some embodiments, the article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from a variety of materials (such as glass or plastic). The article can also include other materials required from a commercial and user perspective, including other buffers, diluents, reagents, tissue culture media, filters, needles, and syringes.

[0198] In some embodiments, the present invention provides a kit comprising a library of receptor cells or reporter cells arranged in a multi-well plate. In some embodiments, the cells are plated and stored frozen.

[0199] Exemplary embodiments

[0200] 1. A multicistronic reporter vector comprising: a promoter operably linked to an open reading frame, wherein the open reading frame comprises two or more cistrons, and wherein expression of the open reading frame in a cell produces separated component polypeptide products from each cistron; wherein each cistron comprises a multiple cloning site (MCS) and a nucleic acid encoding a reporter vector, and wherein each cistron encodes a different reporter polypeptide; and wherein expression of two or more nucleic acids encoding polypeptides inserted into the two or more multiple cloning sites is stoichiometric in nature.

[0201] 2. A multicistronic reporter vector according to embodiment 1, wherein the cistrons are separated from each other by nucleic acids encoding one or more self-cleaving peptides and / or one or more internal ribosome entry sites (IRES).

[0202] 3. A multicistronic reporter vector according to embodiment 2, wherein the one or more self-cleaving peptides are viral self-cleaving peptides.

[0203] 4. A multicistronic reporter vector according to embodiment 3, wherein the one or more viral self-cleaving peptides are one or more 2A peptides.

[0204] 5. The multicistronic reporter vector of embodiment 4, wherein the one or more 2A peptides are T2A peptides, P2A peptides, E2A peptides or F2A peptides.

[0205] 6. A multicistronic reporter vector according to any one of embodiments 2-5, wherein the reporter polypeptide further comprises one or more nucleic acids encoding a peptide linker between one or more of the reporter polypeptides and one or more of the self-cleaving peptides.

[0206] 7. A multicistronic reporter vector according to embodiment 6, wherein the peptide linker comprises the sequence Gly-Ser-Gly.

[0207] 8. A multicistronic reporter vector according to any one of embodiments 1-7, wherein the reporter polypeptide is a fluorescent reporter polypeptide.

[0208] 9. A multicistronic reporter vector according to any one of embodiments 1-8, wherein the reporter polypeptide of each cistron is selected from GFP, EGFP, Emerald, Citrine, Venus, mOrange, mCherry, TagBFP, mTurquoise, Cerulean, UnaG, dsRed, eqFP611, Dronpa, RFP, TagRFP, TdTomato, KFP, EosFP, Dendra, IrisFP, iRFP and smURFP.

[0209] 10. A multicistronic reporter vector according to any one of embodiments 1-9, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron and a second cistron, wherein each cistron comprises, from 5' to 3', a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a viral cleavage peptide.

[0210] 11. A multicistronic reporter vector according to any one of embodiments 1-9, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron and a third cistron, wherein each cistron comprises, from 5' to 3', a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, and the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide.

[0211] 12. A multicistronic reporter vector according to any one of embodiments 1-9, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, a third cistron and a fourth cistron, wherein each cistron comprises, from 5' to 3', a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide, and the third cistron and the fourth cistron are separated by a nucleic acid encoding a third viral cleavage peptide.

[0212] 13. A multicistronic reporter vector according to any one of embodiments 1-9, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, a third cistron and a fourth cistron, wherein each cistron comprises, from 5' to 3', a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide, and the third cistron and the fourth cistron are separated by a nucleic acid encoding an IRES.

[0213] 14. A multicistronic reporter vector according to any one of embodiments 1-13, wherein the vector further comprises one or more inducible elements located between the promoter and the open reading frame.

[0214] 15. The multicistronic reporter vector of embodiment 14, wherein the vector comprises two inducible elements.

[0215] 16. The multicistronic reporter vector of embodiment 14 or 15, wherein the inducible element is a Tet operator 2 (TetO2) inducible element.

[0216] 17. A multicistronic reporter vector according to any one of embodiments 1-16, wherein the promoter is a constitutive promoter.

[0217] 18. A multicistronic reporter vector according to embodiment 17, wherein the constitutive promoter is cytomegalovirus alpha (CMV), thymidine kinase (TK), eF1-α, ubiquitin C (UbC), phosphoglycerate kinase (PGK), CAG promoter, SV40 promoter or human β-actin promoter.

[0218] 19. The polycistronic vector of any one of embodiments 1-16, wherein the promoter is an inducible promoter.

[0219] 20. The polycistronic vector of embodiment 19, wherein the inducible promoter is a tetracycline-responsive promoter.

[0220] 21. A multicistronic reporter vector according to any one of embodiments 1-16, wherein the promoter is a tissue-specific promoter.

[0221] 22. A multi-specific reporter vector according to embodiment 21, wherein the tissue-specific promoter is specific to cells of the heart, blood, muscle, lung, liver, kidney, pancreas, brain or skin.

[0222] 23. The multicistronic reporter vector of any one of embodiments 1-22, further comprising a site-specific recombinase sequence located 3' to the open reading frame.

[0223] 24. A multicistronic reporter vector according to embodiment 23, wherein the vector further comprises a nucleic acid encoding a selectable marker, wherein when the site-specific recombinase sequence is not recombined, the nucleic acid encoding the selectable marker is not operably linked to the promoter, and when the site-specific recombinase sequence is recombined with its target site-specific recombinase sequence, the nucleic acid encoding the selectable marker is operably linked to the promoter.

[0224] 25. The multicistronic reporter vector according to embodiment 24, wherein the site-specific recombinase sequence is a FRT nucleic acid sequence and / or an attP nucleic acid and / or a loxP nucleic acid sequence.

[0225] 26. A multicistronic reporter vector according to embodiment 24 or 25, wherein the selectable marker confers resistance to hygromycin, Zeocin TM , puromycin, neomycin or hygromycin, Zeocin TM , puromycin, blasticidin or neomycin analogs.

[0226] 27. The multicistronic reporter vector of any one of embodiments 1-26, wherein a nucleic acid encoding one or more polypeptides is inserted in-frame into the one or more MCSs.

[0227] 28. A multicistronic reporter vector according to embodiment 27, wherein the one or more polypeptides comprise polypeptides that can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, synthetic lethality, cellular homeostasis, organelle homeostasis or toxic response.

[0228] 29. A multicistronic reporter vector according to any one of embodiments 1-28, which further comprises one, two or three transcription units, wherein the transcription unit comprises a promoter located 5' of an open reading frame comprising two or more cistrons and a nucleic acid encoding a transgene, wherein the reporter vector further comprises a core insulator sequence and a polyA sequence located 3' of the transcription unit and 5' of the open reading frame comprising two or more cistrons.

[0229] 30. A recipient cell for receiving a multicistronic reporter vector, wherein the recipient cell comprises a recombinant nucleic acid integrated into a specific site in the host cell genome, wherein the recombinant nucleic acid comprises a first promoter operably linked to a nucleic acid encoding a fusion polypeptide, wherein the fusion polypeptide comprises a reporter domain and a selectable marker domain, and wherein the nucleic acid comprises a site-specific recombinase nucleic acid sequence located at the 5' end of the nucleic acid encoding the fusion polypeptide.

[0230] 31. A recipient cell according to embodiment 30, wherein the promoter is a constitutive promoter.

[0231] 32. A recipient cell according to embodiment 31, wherein the constitutive promoter is a CMV promoter, a TK promoter, an eF1-α promoter, a UbC promoter, a PGK promoter, a CAG promoter, a SV40 promoter or a human β-actin promoter.

[0232] 33. A recipient cell according to embodiment 32, wherein the promoter is an inducible promoter.

[0233] 34. A recipient cell according to embodiment 33, wherein the inducible promoter is a tetracycline-responsive promoter.

[0234] 35. A recipient cell according to any one of embodiments 30-34, wherein the site-specific recombinase sequence is a FRT nucleic acid sequence and / or an attP nucleic acid sequence and / or a loxP nucleic acid sequence.

[0235] 36. A recipient cell according to any one of embodiments 30-35, wherein the reporter domain of the fusion polypeptide is a fluorescent reporter domain.

[0236] 37. A recipient cell according to any one of embodiments 30-36, wherein the fluorescent reporter domain is selected from GFP, EGFP, Emerald, Citrine, Venus, mOrange, mCherry, TagBFP, mTurquoise, Cerulean, UnaG, dsRed, eqFP611, Dronpa, RFP, TagRFP, TdTomato, KFP, EosFP, Dendra, IrisFP, iRFP and smURFP.

[0237] 38. A recipient cell according to any one of embodiments 30-37, wherein the reporter domain of the fusion polypeptide is an mCherry reporter domain.

[0238] 39. A recipient cell according to any one of embodiments 30-38, wherein the selectable marker domain of the fusion polypeptide confers resistance to hygromycin, Zeocin TM , Puromycin, Blasticidin, Neomycin or Hygromycin, Zeocin TM , puromycin, blasticidin, and neomycin analogs.

[0239] 40. The recipient cell of any one of embodiments 30-39, wherein the integrated recombinant nucleic acid further comprises a nucleic acid encoding a tetracycline repressor polypeptide operably linked to a promoter.

[0240] 41. A recipient cell according to embodiment 40, wherein the promoter is human β-actin promoter or CAG promoter.

[0241] 42. A recipient cell according to any one of embodiments 30-41, wherein the recombinant nucleic acid is integrated into the adeno-associated virus S1 (AAVS1) locus, the chemokine (CC motif) receptor 5 (CCR5) locus, the human homolog of the mouse ROSA26 locus, the hip11 (H11) locus, or the citrate lyase β-like gene locus (CLYBL).

[0242] 43. A recipient cell according to any one of embodiments 30-42, wherein the cell is an immortal cell.

[0243] 44. A recipient cell according to any one of embodiments 30-43, wherein the immortalized cells are HEK293T cells, A549 cells, U2OS cells, RPE cells, NPC1 cells, MCF7 cells, HepG2 cells, HaCat cells, TK6 cells, A375 cells or HeLa cells.

[0244] 45. The recipient cell of any one of embodiments 30-44, wherein the cell is a pluripotent cell, an induced pluripotent stem cell, or a multipotent cell.

[0245] 46. ​​A recipient cell according to any one of embodiments 30-42, wherein the cell is a primary cell.

[0246] 47. A method for producing a recipient cell for receiving a multicistronic reporter vector, the method comprising introducing a recombinant nucleic acid into the cell, wherein the recombinant nucleic acid comprises from 5' to 3'

[0247] a) a first nucleic acid for targeting homologous recombination to a specific site in said cell,

[0248] b) a first promoter,

[0249] c) a site-specific recombinase nucleic acid,

[0250] d) a nucleic acid encoding a first reporter polypeptide and a selectable marker,

[0251] e) a second nucleic acid for targeting homologous recombination to a specific site in said cell,

[0252] f) a second promoter and a nucleic acid encoding a second reporter polypeptide,

[0253] Wherein expression of the first reporter polypeptide but not expression of the second reporter polypeptide indicates targeting of integration of the recombinant nucleic acid to a specific site in the genome of the cell, and expression of the first and second reporter polypeptides indicates random integration in the genome of the cell.

[0254] 48. The method of embodiment 47, wherein the recombinant nucleic acid further comprises a nucleic acid encoding a tetracycline repressor operably linked to a promoter 5' of the second nucleic acid for targeting homologous recombination.

[0255] 49. The method according to embodiment 47 or 48, wherein the recombinant nucleic acid is integrated into the genome of the cell using:

[0256] a) an RNA-guided recombination system comprising a nuclease and a guide RNA,

[0257] b) TALEN endonucleases, or

[0258] c) ZFN endonucleases.

