Stem cell lines containing endogenous marked proteins with differential expression, production methods and their use for cell cycle identification

Abstract

The genic construct comprises: (i) at least a first gene (FUCCIplex) for or a partial fragment of a gene for at least a first expression product the quantity of which in a cell varies in a cell cycle-dependent manner; and at least one of: (ii) at least a second gene for or a partial fragment of a gene for at least a second expression product the quantity of which in a cell varies in a cell structure-dependent manner; (iii) at least a third gene for or a partial fragment of a gene for at least a third expression product the quantity of which in a cell varies in a cell function- dependent manner.

Description

[0001] STEM CELL LINES CONTAINING ENDOGENOUS MARKED PROTEINS WITH DIFFERENTIAL EXPRESSION, PRODUCTION METHODS AND THEIR USE FOR CELL CYCLE IDENTIFICATION

[0002] Technical Field

[0003] The present invention relates to a stem cell line containing endogenous marked proteins with differential expression, production methods and use thereof Background Art

[0004] Pharmaceutical research and development have very low capital efficiency: in fact, as is well known marketing a drug implies large monetary investments and takes up to 10-15 years.

[0005] A typical research and development pipeline starts with 1,000s to 1,000,000s of chemical compounds and goes through several preclinical stages: high- throughput screening (HTS) on simplified in-vitro models to isolate a dozen or so promising compounds and higher-specificity experiments on animal models to test the safety and efficiency of these compounds. Typically, fewer than five compounds are used for human clinical trials and the vast majority of these fails. This is dramatic not only for the company but also for society. Indeed, once on the market, the new drug will be offered at a price capable of covering not only the research costs associated with its development, but also the development of the many failed assays.

[0006] The main factor in the low efficiency of R&D in the pharmaceutical sector is the poor predictivity of the pre-clinical models: traditional in-vitro models do not capture enough of the complexity of human pathophysiology, while animal models simply express genes and proteins that are often different from ours.

[0007] The advent of human pluripotent stem cells (hPSCs) and of protocols for their differentiation into specific cell types has revolutionized the final steps of the preclinical stage by allowing them to complement and / or replace animal models in supporting the crucial decision to advance a potential therapeutic agent to clinical trials.

[0008] In addition, the ability to directly use cells as therapeutic agents in cellular and regenerative medicine or to use patient-derived hPSCs to study personalized gene therapies have increased the palette of available therapeutic options.

[0009] To demonstrate the effectiveness of these new technologies, the U.S. Congress recently decided to authorize the use thereof in the new drug approval process with the FDA modernization act (https: / / www.congress.gov / bill / 117th- congress / senate-bill / 5002).

[0010] While the use of hPSCs in the latest preclinical analyses can prevent unnecessary human and financial costs in clinical trials destined to fail, their use in the HTS phases is still very complicated due to the low scalability of their cost and complexity.

[0011] In fact, generally the typical HTS assay is offered at ~0.1 € / datapoint but culture procedures for hPSCs have much higher costs because they