[0259] 50. A method according to any one of embodiments 47-49, wherein cells that express the first reporter polypeptide but do not express the second reporter polypeptide are selected.

[0260] 51. The method according to any one of embodiments 47-50, wherein the site-specific recombinase nucleic acid is a FRT nucleic acid sequence and / or an attP nucleic acid sequence and / or a loxP nucleic acid sequence.

[0261] 52. A method according to any one of embodiments 47-51, wherein the first reporter polypeptide is a fluorescent polypeptide and the second reporter polypeptide is a different fluorescent polypeptide.

[0262] 53. A method according to any one of embodiments 47-52, wherein the first and second reporter polypeptides are selected from GFP, EGFP, Emerald, Citrine, Venus, mOrange, mCherry, TagBFP, mTurquoise, Cerulean, UnaG, dsRed, eqFP611, Dronpa, RFP, TagRFP, TdTomato, KFP, EosFP, Dendra, IrisFP, iRFP and smURFP.

[0263] 54. A method according to any one of embodiments 47-53, wherein the first reporter polypeptide is an mCherry reporter and the second reporter polypeptide is GFP.

[0264] 55. The method of any one of embodiments 47-54, wherein the selectable marker confers resistance to hygromycin, Zeocin TM , Puromycin, Blasticidin, Neomycin or Hygromycin, Zeocin TM , puromycin, blasticidin, and neomycin analogs.

[0265] 56. A method according to any one of embodiments 47-55, wherein the first promoter is a CMV promoter, a TK promoter, an eF1-α promoter, a UbC promoter, a PGK promoter, a CAG promoter, a SV40 promoter or a human β-actin promoter, and the second promoter is a CMV promoter, a TK promoter, an eF1-α promoter, a UbC promoter, a PGK promoter, a CAG promoter, a SV40 promoter or a human β-actin promoter.

[0266] 57. A method according to any one of embodiments 47-56, wherein the first nucleic acid for targeting homologous recombination and the second nucleic acid for targeting homologous recombination target recombination to the AAVS1 locus, the CCR5 locus, the human homolog of the mouse ROSA26 locus, the H11 locus, or the CLYBL locus.

[0267] 58. The method of any one of embodiments 47-57, wherein the cell is an immortal cell.

[0268] 59. The method according to any one of embodiments 47-58, wherein the immortal cells are HEK293T cells, A549 cells, U2OS cells, RPE cells, NPC1 cells, MCF7 cells, HepG2 cells, HaCat cells, TK6 cells, A375 cells or HeLa cells.

[0269] 60. The method of any one of embodiments 47-59, wherein the cell is a pluripotent cell, an induced pluripotent stem cell, or a multipotent cell.

[0270] 61. A method according to embodiment 60, wherein the induced pluripotent stem cells are WTC-11 cells or NCRM5 cells.

[0271] 62. The method of any one of embodiments 47-58, wherein the cell is a primary cell.

[0272] 63. A recipient cell line produced using the method of any one of embodiments 47-62.

[0273] 64. A multi-reporter cell comprising a recipient cell according to any one of embodiments 30-46, wherein the multicistronic reporter vector according to embodiment 27 or 28 has been integrated into the genome of the recipient cell.

[0274] 65. The multi-reporter cell of embodiment 64, wherein the multicistronic reporter vector has been integrated into the AAVS1 locus of the recipient cell.

[0275] 66. A multi-reporter cell comprising a multicistronic reporter construct, wherein the multicistronic reporter construct comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises two or more cistrons; wherein each cistron comprises a nucleic acid encoding a different transgene product fused to a different reporter polypeptide, wherein expression of the open reading frame in the cell produces separate component polypeptide products from each cistron; and wherein expression of the transgene products is stoichiometric in nature.

[0276] 67. The multi-reporter cell of embodiment 66, wherein the cistrons are separated from each other by nucleic acids encoding one or more self-cleaving peptides and / or one or more internal ribosome entry sites (IRES).

[0277] 68. A multi-reporter cell according to embodiment 66 or 68, wherein each of the reporter polypeptides is a fluorescent reporter polypeptide.

[0278] 69. A multi-reporter cell according to any one of embodiments 66-68, wherein the fluorescent reporter domain is selected from GFP, EGFP, Emerald, Citrine, Venus, mOrange, mCherry, TagBFP, mTurquoise, Cerulean, UnaG, dsRed, eqFP611, Dronpa, RFP, TagRFP, TdTomato, KFP, EosFP, Dendra, IrisFP, iRFP and smURFP.

[0279] 70. The multi-reporter cell of any one of embodiments 66-69, wherein the one or more self-cleaving peptides are one or more 2A peptides.

[0280] 71. A multi-reporter cell according to any one of embodiments 66-70, wherein the nucleic acid encoding the transgene product fused to the reporter polypeptide further comprises one or more nucleic acids encoding a peptide linker between the reporter polypeptide and the viral self-cleaving peptide.

[0281] 72. A multi-reporter cell according to embodiment 71, wherein the peptide linker comprises the sequence Gly-Ser-Gly.

[0282] 73. A multi-reporter cell according to any one of embodiments 66-72, wherein the multi-cistronic reporter vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, a third cistron and a fourth cistron, wherein each cistron comprises from 5' to 3' a nucleic acid encoding a transgene product fused to a fluorescent reporter polypeptide and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide, and the third cistron and the fourth cistron are separated by a nucleic acid encoding an IRES.

[0283] 74. The multi-reporter cell of any one of embodiments 66-73, wherein the multicistronic reporter vector further comprises one or more inducible elements located between the promoter and the open reading frame.

[0284] 75. The multi-reporter cell of embodiment 74, wherein the inducible element is a Tet operator 2 (TetO2) inducible element.

[0285] 76. The multi-reporter cell of any one of embodiments 66-75, wherein the promoter is a constitutive promoter.

[0286] 77. A multi-reporter cell according to embodiment 76, wherein the constitutive promoter is a CMV promoter, a TK promoter, an eF1-α promoter, a UbC promoter, a PGK promoter, a CAG promoter, a SV40 promoter or a human β-actin promoter.

[0287] 78. The multi-reporter cell of any one of embodiments 66-75, wherein the promoter is a tissue-specific promoter.

[0288] 79. The multi-reporter cell of embodiment 78, wherein the tissue-specific promoter is specific for cells of the heart, blood, muscle, lung, liver, kidney, pancreas, brain, or skin.

[0289] 80. The multi-reporter cell of any one of embodiments 66-75, wherein the promoter is an inducible promoter.

[0290] 81. The multi-reporter cell of embodiment 80, wherein the inducible promoter is a TRE promoter.

[0291] 82. The multi-reporter cell of any one of embodiments 66-81, wherein the one or more transgene products comprise a polypeptide that can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, cellular homeostasis, organelle homeostasis, or a toxic response.

[0292] 83. A multi-reporter cell according to embodiment 82, wherein the analysis is performed on a single cell.

[0293] 84. A multi-reporter cell according to any one of embodiments 64-83, wherein the reporter polypeptide can be visualized by microscopy, high-throughput microscopy, fluorescence activated cell sorting (FACS), luminescence, or using a plate reader.

[0294] 85. A multi-reporter cell according to any one of embodiments 66-84, which further comprises one, two or three transcription units, wherein the transcription unit comprises a promoter located 5' of an open reading frame comprising two or more cistrons and a nucleic acid encoding a transgene, wherein the reporter vector further comprises a core insulator sequence located 3' of the transcription unit and 5' of the open reading frame comprising two or more cistrons.

[0295] 86. A method for producing a multi-reporter cell, the method comprising introducing the multicistronic reporter vector according to embodiment 27 or 28 into a recipient cell according to any one of embodiments 33-49.

[0296] 87. A method according to embodiment 86, wherein the recombinase-associated nucleic acid sequence is a FRT nucleic acid sequence and the recipient cell contains the flp recombinase.

[0297] 88. The method of embodiment 87, wherein the recombinase-associated nucleic acid is attP and the recipient cell comprises Bxb1 recombinase, PhiC31 recombinase or R4 recombinase.

[0298] 89. A method according to embodiment 87, wherein the recombinase-associated nucleic acid sequence is a loxP nucleic acid sequence, and the recipient cell contains a CRE recombinase.

[0299] 90. A library of multiple reporter vectors, wherein the library comprises the multicistronic reporter vectors of any one of embodiments 1-29, comprising nucleic acids encoding different transgene products fused to a reporter polypeptide, or wherein the library comprises a plurality of reporter cells of any one of embodiments 64-85, wherein when introduced into cells, two or more of the different transgene products on each vector are expressed essentially stoichiometrically.

[0300] 91. A library according to embodiment 90, wherein the reporter vector encodes two or more transgenes, which encode polypeptides that can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, cellular homeostasis, organelle homeostasis, or toxicity responses.

[0301] 92. The library of embodiment 90 or 91, wherein the biological pathway is a disease-associated pathway.

[0302] 93. A library according to embodiment 92, wherein the disease is cancer, cardiovascular disease, neurodegenerative disease or autoimmune disease.

[0303] 94. The library of embodiment 92 or 93, wherein the biological pathway is a pathway associated with a toxic response mechanism within the cell.

[0304] 95. A library according to embodiment 92 or 93, wherein the biological pathway is a pathway related to cell proliferation, cell differentiation, cell death, apoptosis, autophagy, DNA damage and repair, oxidative stress, chromatin / epigenetics, MAPK signaling, PI3K / Akt signaling, translation control, cell cycle and checkpoint control, cellular metabolism, developmental and differentiation signaling, immunology and inflammatory signaling, tyrosine kinase signaling, vesicle trafficking, cytoskeletal regulation or ubiquitin pathways.

[0305] 96. The library of any one of embodiments 90-95, wherein each polycistronic vector comprising a transgene for analysis of a specific single biological pathway, a specific crosstalk between two or more biological pathways, synthetic lethality, a specific cellular homeostasis, a specific organelle homeostasis, a specific toxic response, a specific tissue comprises a common transgene product fused to a different reporter polypeptide for each polycistronic reporter vector.

[0306] 97. The library of any one of embodiments 90-96, wherein each polycistronic vector comprising a transgene for analyzing a specific single biological pathway, a specific crosstalk between two or more biological pathways, a specific cellular homeostasis, a specific organelle homeostasis, or a specific toxic response comprises a common transgene product fused to a reporter polypeptide.

[0307] 98. A library of recipient cells for receiving a polycistronic reporter vector, wherein the library comprises the recipient cells according to embodiments 30-46.

[0308] 99. A multiple reporter cell library, wherein each cell in the library comprises a multicistronic reporter vector comprising nucleic acids encoding different transgene products fused to a reporter polypeptide, wherein when introduced into the cell, the different nucleic acids encoding different transgene products on each vector are expressed essentially stoichiometrically.

[0309] 100. The library of embodiment 98 or 99, wherein the library comprises different immortalized cells.

[0310] 101. A library according to embodiment 100, wherein the library comprises one or more of HEK293T cells, A549 cells, U2OS cells, RPE cells, NPC1 cells, MCF7 cells, HepG2 cells, HaCat cells, TK6 cells, A375 cells or HeLa cells.

[0311] 102. The library of embodiment 98 or 99, wherein the library comprises different pluripotent, multipotent and / or progenitor cells.

[0312] 103. A library according to embodiment 102, wherein the different multipotent or pluripotent cells include one or more of induced pluripotent stem cells, multipotent cells, hematopoietic cells, endothelial progenitor receptor cells, mesenchymal progenitor cells, neural progenitor cells, osteochondrogenic progenitor cells, lymphoid progenitor cells or pancreatic progenitor cells.

[0313] 104. The library of embodiment 98 or 99, wherein after introduction of the multicistronic reporter vector, the multi-reporter cell library of pluripotent or multipotent cells is differentiated.

[0314] 105. The library of embodiment 98 or 99, wherein the library comprises different primary cells.

[0315] 106. A library according to embodiment 105, wherein the primary cells comprise one or more of cardiomyocytes, muscle cells, lung cells, liver cells, kidney cells, pancreatic cells, neurons or tumor cells.

[0316] 107. A library according to any one of embodiments 99-106, wherein each cell in the library contains the same multicistronic reporter vector.

[0317] 108. A cell library according to any one of embodiments 99-107, wherein the cells in the library contain different polycistronic reporter vectors.

[0318] 109. A cell library according to embodiment 108, wherein different multicistronic reporter vectors are introduced into isogenic recipient cells.

[0319] 110. A cell library according to any one of embodiments 99-109, wherein the reporter vector encodes one or more transgenes and the one or more polypeptides comprise polypeptides that can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, cellular homeostasis, or phenotypic characteristics.

[0320] 111. The library of embodiment 110, wherein the biological pathway is a disease-associated pathway.

[0321] 112. A library according to embodiment 111, wherein the disease is cancer, cardiovascular disease, neurodegenerative disease or autoimmune disease.

[0322] 113. A library according to any of embodiments 110, wherein the biological pathway is a pathway associated with a toxic response mechanism within the cell.

[0323] 114. A library according to any one of embodiments 110-113, wherein the biological pathway is a pathway related to cell proliferation, cell differentiation, cell death, apoptosis, autophagy, DNA damage and repair, oxidative stress, chromatin / epigenetics, MAPK signaling, PI3K / Akt signaling, translation control, cell cycle and checkpoint control, cellular metabolism, developmental and differentiation signaling, immunology and inflammation signaling, tyrosine kinase signaling, vesicle trafficking, cytoskeletal regulation, or an ubiquitin pathway.

[0324] 115. The library of any one of embodiments 99-114, wherein each polycistronic vector comprising a transgene for analyzing a specific single biological pathway, a specific crosstalk between two or more biological pathways, synthetic lethality, a specific cellular homeostasis, a specific organelle homeostasis, a specific toxic response, a specific tissue, or a specific phenotypic characteristic comprises a common transgene encoding a polypeptide fused to a different reporter polypeptide.

[0325] 116. The library of any one of embodiments 99-115, wherein each polycistronic vector comprising a transgene for analyzing a specific single biological pathway, a specific crosstalk between two or more biological pathways, a specific cellular homeostasis, a specific organelle homeostasis, a specific toxic response, or a specific phenotypic characteristic comprises a common transgene encoding a polypeptide fused to a reporter polypeptide.

[0326] 117. A kit comprising one or more multicistronic reporter vectors according to any one of embodiments 1-29.

[0327] 118. A kit comprising one or more recipient cells according to any one of embodiments 30-46.

[0328] 119. A kit comprising one or more multicistronic reporter vectors according to any one of embodiments 1-29 and one or more recipient cells according to any one of embodiments 30-46.

[0329] 120. A kit comprising one or more multi-reporter cells according to any one of embodiments 64-85.

[0330] 121. A kit according to any one of embodiments 118-120, wherein the kit comprises a library of receptor cells and / or reporter cells arranged in a multi-well plate.

[0331] 122. A kit according to embodiment 121, wherein the cells in the multi-well plate are cryopreserved.

[0332] 123. A method of analyzing two or more polypeptides in a living cell, the method comprising determining the expression of the two or more transgenes and / or the location of the two or more transgene products of a multi-reporter cell according to any one of embodiments 64-85 or a cell comprising a multicistronic vector according to any one of embodiments 1-29.

[0333] 124. A method according to embodiment 123, wherein the method is used to analyze a single biological pathway, crosstalk between two or more biological pathways, synthetic lethality, cellular homeostasis, organelle homeostasis, or a toxic response.

[0334] 125. The method of embodiment 123 or 124, wherein the expression of the two or more transgenes and / or the location of the two or more transgene products is determined at one or more time points.

[0335] 126. The method of embodiment 125, wherein the expression of the two or more transgenes and / or the location of the two or more transgene products is determined at one or more time points of 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 4 days, 7 days, 14 days, 21 days, 30 days, 1 month, 3 months, 6 months, 9 months, 1 year, or more.

[0336] 127. The method of any one of embodiments 123-126, wherein the cell is prepared by the method of any one of embodiments 86-89.

[0337] 128. A method according to any one of embodiments 123-126, wherein the cells are a cell library according to embodiments 90-116.

[0338] 129. The method of embodiment 129, wherein the cell is derived from an isogenic recipient cell.

[0339] 130. A method according to embodiment 128 or 129, wherein the cells or the libraries are pooled prior to analysis.

[0340] 131. A method for measuring the effect of an agent on the spectrum of two or more polypeptides in a living cell, the method comprising subjecting a multi-reporter cell according to any one of embodiments 64-85 to the agent and determining the expression of the two or more transgenes and / or the location of the two or more transgene products in the cell in response to the agent.

[0341] 132. The method of embodiment 131, wherein the agent is a drug or a drug candidate.

[0342] 133. The method of embodiment 131 or 132, wherein the method is a toxicology screen.

[0343] 134. The method of any one of embodiments 131-133, wherein the expression of the two or more transgenes and / or the location of the two or more transgene products is determined in a multiple reporter cell library.

[0344] 135. The method of any one of embodiments 131-134, wherein the spectrum is obtained using a single cell.

[0345] 136. The method of any one of embodiments 131-135, wherein the expression of the two or more transgenes and / or the location of the two or more transgene products is measured by microscopy, high throughput microscopy, fluorescence activated cell sorting (FACS), luminescence, use of a plate reader, mass spectrometry, or deep sequencing.

[0346] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise explicitly stated, each feature disclosed is only an example of a series of equivalent or similar features.

[0347] Further details of the present invention are illustrated by the following non-limiting examples.The disclosures of all references in this specification are expressly incorporated herein by reference.

[0348] Example

[0349] Example 1. Development of monoclonal recipient cells

[0350] Genome editing tools have been optimized to generate monoclonal cell lines containing "acceptor sites". Use of the acceptor site enables reliable, rapid, and consistent site-specific integration of selected reporter constructs. Figure 1A , lower figure) contains 1) two AAVS1 sequences that direct the integration of the receptor site into the AAVS1 locus; 2) a fluorescent marker (mCherry) visible by microscopy; 3) an antibiotic (bleomycin-Thermo Fisher) resistance selection marker; 4) an FRT site for site-specific recombination of the reporter construct by flippase (Flp); 5) a constitutive promoter SV40 that drives bleomycin (Thermo Scientific TM) resistance gene and mCherry expression, enabling antibiotic selection and fluorescence screening to identify positive receptor cell clones, respectively; 6) human b-actin promoter driving tetracycline repressor protein (TetR).

[0351] Using a Cas9-mediated genome editing protocol, the acceptor site was stably integrated into the genome of the target cell. According to the design of the acceptor site, the AAVS1 locus was used as the integration site. The acceptor site was synthesized and cloned into the AAVS1-donor vector (GeneCopoeia TM ) and co-transfected with Cas9:sgRNA(AAVS1), a guide RNA (gRNA) complementary to the target AAVS1 sequence, to obtain stable integration at a single integration site (IS), the AAVS1 locus located between exons 1 and 2 at the PPP1R12C locus on chromosome 19. Transfection and junction PCR combined with Southern blot analysis were used to confirm the correct recipient site integration obtained in recipient cell clones after selection.

[0352] The receptor loci were transfected into HEK 293T, U2OS, A549, HeLa, RPE, and NPC1 cells (plated in 2-4 96-well plates). Lipofectamine 3000 (Life Technologies TM ) and following the manufacturer's recommended protocol, HEK 293T, U2OS, A549 and Hela cells were successfully transfected with efficiencies exceeding 60% (as estimated by the number of mCherry positive cells). Using Lipofectamine and the same protocol, RPE and NPC1 cell lines showed very low (<2%) transfection efficiencies.

[0353] Also tried using Transfection system (Thermo Scientific TM ) RPE and NPC1 cell lines were transfected by electroporation under 4 different conditions of varying cell density, construct concentration and applied voltage. One of the test conditions (1350V, 20ms pulse, two pulses, 10 μl at the tip) resulted in a high transfection efficiency (>60%) for RPE cells, but the transfection of the NPC1 system was unsuccessful.

[0354] After enrichment with U2OS, HEK293T, RPE, A549 and HeLa clones of receptor site integration using bleomycin selection, single colonies of each cell line were produced, and integration at AAVS1 locus was screened by PCR and random integration; and site-specific mono / biallelic integration was screened by Southern blot. mCherry positive and bleomycin-resistant clones were plated in 3-5 96-well plates with 1-2 cells / well, and were screened using connection PCR. As assessed by connection PCR, approximately 58%-91% of clones were confirmed to have site-specific integration (Table 1).

[0355] Table 1

[0356]

[0357] Two probes were designed for Southern blotting: 5' probe ( Figure 1A , upper panel), to confirm monoallelic integration of the recipient site at the AAVS1 locus; and an internal probe ( Figure 1A , bottom), to confirm single copy integration in the genome without random integration. After digestion of genomic DNA with EcoRI, the 5' probe will detect a single band at 2.8 kb in the case of integration in both alleles, or a single band at 8.2 kb in the case of no integration. Samples with monoallelic integration have 2 bands ( Figure 1C ). It was determined that the non-radioactive Southern blotting protocol was not sensitive enough for screening. For each cell line, at least 9 PCR positive clones were selected for screening with the 5' probe. The genomic DNA of clones confirmed to have monoallelic integration with the 5' probe was then probed with an internal probe to identify clones with random integration without the receptor site in other genomic loci (identified by the presence of a single band at 10 Kb) ( Figure 1D ).

[0358] In the absence of random integration, site-specific receptor sites were obtained in RPE cells with an efficiency of 56%. For A549 and Hela, 21 and 13 clones were screened by PCR and southern blotting, and only 1 clone had a single copy integration of the receptor site at the AAVS1 locus, corresponding to an overall recombination efficiency of 5% and 8%, respectively. However, Southern blotting screening of 9 U2OS and 13 HEK293T clones failed to identify any clones with a single copy integration of the receptor site at the correct locus (Table 1).

[0359] Example 2. Development of a polycistronic platform

[0360] The tetracistronic reporter was designed to incorporate a constitutive promoter (CMV) driving the expression of a single open reading frame (ORF) containing three multiple cloning sites (MCS) separated by two unique viral 2A self-cleaving peptides and a fourth MCS separated by an internal ribosome entry site (IRES) element that allows translation to start in the middle of the mRNA sequence ( Figure 2 ). The 2A self-cleaving peptide allows multiple proteins to be encoded as a polyprotein, which is separated into component proteins during translation. The 2A peptide sequence impairs normal peptide bond formation through the ribosome skipping mechanism. From the family of peptide 2A cleavage sequences, the P2A and T2A versions were identified as the best candidates because these versions have been shown to exhibit the most efficient cleavage effects (Kim et al., PLOS One, 2011). In order to improve the cleavage efficiency of the viral peptide, a Gly-Ser-Gly linker ( Figure 2 ). Immediately downstream of the promoter are 2 copies of the Tet operator 2 (TetO2), to which TetR (expressed from the receptor site) binds, thereby inhibiting the expression of the target gene. Gene expression is induced by the addition of doxycycline (a tetracycline analog). In addition, the FRT sequence is fused to a promoterless hygromycin resistance gene that can only be expressed when correct gene targeting occurs. The reporter construct is assembled using InFusion. After cloning, the vector is verified by Sanger sequence. After the reporter is completed, the sequence is verified again to ensure that 3 or 4 reporters have been inserted.

[0361] To test localization or activation of the reporter, cells are transiently transfected and localization assessed by microscopy, or if it is a biosensor, cells are treated with a compound that activates the corresponding pathway to verify whether the biosensor responds.

[0362] Some proteins that are disturbed by the presence of extra amino acids in C' cannot be placed in the first or second position because 2A cleavage will leave extra amino acids on the C' of the protein. The synthesized nucleotide sequence is cloned into the pCDNA5 / FRT vector backbone. The FRT target site is connected to the mCherry reporter gene to allow fluorescent screening. This vector is designed to be recombined into a recipient cell line that is generated when co-transfected with a second vector (pOG44, Thermo Fisher Scientific) containing a flippase (Flp).

[0363] The polycistronic vector was co-transfected with pOG44 into a recipient cell line, and transfection conditions were optimized to produce a multicolor stable reporter cell line that can be used as the basis for assay development and disease modeling. The recombinase used, flippase (Flp), has a recombination rate of 1 in 10-6 cells (O'Gorman, S., Fox, DT & Wahl, GM Science 251, 1351-1355 (1991)).

[0364] After generating a stable reporter cell line, the effect of position within the polycistronic vector was evaluated. Multiple rounds of optimization were used to determine the best construct that would produce stable balanced expression of a functional fluorescently labeled protein of interest in the recipient cell line. Such cell lines would serve as a useful basis for assays based on live cell microscopy.

[0365] Example 3. Multiparameter visualization of RAS-MAPK signaling in living cells.

[0366] Spatiotemporal regulation of Ras / MAPK signaling proteins in cells Figure 3 ) is an important aspect of its signaling behavior. Ras / MAPK signaling proteins are localized in different cellular compartments depending on the pathway activation state. New assays that comprehensively probe the dynamic activity of the entire pathway in intact cells can help identify and optimize the next generation of inhibitors that circumvent resistance mechanisms. This article describes a novel method for monitoring the cellular activity of putative inhibitors in a high-throughput compatible multiplex assay that reports the dynamic activity of the entire Ras / MAPK pathway in living cells. To monitor the survival of the pathway, a stable cell line that balances the expression of fluorescent marker proteins was used.

[0367] To generate two-color cell lines, CRaf and mutant KRas (G12C) constructs with N-terminal fluorescent protein tags were cloned into a tetracycline-inducible bicistronic expression vector. (Promega TM ) Stable cell lines were generated by co-transfecting Flp-In TREx 293 cells with a bicistronic vector and a Flp-recombinase expression plasmid followed by selection with hygromycin B.

[0368] Two color stable cell lines were plated overnight in 96-well glass bottom plates and treated with 1 μg / ml doxycycline to induce fluorescent reporter expression. Cells were treated with different compounds for four or nine hours. TM Epi-fluorescence Nikon for incubator systems TM Cells were imaged using a microscope and Plan Fluor TM 40x objective lens and Software (NikonTM ) to capture images. A similar procedure (using N-terminally tagged KRas(G12C), CRaf, Mek, and Erk cloned into a polycistronic construct) was used to generate a monoclonal, inducible, 4-color cell line with approximately balanced expression levels of the reporter constructs ( Figure 4 and Figure 5 , control).

[0369] A 4-color system was used to visualize the MAPK pathway in living cells. The HEK293 multicolor stable cell line expressing KRas(G12C), C-Raf, Mek, and Erk reporters showed that the 4 proteins were correctly localized at the plasma membrane, plasma membrane and cytoplasm, cytoplasm, and nucleus, respectively ( Figure 5 ). Monitoring cells under a microscope allowed visualization of responses to inhibitors of the Ras-MAPK pathway. To monitor responses to inhibitors, cells were treated with GDC-0879, FR180204, lovastatin, and PD0325901 for four or nine hours after protein induction. Representative images of the different treatments are shown in Figure 5 middle.

[0370] GDC-0879 is an ATP-mimetic Raf inhibitor that binds to the active CRaf conformation and induces CRaf plasma membrane targeting. Lovastatin is an HMG-CoA antagonist that blocks the processing and membrane localization of Ras proteins via inhibition of prenylation. FR180204 is an Erk inhibitor that prevents the catalytic activity of Erk, but cannot prevent the phosphorylation of Erk. Treatment with GDC-0879 (a Raf inhibitor) shows that CRaf is significantly enriched in the plasma membrane after compound treatment, while other proteins remain in the same position relative to the control. Treatment with FR180204 (an Erk inhibitor) shows that the CRaf level on the plasma membrane is moderately increased. This can be explained by the release of negative feedback triggered by active Erk, which directly acts on C-Raf. Treatment with lovastatin (which affects KRas inactivation) results in the absence of KRas on the membrane, so C-Raf is not enriched on the membrane, and the downstream effector mCherry-Erk of the MAPK pathway changes its location in the cytoplasm. Treatment with PD0325901, a Mek inhibitor, resulted in a redistribution of the downstream effector Erk (from the nucleus to the cytoplasm), indicating that Erk was inactive. As expected, none of the Mek upstream proteins (KRas and C-Raf) showed altered localization.

[0371] Lovastatin caused a significant displacement of KRas relative to the cell membrane (***p<0.0001). TA-155 treatment caused a similar displacement of KRas relative to the cell membrane (***p<0.0001), validating this molecule as an effector of the pathway.

[0372] To further demonstrate the utility of the multiplex assay, a library with 23 different reporter cells was generated using a library of 24 MAPK vectors ( Fig. 6A The MAPK library consisted of a collection of polycistronic constructs containing 4 proteins: 3 fluorescently labeled proteins - mcherry-Erk, mCerulean-Raf (isoforms and mutants), Venus-Ras (isoforms and mutants) and additionally the unlabeled protein Mek. Unlabeled Mek and mCherry-Erk were fixed in all constructs. Each of the Ras and Raf wild type or isoforms (K-, H-, N-Ras and A-, B-, C-Raf) and mutants (K-Ras G12C 、K-Ras G12D 、H-Ras Q61L and B-Raf V600E ) were combined in a MAPK construct. MAPK reporter constructs are denoted "MAPK R" and are numbered from "1-24". (Promega TM ) The collection of 24 vectors was recombined with Bxb1 in U2OS A -Tet The reporter cell lines were generated in the recipient cell lines. The cells were selected with 75ug / ml hygromycin B for 3 days. After selection, the cells were expanded and verified by testing tetracycline induction and plated in 96 glass bottom plates for imaging to verify the presence of the 3 fluorescent marker proteins. All reporter cell lines were treated with 1μg / ml doxycycline for 12-16 hours to induce the expression of fluorescent reporters. TM Epi-fluorescence Nikon for incubator systems TM Cells were imaged using a microscope and Plan Fluor TM 40x objective lens and Software (Nikon TM )Acquisition image( Figure 6B ).

[0373] Three MAPK reporter cell lines were used to visualize the MAPK pathway in living cells and analyze the dose response of MAPK inhibitors. U2OS MAPKR19, R20 and R21 multicolor stable cell lines (wt, G12C and G12D, respectively) expressing mCerulean-CRaf, unlabeled Mek, mCherry-Erk and Venus-KRas were plated in glass-bottom 96-well plates. Twenty-four hours after cell plating, cells were treated with 1 μg / ml of doxycycline for 5h to induce fluorescent reporter expression. Monitoring cells under a microscope allows visualization of the response to Ras-MAPK pathway inhibitors. Two inhibitors were used in the assay: trametinib (a Mek1 inhibitor) and PD 0329501 (a Mek1 / 2 inhibitor). Trametinib is a reversible allosteric inhibitor of MEK1 / 2 that binds to serine 12 in the activation loop. One hour after compound addition, inhibitors were tested in duplicate with four-point ten-fold serial dilutions starting at 10 μM and data were collected. Images were collected in a Nikon Ti-E inverted microscope equipped with a motorized stage with autofocus and an OkoLab stage-top environmental control chamber (supplied with CO2 and temperature controlled for live cell imaging). Four positions per well ( Figure 7B ).

[0374] Subcellular localization of mCherry-Erk was used as a measure of MAPK pathway activation; cells with activated MAPK signaling showed nuclear localization of Erk, while cells with inactivated or inhibited MAPK signaling showed nuclear exclusion of Erk. For each assay, images were analyzed using CellProfiler (open source software for image analysis, Kamensty, L. et al., Bioinformatics (2011)) to quantify the ratio of nuclear to cytoplasmic Erk fluorescence intensity, and the resulting data were further processed to determine compound IC 50 .

[0375] Image analysis uses open source software CellProfiler.After importing the image, the nucleus is identified as the main object from the Hoescht image.Use the proliferation from the main object, the cytoplasm of each cell is identified as the secondary object from the K-Ras image.The cytoplasm area is deducted from the nuclear area of ​​each cell to obtain the final cytoplasm mask.The mCherry intensity of the nucleus and cytoplasm of each cell is calculated from the original mCherry-Erk image, and the nuclear-cytoplasmic ratio of the mCherry intensity of each cell is calculated, and the output is an HDF5 file for analysis.The data analyzed from CellProfiler are exported to Python programming language (Python Software Foundation, World Wide Web website python.org) for further calculations.

[0376] To quantify the degree of inhibition (mCherry-Erk cytoplasmic localization) of each compound, we used Gaussian kernel density estimation performed by the SciPy library of Python open source software. Untreated (vehicle) and maximally inhibited (highest concentration of the strongest inhibitor) control values ​​for each cell line were used to generate a density map of the distribution of values. The data from each well were plotted against the control density map, and the probability of each cell being classified as inhibited or not inhibited was used to determine the percentage of inhibition for each compound at 4 different concentrations. The values ​​were exported to GraphPad Prism 7.0 software (La Jolla, CA, USA), and we used nonlinear regression to fit the data and calculate the concentration at which we observed 50% activity of the inhibitor (IC 50 )( Figure 7B and 7C ).

[0377] Trametinib activity was detected in all 3 cell lines ( Fig. 7A ), for cells expressing K-Ras(wt), IC 50 The value is 9 nM; for cells expressing K-Ras(G12C) and K-Ras(G12D), the IC 50 The values ​​were 55nM and 28nM, respectively. 50 ( Figure 7C ).

[0378] PD 0329501 activity was detected in all 3 cell lines ( Fig. 7A ), for cells expressing K-Ras(wt), IC 50 The value is 9 nM; for cells expressing K-Ras(G12C) and K-Ras(G12D), the IC 50 The values ​​were 13nM and 76nM, respectively. 50( Figure 7C ).

[0379] These results demonstrate that multiplexing allows monitoring, measuring, and comparing the activity, dose response, and determination of IC of MAPK inhibitors in different KRas contexts. 50 .

[0380] Example 4. Optimization of receptor sites

[0381] The receptor sites were further optimized to be more adaptable and allow for more efficient discrimination between targeted and non-targeted integration. Figure 1E As shown, the updated acceptor site includes, in addition to the FRT site, 1) a CMV-driven GFP element and 2) an attP site for recombination using the Bxb1 recombinase. The acceptor site was redesigned to contain GFP driven by the CMV promoter (CMV-GFP) after the AAVS1 right homologous recombination arm ( Figure 1E ), which makes it easier to distinguish between targeted integration at the AAVS1 locus or random integration in the genome. Cells with randomly integrated redesigned receptor sites will emit green fluorescence. Cells with integrated redesigned receptor sites (targeted or randomly integrated) will emit red fluorescence due to the expression of mCherry. Therefore, cells with targeted integration will only be red, while cells with any random integration will also express GFP and be red and green. This second method allows the use of fluorescence microscopy to identify cells without random integration, or the use of FACS to sort cells without recombination, without having to perform southern blot screening. ( Figure 1F ). The attP site enables site-specific recombination in the acceptor site.

[0382] Using the established Cas9-mediated genome editing protocol, the new receptor site is stably integrated into the integration site within the genome of the target cell. Regarding the design of the receptor site, the safe harbor AAVS1 locus is used as the integration site according to the design of the receptor site. The receptor site is synthesized and cloned into the AAVS1-donor vector (GeneCopoeia) and co-transfected with Cas9:sgRNA (AAVS1) (a guide RNA (gRNA) complementary to the target AAVS1 sequence) to obtain stable integration at a single integration site (IS) (i.e., the AAVS1 locus located between exons 1 and 2 of the PPP1R12C locus on chromosome 19). Transfection and stable integration are performed using RNA-guided Cas9-CRISPR-mediated genome editing, followed by antibiotic selection using bleomycin. After clonal cell growth, 2 ligation PCRs ( Figure 1A) identify the monoallelic integration of the receptor site. Primer pairs are designed to detect the single PCR product band at the insertion site (IS). The second pair of primers amplifies the PCR product from the allele where integration occurs. The cloned cells with monoallelic integration should show amplification twice PCR. Next, in order to determine the copy number integration at the correct site, a droplet digital PCR (ddPCR) is performed using a specific probe for the mCherry gene. The probe for the housekeeping gene RPP30 with 2 copies in the human genome is used to quantify the relative copy number in the sample (Table 2).

[0383] Table 2

[0384]

[0385] In the absence of random integration, site-specific, single-copy, monoallelic integration of the optimized receptor site was obtained in HepG2, U2OS, A549, and A375 cells with efficiencies of 26%, 16%, 10%, and 14%, respectively. 43 HepG2 clones were screened by PCR and ddPCR; 11 of them had single-copy integration at the correct locus. A total of 61 U2OS clones were screened by PCR and ddPCR, and 10 of them had single integration at the AAVs1 locus. For A549 cells, a total of 20 clones were screened by PCR and ddPCR, and 2 clones had single-copy integration of the receptor site at the AAVS1 locus, corresponding to an overall recombination efficiency of 10%. For A375 cells, a total of 73 clones were screened, and 10 had single-copy integration at the AAVS1 locus. In the case of U2OS and A549, previous screening by PCR and Southern blotting failed to identify any colonies with single-copy integration of the receptor site at the correct locus. Therefore, the optimized recipient cell platform is much more efficient for the generation of recipient cells.

[0386] Five quality control steps are performed to select a complete integration site and a fully functional recipient cell line with optimal performance (Table 3). The first step is to sequence the genomic DNA at the 5' and 3' insertion sites in the AAVS1 genomic locus of the recipient cell line to confirm that the recombination process will not trigger any small insertions, deletions or mutations flanking the recombination site. The second step is to assess the homogeneity of the recipient cells to ensure that they come from a single cell - flow cytometry analysis is performed for this purpose and the coefficient of variation (CV) of the mCherry fluorophore is calculated (step 2-CV (mCherry) = 51%). In order to determine whether the recipient cells contain functional tetracycline regulation, under tetracycline regulation, the cells are co-transfected with BxB1 and a dual-color multicistronic construct containing MTS-Venus, H2B-TagBFP. The reporter cells are selected for 3 days with hygromycin B and grown for 6-10 days. The number of colonies formed is scored (step 4). After selection, the cells are treated with 1 μg / ml doxycycline for 12-15h. The next validation and QC steps were performed using flow cytometry analysis. For step 3, induced cells were compared to uninduced cells to verify the loss of mCherry and the acquisition of Venus and TagBFP fluorescence. To determine whether the variability of protein expression in the polyclonal population after reporter integration was similar to the variability of protein expression in the recipient cell line population, the CV of the fluorescence intensity of each fluorophore present in the polycistronic vector was determined (step 4-CV (TagBFP) = 67% and CV (Venus) = 69%) and compared with the parental CV. In order to verify whether the expression of different proteins placed on the receptor site is homogeneous and whether the payload is intact, we determined the correlation coefficient (r) based on the flow cytometry values ​​of the fluorescence intensity of the expressed proteins (correlation coefficient r = 0.89, step 5).

[0387] Table 3

[0388]

[0389] Only recipient cell lines that passed all checkpoints were selected. An example of the results for one recipient cell line is shown in Figures 8A-8C middle.

[0390] Example 5. Optimization of the polycistronic platform

[0391] Several variants of the polycistronic platform have been developed, including one that contains: (1) a plug-and-play recombinase site; an attB site for Bxb1-specific serine-type recombination with attP sequences in recipient cell lines and an FRT site for recombination using a tyrosine-family site-specific recombinase flippase (frt); (2) a plug-and-play promoter; (3) a plug-and-play resistance marker; and (4) three 2A peptidase sequences upstream of the IRES sequence to allow expression of up to four genes from the same promoter ( Fig.18 ). While the polycistronic platform contains a single transcription unit (TU) in which all transcripts are driven by a single promoter; the universal platform includes, in addition to the polycistronic unit described above, the option to integrate up to two of three different TUs by adding core insulator sequences (four core elements of the chicken hypersensitive site) to avoid promoter interference ( Fig. 9 , figure below).

[0392] By co-transfection with the highly efficient serine recombinase Bxb1, efficient recombination of the reporter vector can be achieved (Duportet, X. et al., NAS (2014)). Recombination is accompanied by loss of mCherry transcription, which is due to the displacement of mCherry relative to the "acceptor site" promoter.

[0393] Example 6. Multiplexed high-content assay for predictive toxicology studies to measure cellular stress mechanisms

[0394] Toxicity is a major concern in the pharmaceutical industry as well as for environmental chemicals. Both drug candidates and approved drugs face the problem of induced cytotoxicity. New high-throughput assay platforms are needed that rapidly pinpoint the mechanism of action and toxicity potential of compounds and monitor multiple aspects of toxicity. Herein, a set of multicolor cellular stress reporters stably integrated into cell lines relevant to compound toxicity assessment is described. The resulting set of multicolor stable reporter cell lines can be used as the basis for multiplexed assays of toxicity.

[0395] U2OS and HepG2 recipient cells were transfected with a vector containing the recombinase Bxb1 and a vector constitutively expressing TagBFP ("test-reporter") to determine optimal recombination conditions: transfection method; recombinase:reporter ratio; drug selection addition; cell plating number. The test-reporter vector allows rapid quantification of transfection efficiency and recombination efficiency by flow cytometric analysis (FACS).

[0396] Transfection efficiency is calculated by the number of TagBFP+ cells in the total population. Recombination efficiency is calculated by the ratio between the number of cells with site-specific integration (mCherry- and TagBFP+) and the total number of cells transfected (TagBFP+). Cells in which site-specific recombination has occurred will be TagBFP+, because TagBFP will be expressed by the CAG promoter from the test-reporter; and will be mCherry-, because the recombination of the receptor site will shift its start codon. Several conditions were tested.

[0397] The construct containing the recombinase Bxb1 and the polycistronic Tox reporter (named U2OS A :Tox ORG ) co-transfected U2OS recipient cells, the reporter carried three fluorescent marker proteins: MTS-Venus, H2B-TagBFP and mCherry-LC3 ( Fig. 10A ). After 3 days of transfection and 3 days of hygromycin treatment, surviving cells were grown for an additional 10 days without any selection prior to FACS analysis. As expected, we obtained highly homogenous expression of the three fluorescent proteins. We observed that the variability of expression in the post-integration polyclonal population, as measured by the coefficient of variation (CV), Fig. 10B mCherry: CV = 69%; TgBFP: CV = 67%, Venus: CV = 73%) was similar to the variability of expression in the monoclonal basal cell line population (mCherry: CV = 68%). We also observed that the expression of transgenes placed with the same payload was highly correlated (correlation coefficient r = 0.81, Fig. 10C ).

[0398] The construct containing the recombinase Bxb1 and the polycistronic Tox reporter (named U2OS A :Tox DUPR) were co-transfected into U2OS recipient cells, and the reporter carried three fluorescently labeled proteins: 53BP1-mCerulean, H2B-mCherry, which bind to double-strand breaks in DNA, and XBP1Δ-Venus, which is a UPR stress-related sensor. Once the cells were selected and expanded, they were assayed to determine the dose response of etoposide and neocarcinostatis (NCS). Etoposide inhibits DNA synthesis and has very high activity on cells in the late S and G2 phases of the cell cycle, inducing double-strand and single-strand breaks of DNA in intact cells. NCS inhibits DNA synthesis, G2 cycle arrest, and cell apoptosis. Both compounds are inducers of DNA damage and therefore increase the number of 53BP1 nuclear foci. The cells were plated in glass-bottomed 384-well plates. In order to evaluate the specificity of the reporter, three other compounds were used as controls together with the DNA inducer. Thapsigargin induces UPR-related stress (Ca) by inhibiting the uptake of calcium into the ER. 2+ is a chaperone cofactor, thus inhibiting protein folding), tunicamycin also participates in UPR-related stress by inhibiting newly synthesized protein glycosylation, and aphidicolin inhibits DNA replication in eukaryotes. All compounds were tested in quadruplicate at 8 hours after compound addition with four-point ten-fold serial dilutions, and data were collected. Images were collected in a Nikon Ti-E inverted microscope equipped with an electric stage with an autofocus device and an OkoLab stage-top environmental control chamber (supplied with CO2 and temperature controlled for live cell imaging) ( Fig.11A ).

[0399] The amount of 53BP1-mCerulean was used as a measure of double-strand breaks and DNA damage. For each assay, images were analyzed using the open source software CellProfiler to quantify the number of foci per nucleus, and the resulting data were further processed to determine compound EC values. 50 .

[0400] Image analysis was performed using open source software CellProfiler. After importing the image, the nucleus was identified as the main object from the H2B-mCherry image, then the mCerulean structure was enhanced, and the detection of these enhanced structures was used as the object. The nucleus was associated with the foci, and the number of progeny (foci) of each nucleus was counted. The data from CellProfiler were exported to the Python programming language for further calculations.

[0401] Using Python, the data were grouped by well and the average number of foci / nucleus was determined. Wells corresponding to the same condition were grouped. All data were exported to GraphPad Prism 7.0 software to generate graphs. Nonlinear regression was used to fit the data and calculate the concentration at which we observed 50% activity of the enhancer (EC 50 )( Fig. 11B ).

[0402] Etoposide activity through U2OS A :Tox DUPR Reporter cells to detect ( Fig.11A and 11B ), E.C. 50 The value is 161nM.

[0403] NCS activity through U2OS A :Tox DUPR Reporter cells to detect ( Fig.11A and 11B ), EC50 value was 257ng / ml.

[0404] Tunicamycin, thapsigargin, and aphidicolin did not show any effect on the number of 53BP1 foci, demonstrating that the number of 53BP1 foci is specifically associated with DNA damage.

[0405] In U2OS A :Tox DUPR 53BP1-mCerulean included in the reporter cells is a functional and specific reporter of DNA damage and double-strand breaks. DUPR Performance measurements of reporter cells confirm that the reporter and the assay are suitable for high-throughput assays ( Figures 12A-12E The Z factor (Z') of the assay was > 0.5, validating its suitability for a high-throughput quantitative assay to monitor, measure and compare compound activity, dose response and determine the EC of compounds that induce DNA damage. 50 .

[0406] U2OS A :Tox DUPR and U2OS A :Tox ORG Combined with a pool of 5 biosensors and phenotypic readouts (MTS-Venus, H2B-TagBFP, mCherry-LC3, 53BP1-cerulean, XBP1-Venus, and H2B-mCherry) in a pooled assay where DNA was differentially labeled in each of the two reporter cell lines ( Fig.13 ).

[0407] U2OS A Tox ORG and Tox DUPR The two reporter cell lines were mixed 1:1 and plated in 384-well plates 24h before adding the compounds. Two DNA damage inducers were tested: etoposide and neocarcinostatis (NCS). Etoposide inhibits DNA synthesis and has very high activity on cells in the late S and G2 phases of the cell cycle, inducing double-strand and single-strand breaks of DNA in intact cells. NCS inhibits DNA synthesis, G2 cycle arrest, and apoptosis. Both compounds are inducers of double-strand breaks of DNA and therefore increase the number of 53BP1 nuclear foci. Eight hours after adding the compounds, images were collected in a Nikon Ti-E inverted microscope equipped with a motorized stage with autofocus and an OkoLab stage top environmental control chamber (supplied with CO2 and temperature controlled for live cell imaging) ( Fig.14 ). When compared to vehicle control wells, an increase in Tox D-UPR A clear increase in the number of foci / nuclei in reporter cells. Fig. 11B , E.C. 50 NCS = 257 ng / μl) or combined with other reporter cell lines ( Fig.14 EC 50 NCS = 376 ng / μl) and obtained similar EC 50 .

[0408] Pooling assays demonstrated that mixing reporter cell lines with different reporters allowed unambiguous inferences about the mechanism of compound toxicity.

[0409] Three 3-color reporter vectors were developed that encompass three stress signaling pathways (unfolded protein response, DNA damage, oxidative stress, and p53-dependent stress response) combined with an aggregation of seven biosensors and phenotypic readouts that can be used individually or combined to fingerprint the mechanisms of compound toxicity.

[0410] An additional platform for toxic compound assessment was developed. Using the above method, a four-color reporter vector ( Fig.15 ), said phenotypic markers can be used alone or in combination to fingerprint the mechanism of compound toxicity.

[0411] Example 7. Generation of multicolor iPS cell lines

[0412] A robust targeting strategy was developed that uses RNA-guided genome engineering tools mediated by Cas9 to target the “acceptor site” ( Fig.16A ) was introduced into the endogenous AAVS1 locus of the iPSC line. The acceptor site contained: (1) an attP site for recombination by the serine recombinase Bxb1; (2) an mCherry fluorescent marker to confirm integration at the acceptor site; (3) an antibiotic resistance gene driven by the cytomegalovirus / chicken β-actin promoter (CAG) promoter to enable cell selection; and (4) a GFP gene driven by the CMV promoter downstream of the homologous recombination region to enable rapid differentiation between random and targeted integration (cells with random integration will emit green fluorescence due to GFP expression, while cells with targeted integration will not emit green fluorescence due to the loss of CMV-GFP) (see Fig. 16B ). After antibiotic selection, clones were generated from single cells and manually isolated. PCR was used to identify monoallelic integration of the receptor site at the correct locus in these clones, and then droplet digital PCR was performed to confirm single copy integration (Table 3). PCR-based integration identification is commonly used in the art, and the method is well known and conventional. As explained in Examples 1-3, this strategy was used to generate a group of human immortalized recipient cells, and to establish two iPSC recipient systems with single copy integration of the receptor site (Table 3).

[0413] Table 3.

[0414]

[0415] The present disclosure relates to the following embodiments:

[0416] 1. A multicistronic reporter vector comprising:

[0417] a promoter operably linked to an open reading frame, wherein the open reading frame comprises two or more cistrons, and wherein expression of the open reading frame in a cell produces separate component polypeptide products from each cistron;

[0418] wherein each cistron comprises a multiple cloning site (MCS) and a nucleic acid encoding a reporter vector, and wherein each cistron encodes a different reporter polypeptide;

[0419] and

[0420] The expression of the two or more nucleic acids encoding polypeptides inserted into the two or more multiple cloning sites is essentially stoichiometric.

[0421] 2. A multicistronic reporter vector according to item 1, wherein the cistrons are separated from each other by nucleic acids encoding one or more self-cleaving peptides and / or one or more internal ribosome entry sites (IRES).

[0422] 3. A multicistronic reporter vector according to item 2, wherein the one or more self-cleaving peptides are viral self-cleaving peptides.

[0423] 4. A multicistronic reporter vector according to item 3, wherein the one or more viral self-cleaving peptides are one or more 2A peptides.

[0424] 5. The multicistronic reporter vector according to item 4, wherein the one or more 2A peptides are T2A peptides, P2A peptides, E2A peptides or F2A peptides.

[0425] 6. A multicistronic reporter vector according to any one of items 2 to 5, wherein the reporter polypeptide further comprises one or more nucleic acids encoding a peptide linker between one or more of the reporter polypeptides and one or more of the self-cleaving peptides.

[0426] 7. A multicistronic reporter vector according to item 6, wherein the peptide linker comprises the sequence Gly-Ser-Gly.

[0427] 8. A multicistronic reporter vector according to any one of items 1-7, wherein the reporter polypeptide is a fluorescent reporter polypeptide.

[0428] 9. A multicistronic reporter vector according to any one of items 1-8, wherein the reporter polypeptide of each cistron is selected from GFP, EGFP, Emerald, Citrine, Venus, mOrange, mCherry, TagBFP, mTurquoise, Cerulean, UnaG, dsRed, eqFP611, Dronpa, RFP, TagRFP, TdTomato, KFP, EosFP, Dendra, IrisFP, iRFP and smURFP.

[0429] 10. A multicistronic reporter vector according to any one of items 1-9, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron and a second cistron, wherein each cistron comprises, from 5' to 3', a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a viral cleavage peptide.

[0430] 11. A multicistronic reporter vector according to any one of items 1-9, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron and a third cistron, wherein each cistron comprises from 5' to 3' a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, and the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide.

[0431] 12. A multicistronic reporter vector according to any one of items 1-9, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, a third cistron and a fourth cistron, wherein each cistron comprises from 5' to 3' a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide, and the third cistron and the fourth cistron are separated by a nucleic acid encoding a third viral cleavage peptide.

[0432] 13. A multicistronic reporter vector according to any one of items 1-9, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, a third cistron and a fourth cistron, wherein each cistron comprises from 5' to 3' a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide, and the third cistron and the fourth cistron are separated by a nucleic acid encoding an IRES.

[0433] 14. A multicistronic reporter vector according to any one of items 1 to 13, wherein the vector further comprises one or more inducible elements located between the promoter and the open reading frame.

[0434] 15. A multicistronic reporter vector according to item 14, wherein the vector comprises two inducible elements.

[0435] 16. A multicistronic reporter vector according to item 14 or 15, wherein the inducible element is a Tet operator 2 (TetO2) inducible element.

[0436] 17. A multicistronic reporter vector according to any one of items 1 to 16, wherein the promoter is a constitutive promoter.

[0437] 18. A multicistronic reporter vector according to item 17, wherein the constitutive promoter is cytomegalovirus alpha (CMV), thymidine kinase (TK), eF1-α, ubiquitin C (UbC), phosphoglycerate kinase (PGK), CAG promoter, SV40 promoter or human β-actin promoter.

[0438] 19. A multicistronic vector according to any one of items 1 to 16, wherein the promoter is an inducible promoter.

[0439] 20. The polycistronic vector according to item 19, wherein the inducible promoter is a tetracycline-responsive promoter.

[0440] 21. A multicistronic reporter vector according to any one of items 1-16, wherein the promoter is a tissue-specific promoter.

[0441] 22. A multi-specific reporter vector according to claim 21, wherein the tissue-specific promoter is specific to cells of the heart, blood, muscle, lung, liver, kidney, pancreas, brain or skin.

[0442] 23. A multicistronic reporter vector according to any one of items 1 to 22, further comprising a site-specific recombinase sequence located 3' to the open reading frame.

[0443] 24. A multicistronic reporter vector according to item 23, wherein the vector further comprises a nucleic acid encoding a selectable marker, wherein when the site-specific recombinase sequence is not recombined, the nucleic acid encoding the selectable marker is not operably linked to the promoter, and when the site-specific recombinase sequence is recombined with its target site-specific recombinase sequence, the nucleic acid encoding the selectable marker is operably linked to the promoter.

[0444] 25. The multicistronic reporter vector according to item 24, wherein the site-specific recombinase sequence is a FRT nucleic acid sequence and / or an attP nucleic acid and / or a loxP nucleic acid sequence.

[0445] 26. A multicistronic reporter vector according to item 24 or 25, wherein the selectable marker confers resistance to hygromycin, Zeocin TM , puromycin, neomycin or hygromycin, Zeocin TM , puromycin, blasticidin or neomycin analogs.

[0446] 27. A multicistronic reporter vector according to any one of items 1 to 26, wherein a nucleic acid encoding one or more polypeptides is inserted in-frame into the one or more MCSs.

[0447] 28. A multicistronic reporter vector according to item 27, wherein the one or more polypeptides comprise polypeptides that can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, synthetic lethality, cellular homeostasis, organelle homeostasis or toxic responses.

[0448] 29. A multicistronic reporter vector according to any one of items 1-28, which further comprises one, two or three transcription units, wherein the transcription unit comprises a promoter located 5' of an open reading frame comprising two or more cistrons and a nucleic acid encoding a transgene, wherein the reporter vector further comprises a core insulator sequence and a polyA sequence located 3' of the transcription unit and 5' of the open reading frame comprising two or more cistrons.

[0449] 30. A recipient cell for receiving a multicistronic reporter vector, wherein the recipient cell comprises a recombinant nucleic acid integrated into a specific site in the host cell genome, wherein the recombinant nucleic acid comprises a first promoter operably linked to a nucleic acid encoding a fusion polypeptide, wherein the fusion polypeptide comprises a reporter domain and a selectable marker domain, and wherein the nucleic acid comprises a site-specific recombinase nucleic acid sequence located at the 5' end of the nucleic acid encoding the fusion polypeptide.

[0450] 31. A recipient cell according to item 30, wherein the promoter is a constitutive promoter.

[0451] 32. A recipient cell according to item 31, wherein the constitutive promoter is a CMV promoter, a TK promoter, an eF1-α promoter, a UbC promoter, a PGK promoter, a CAG promoter, a SV40 promoter or a human β-actin promoter.

[0452] 33. A recipient cell according to item 32, wherein the promoter is an inducible promoter.

[0453] 34. A recipient cell according to item 33, wherein the inducible promoter is a tetracycline-responsive promoter.

[0454] 35. The recipient cell according to any one of items 30 to 34, wherein the site-specific recombinase sequence is a FRT nucleic acid sequence and / or an attP nucleic acid sequence and / or a loxP nucleic acid sequence.

[0455] 36. A recipient cell according to any one of items 30-35, wherein the reporter domain of the fusion polypeptide is a fluorescent reporter domain.

[0456] 37. A recipient cell according to any one of items 30-36, wherein the fluorescent reporter domain is selected from GFP, EGFP, Emerald, Citrine, Venus, mOrange, mCherry, TagBFP, mTurquoise, Cerulean, UnaG, dsRed, eqFP611, Dronpa, RFP, TagRFP, TdTomato, KFP, EosFP, Dendra, IrisFP, iRFP and smURFP.

[0457] 38. A recipient cell according to any one of items 30-37, wherein the reporter domain of the fusion polypeptide is an mCherry reporter domain.

[0458] 39. A recipient cell according to any one of items 30-38, wherein the selectable marker domain of the fusion polypeptide confers resistance to hygromycin, Zeocin TM , Puromycin, Blasticidin, Neomycin or Hygromycin, Zeocin TM , puromycin, blasticidin, and neomycin analogs.

[0459] 40. The recipient cell according to any one of items 30-39, wherein the integrated recombinant nucleic acid further comprises a nucleic acid encoding a tetracycline repressor polypeptide operably linked to a promoter.

[0460] 41. A recipient cell according to item 40, wherein the promoter is human β-actin promoter or CAG promoter.

[0461] 42. A recipient cell according to any one of items 30-41, wherein the recombinant nucleic acid is integrated into the adeno-associated virus S1 (AAVS1) locus, the chemokine (CC motif) receptor 5 (CCR5) locus, the human homolog of the mouse ROSA26 locus, the hip11 (H11) locus or the citrate lyase β-like gene locus (CLYBL).

[0462] 43. A recipient cell according to any one of items 30-42, wherein the cell is an immortal cell.

[0463] 44. A recipient cell according to any one of items 30-43, wherein the immortalized cells are HEK293T cells, A549 cells, U2OS cells, RPE cells, NPC1 cells, MCF7 cells, HepG2 cells, HaCat cells, TK6 cells, A375 cells or HeLa cells.

[0464] 45. A recipient cell according to any one of items 30-44, wherein the cell is a pluripotent cell, an induced pluripotent stem cell or a multipotent cell.

[0465] 46. ​​A recipient cell according to any one of items 30-42, wherein the cell is a primary cell.

[0466] 47. A method for producing a recipient cell for receiving a multicistronic reporter vector, the method comprising introducing a recombinant nucleic acid into the cell, wherein the recombinant nucleic acid comprises from 5' to 3'

[0467] a) a first nucleic acid for targeting homologous recombination to a specific site in said cell,

[0468] b) a first promoter,

[0469] c) a site-specific recombinase nucleic acid,

[0470] d) a nucleic acid encoding a first reporter polypeptide and a selectable marker,

[0471] e) a second nucleic acid for targeting homologous recombination to a specific site in said cell,

[0472] f) a second promoter and a nucleic acid encoding a second reporter polypeptide,

[0473] Wherein expression of the first reporter polypeptide but not expression of the second reporter polypeptide indicates targeting of integration of the recombinant nucleic acid to a specific site in the genome of the cell, and expression of the first and second reporter polypeptides indicates random integration in the genome of the cell.

[0474] 48. A method according to item 47, wherein the recombinant nucleic acid further comprises a nucleic acid encoding a tetracycline repressor operably linked to a promoter 5' of the second nucleic acid for targeting homologous recombination.

[0475] 49. The method according to item 47 or 48, wherein the recombinant nucleic acid is integrated into the genome of the cell using:

[0476] a) RNA-guided recombination system comprising a nuclease and a guide RNA

[0477] b) TALEN endonucleases, or

[0478] c) ZFN endonucleases.

[0479] 50. A method according to any one of items 47-49, wherein cells are selected that express the first reporter polypeptide but do not express the second reporter polypeptide.

[0480] 51. The method according to any one of items 47-50, wherein the site-specific recombinase nucleic acid is a FRT nucleic acid sequence and / or an attP nucleic acid sequence and / or a loxP nucleic acid sequence.

[0481] 52. A method according to any one of items 47-51, wherein the first reporter polypeptide is a fluorescent polypeptide and the second reporter polypeptide is a different fluorescent polypeptide.

[0482] 53. A method according to any one of items 47-52, wherein the first and second reporter polypeptides are selected from GFP, EGFP, Emerald, Citrine, Venus, mOrange, mCherry, TagBFP, mTurquoise, Cerulean, UnaG, dsRed, eqFP611, Dronpa, RFP, TagRFP, TdTomato, KFP, EosFP, Dendra, IrisFP, iRFP and smURFP.

[0483] 54. A method according to any one of items 47-53, wherein the first reporter polypeptide is an mCherry reporter and the second reporter polypeptide is GFP.

[0484] 55. A method according to any one of items 47-54, wherein the selectable marker confers resistance to hygromycin, Zeocin TM , Puromycin, Blasticidin, Neomycin or Hygromycin, Zeocin TM , puromycin, blasticidin, and neomycin analogs.

[0485] 56. A method according to any one of items 47-55, wherein the first promoter is a CMV promoter, a TK promoter, an eF1-α promoter, a UbC promoter, a PGK promoter, a CAG promoter, a SV40 promoter or a human β-actin promoter, and the second promoter is a CMV promoter, a TK promoter, an eF1-α promoter, a UbC promoter, a PGK promoter, a CAG promoter, a SV40 promoter or a human β-actin promoter.

[0486] 57. A method according to any one of items 47-56, wherein the first nucleic acid for targeting homologous recombination and the second nucleic acid for targeting homologous recombination target the recombination to the AAVS1 locus, the CCR5 locus, the human homolog of the mouse ROSA26 locus, the H11 locus or the CLYBL locus.

[0487] 58. A method according to any one of items 47-57, wherein the cells are immortal cells.

[0488] 59. A method according to any one of items 47-58, wherein the immortal cells are HEK293T cells, A549 cells, U2OS cells, RPE cells, NPC1 cells, MCF7 cells, HepG2 cells, HaCat cells, TK6 cells, A375 cells or HeLa cells.

[0489] 60. A method according to any one of items 47-59, wherein the cell is a pluripotent cell, an induced pluripotent stem cell or a multipotent cell.

[0490] 61. A method according to item 60, wherein the induced pluripotent stem cells are WTC-11 cells or NCRM5 cells.

[0491] 62. A method according to any one of items 47-58, wherein the cell is a primary cell.

[0492] 63. A recipient cell line produced using the method according to any one of items 47-62.

[0493] 64. A multi-reporter cell comprising a recipient cell according to any one of items 30-46, wherein the multicistronic reporter vector according to item 27 or 28 has been integrated into the genome of the recipient cell.

[0494] 65. A multi-reporter cell according to item 64, wherein the multicistronic reporter vector has been integrated into the AAVS1 locus of the recipient cell.

[0495] 66. A multi-reporter cell comprising a multicistronic reporter construct, wherein the multicistronic reporter construct comprises

[0496] a promoter operably linked to an open reading frame, wherein the open reading frame comprises two or more cistrons;

[0497] wherein each cistron comprises a nucleic acid encoding a different transgene product fused to a different reporter polypeptide, wherein expression of the open reading frame in the cell produces separate component polypeptide products from each cistron; and

[0498] wherein expression of the transgene product is stoichiometric in nature.

[0499] 67. A multi-reporter cell according to item 66, wherein the cistrons are separated from each other by nucleic acids encoding one or more self-cleaving peptides and / or one or more internal ribosome entry sites (IRES).

[0500] 68. A multi-reporter cell according to item 66 or 67, wherein each of the reporter polypeptides is a fluorescent reporter polypeptide.

[0501] 69. A multi-reporter cell according to any one of items 66-68, wherein the fluorescent reporter domain is selected from GFP, EGFP, Emerald, Citrine, Venus, mOrange, mCherry, TagBFP, mTurquoise, Cerulean, UnaG, dsRed, eqFP611, Dronpa, RFP, TagRFP, TdTomato, KFP, EosFP, Dendra, IrisFP, iRFP and smURFP.

[0502] 70. The multi-reporter cell according to any one of items 66-69, wherein the one or more self-cleaving peptides are one or more 2A peptides.

[0503] 71. A multi-reporter cell according to any one of items 66-70, wherein the nucleic acid encoding the transgene product fused to the reporter polypeptide further comprises one or more nucleic acids encoding a peptide linker between the reporter polypeptide and the viral self-cleaving peptide.

[0504] 72. A multi-reporter cell according to item 71, wherein the peptide linker comprises the sequence Gly-Ser-Gly.

[0505] 73. A multi-reporter cell according to any one of items 66-72, wherein the multi-cistronic reporter vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron, a second cistron, a third cistron and a fourth cistron, wherein each cistron comprises from 5' to 3' a nucleic acid encoding a transgenic product fused to a fluorescent reporter polypeptide and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a first viral cleavage peptide, the second cistron and the third cistron are separated by a nucleic acid encoding a second viral cleavage peptide, and the third cistron and the fourth cistron are separated by a nucleic acid encoding an IRES.

[0506] 74. A multi-reporter cell according to any one of items 66-73, wherein the multicistronic reporter vector further comprises one or more inducible elements located between the promoter and the open reading frame.

[0507] 75. A multi-reporter cell according to item 74, wherein the inducible element is a Tet operator 2 (TetO2) inducible element.

[0508] 76. A multi-reporter cell according to any one of items 66-75, wherein the promoter is a constitutive promoter.

[0509] 77. A multi-reporter cell according to item 76, wherein the constitutive promoter is a CMV promoter, a TK promoter, an eF1-α promoter, a UbC promoter, a PGK promoter, a CAG promoter, a SV40 promoter or a human β-actin promoter.

[0510] 78. A multi-reporter cell according to any one of items 66-75, wherein the promoter is a tissue-specific promoter.

[0511] 79. A multi-reporter cell according to item 78, wherein the tissue-specific promoter is specific for cells of the heart, blood, muscle, lung, liver, kidney, pancreas, brain or skin.

[0512] 80. A multi-reporter cell according to any one of items 66-75, wherein the promoter is an inducible promoter.

[0513] 81. A multi-reporter cell according to item 80, wherein the inducible promoter is a TRE promoter.

[0514] 82. A multi-reporter cell according to any one of items 66-81, wherein the one or more transgenic products comprise a polypeptide that can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, cellular homeostasis, organelle homeostasis, or a toxic response.

[0515] 83. A multi-reporter cell according to item 82, wherein the analysis is performed on a single cell.

[0516] 84. A multi-reporter cell according to any one of items 64-83, wherein the reporter polypeptide can be visualized by microscopy, high-throughput microscopy, fluorescence activated cell sorting (FACS), luminescence or using a plate reader.

[0517] 85. A multi-reporter cell according to any one of items 66-84, which further comprises one, two or three transcription units, wherein the transcription unit comprises a promoter located 5' of an open reading frame comprising two or more cistrons and a nucleic acid encoding a transgene, wherein the reporter vector further comprises a core insulator sequence located 3' of the transcription unit and 5' of the open reading frame comprising two or more cistrons.

[0518] 86. A method for producing a multi-reporter cell, the method comprising introducing the multicistronic reporter vector according to item 27 or 28 into a recipient cell according to any one of items 33-49.

[0519] 87. A method according to item 86, wherein the recombinase-associated nucleic acid sequence is a FRT nucleic acid sequence and the recipient cell contains the flp recombinase.

[0520] 88. A method according to item 87, wherein the recombinase-associated nucleic acid is attP, and the recipient cell contains Bxb1 recombinase, PhiC31 recombinase or R4 recombinase.

[0521] 89. A method according to item 87, wherein the recombinase-associated nucleic acid sequence is a loxP nucleic acid sequence, and the recipient cell contains a CRE recombinase.

[0522] 90. A library of multiple reporter vectors, wherein the library comprises a multicistronic reporter vector according to any one of items 1-29, which comprises nucleic acids encoding different transgenic products fused to a reporter polypeptide, or wherein the library comprises a plurality of reporter cells according to any one of items 64-85, wherein when introduced into the cells, two or more of the different transgenic products on each vector are expressed essentially stoichiometrically.

[0523] 91. A library according to item 90, wherein the reporter vector encodes two or more transgenes, which encode polypeptides that can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, cellular homeostasis, organelle homeostasis or toxicity responses.

[0524] 92. A library according to item 90 or 91, wherein the biological pathway is a disease-associated pathway.

[0525] 93. A library according to item 92, wherein the disease is cancer, cardiovascular disease, neurodegenerative disease or autoimmune disease.

[0526] 94. A library according to item 92 or 93, wherein the biological pathway is a pathway associated with a toxic response mechanism within the cell.

[0527] 95. A library according to item 92 or 93, wherein the biological pathway is a pathway associated with cell proliferation, cell differentiation, cell death, apoptosis, autophagy, DNA damage and repair, oxidative stress, chromatin / epigenetics, MAPK signaling, PI3K / Akt signaling, translation control, cell cycle and checkpoint control, cellular metabolism, developmental and differentiation signaling, immunology and inflammatory signaling, tyrosine kinase signaling, vesicle trafficking, cytoskeletal regulation or ubiquitin pathways.

[0528] 96. A library according to any one of items 90-95, wherein each polycistronic vector containing a transgene for analyzing a specific single biological pathway, a specific crosstalk between two or more biological pathways, synthetic lethality, a specific cellular homeostasis, a specific organelle homeostasis, a specific toxic response, or a specific tissue comprises a common transgene product fused to a different reporter polypeptide for each polycistronic reporter vector.

[0529] 97. A library according to any one of items 90-96, wherein each polycistronic vector containing a transgene for analyzing a specific single biological pathway, a specific crosstalk between two or more biological pathways, a specific cellular homeostasis, a specific organelle homeostasis, or a specific toxic response comprises a common transgene product fused to a reporter polypeptide.

[0530] 98. A library of recipient cells for receiving a multicistronic reporter vector, wherein the library comprises the recipient cells according to items 30-46.

[0531] 99. A multiple reporter cell library, wherein each cell in the library comprises a multicistronic reporter vector comprising nucleic acids encoding different transgene products fused to a reporter polypeptide, wherein when introduced into the cell, the different nucleic acids encoding different transgene products on each vector are expressed essentially stoichiometrically.

[0532] 100. The library according to item 98 or 99, wherein the library comprises different immortalized cells.

[0533] 101. A library according to item 100, wherein the library comprises one or more of HEK293T cells, A549 cells, U2OS cells, RPE cells, NPC1 cells, MCF7 cells, HepG2 cells, HaCat cells, TK6 cells, A375 cells or HeLa cells.

[0534] 102. A library according to item 98 or 99, wherein the library comprises different multipotent, pluripotent and / or progenitor cells.

[0535] 103. A library according to item 102, wherein the different multipotent or pluripotent cells comprise one or more of induced pluripotent stem cells, multipotent cells, hematopoietic cells, endothelial progenitor receptor cells, mesenchymal progenitor cells, neural progenitor cells, osteochondrogenic progenitor cells, lymphoid progenitor cells or pancreatic progenitor cells.

[0536] 104. A library according to item 98 or 99, wherein after introduction of the multicistronic reporter vector, the multi-reporter cell library of pluripotent or multipotent cells is differentiated.

[0537] 105. A library according to item 98 or 99, wherein the library comprises different primary cells.

[0538] 106. A library according to item 105, wherein the primary cells comprise one or more of cardiomyocytes, muscle cells, lung cells, liver cells, kidney cells, pancreatic cells, neurons or tumor cells.

[0539] 107. A library according to any one of items 99-106, wherein each cell in the library contains the same multicistronic reporter vector.

[0540] 108. A cell library according to any one of items 99-107, wherein the cells in the library contain different polycistronic reporter vectors.

[0541] 109. A cell library according to item 108, wherein different multicistronic reporter vectors are introduced into isogenic recipient cells.

[0542] 110. A cell library according to any one of items 99-109, wherein the reporter vector encodes one or more transgenes and the one or more polypeptides comprise polypeptides that can be used to analyze a single biological pathway, crosstalk between two or more biological pathways, cellular homeostasis or phenotypic characteristics.

[0543] 111. A library according to item 110, wherein the biological pathway is a disease-associated pathway.

[0544] 112. A library according to item 111, wherein the disease is cancer, cardiovascular disease, neurodegenerative disease or autoimmune disease.

[0545] 113. A library according to any one of item 110, wherein the biological pathway is a pathway associated with a toxic response mechanism within the cell.

[0546] 114. A library according to any one of items 110-113, wherein the biological pathway is a pathway associated with cell proliferation, cell differentiation, cell death, apoptosis, autophagy, DNA damage and repair, oxidative stress, chromatin / epigenetics, MAPK signaling, PI3K / Akt signaling, translation control, cell cycle and checkpoint control, cellular metabolism, developmental and differentiation signaling, immunology and inflammatory signaling, tyrosine kinase signaling, vesicle transport, cytoskeletal regulation or ubiquitin pathways.

[0547] 115. A library according to any one of items 99-114, wherein each polycistronic vector containing a transgene for analyzing a specific single biological pathway, a specific crosstalk between two or more biological pathways, synthetic lethality, a specific cellular homeostasis, a specific organelle homeostasis, a specific toxic response, a specific tissue or a specific phenotypic characteristic comprises a common transgene encoding a polypeptide fused to a different reporter polypeptide.

[0548] 116. A library according to any one of items 99-115, wherein each polycistronic vector containing a transgene for analyzing a specific single biological pathway, a specific crosstalk between two or more biological pathways, a specific cellular homeostasis, a specific organelle homeostasis, a specific toxic response or a specific phenotypic characteristic comprises a common transgene encoding a polypeptide fused to a reporter polypeptide.

[0549] 117. A kit comprising one or more multicistronic reporter vectors according to any one of items 1-29.

[0550] 118. A kit comprising one or more recipient cells according to any one of items 30-46.

[0551] 119. A kit comprising one or more multicistronic reporter vectors according to any one of items 1-29 and one or more recipient cells according to any one of items 30-46.

[0552] 120. A kit comprising one or more multi-reporter cells according to any one of items 64-85.

[0553] 121. A kit according to any one of items 118-120, wherein the kit comprises a library of receptor cells and / or reporter cells arranged in a multi-well plate.

[0554] 122. A kit according to item 121, wherein the cells in the multiwell plate are cryopreserved.

[0555] 123. A method for analyzing two or more polypeptides in living cells, the method comprising determining the expression of the two or more transgenes and / or the location of the two or more transgene products in a multi-reporter cell according to any one of items 64-85 or a cell comprising a multicistronic vector according to any one of items 1-29.

[0556] 124. A method according to item 123, wherein the method is used to analyze a single biological pathway, crosstalk between two or more biological pathways, synthetic lethality, cellular homeostasis, organelle homeostasis or a toxic response.

[0557] 125. A method according to item 123 or 124, wherein the expression of the two or more transgenes and / or the location of the two or more transgene products is determined at one or more time points.

[0558] 126. A method according to claim 125, wherein the expression of the two or more transgenes and / or the location of the two or more transgene products is determined at one or more time points of 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 4 days, 7 days, 14 days, 21 days, 30 days, 1 month, 3 months, 6 months, 9 months, 1 year or more.

[0559] 127. A method according to any one of items 123-126, wherein the cell is prepared by a method according to any one of items 86-89.

[0560] 128. A method according to any one of items 123-126, wherein the cells are a cell library according to items 90-116.

[0561] 129. A method according to claim 128, wherein the cell is derived from an isogenic recipient cell.

[0562] 130. A method according to item 128 or 129, wherein the cells or the libraries are pooled prior to analysis.

[0563] 131. A method for measuring the effect of an agent on the spectrum of two or more polypeptides in a living cell, the method comprising subjecting a multi-reporter cell according to any one of items 64-85 to the agent and determining the expression of the two or more transgenes and / or the location of the two or more transgene products in the cell in response to the agent.

[0564] 132. A method according to claim 131, wherein the agent is a drug or a drug candidate.

[0565] 133. A method according to item 131 or 132, wherein the method is a toxicology screening.

[0566] 134. A method according to any one of items 131-133, wherein the expression of the two or more transgenes and / or the location of the two or more transgene products are determined in a multi-reporter cell library.

[0567] 135. A method according to any one of items 131-134, wherein the spectrum is obtained using a single cell.

[0568] 136. A method according to any one of items 131-135, wherein the expression of the two or more transgenes and / or the location of the two or more transgene products is measured by microscopy, high-throughput microscopy, fluorescence-activated cell sorting (FACS), luminescence, use of a plate reader, mass spectrometry or deep sequencing.

Claims

1. A multicistronic reporter vector comprising: a promoter operably linked to an open reading frame, wherein the open reading frame comprises two or more cistrons, and wherein expression of the open reading frame in a cell produces separate component polypeptide products from each cistron; wherein each cistron comprises a multiple cloning site (MCS) and a nucleic acid encoding a reporter vector, and wherein each cistron encodes a different reporter polypeptide; and The expression of the two or more nucleic acids encoding polypeptides inserted into the two or more multiple cloning sites is essentially stoichiometric.

2. The multicistronic reporter vector according to claim 1, wherein the cistrons are separated from each other by nucleic acids encoding one or more self-cleaving peptides and / or one or more internal ribosome entry sites (IRES).

3. The multicistronic reporter vector of claim 2, wherein the one or more self-cleaving peptides are viral self-cleaving peptides.

4. The multicistronic reporter vector of claim 3, wherein the one or more viral self-cleaving peptides are one or more 2A peptides.

5. The multicistronic reporter vector of claim 4, wherein the one or more 2A peptides are T2A peptides, P2A peptides, E2A peptides or F2A peptides.

6. The multicistronic reporter vector according to any one of claims 2 to 5, wherein the reporter polypeptide further comprises one or more nucleic acids encoding a peptide linker between one or more of the reporter polypeptides and one or more of the self-cleaving peptides.

7. The multicistronic reporter vector of claim 6, wherein the peptide linker comprises the sequence Gly-Ser-Gly.

8. The multicistronic reporter vector according to any one of claims 1 to 7, wherein the reporter polypeptide is a fluorescent reporter polypeptide.

9. The multicistronic reporter vector according to any one of claims 1 to 8, wherein the reporter polypeptide of each cistron is selected from GFP, EGFP, Emerald, Citrine, Venus, mOrange, mCherry, TagBFP, mTurquoise, Cerulean, UnaG, dsRed, eqFP611, Dronpa, RFP, TagRFP, TdTomato, KFP, EosFP, Dendra, IrisFP, iRFP and smURFP.

10. A multicistronic reporter vector according to any one of claims 1-9, wherein the vector comprises a promoter operably linked to an open reading frame, wherein the open reading frame comprises a first cistron and a second cistron, wherein each cistron comprises, from 5' to 3', a nucleic acid containing an MCS, a nucleic acid encoding a reporter polypeptide, and a nucleic acid encoding a linker peptide; wherein the first cistron and the second cistron are separated by a nucleic acid encoding a viral cleavage peptide.

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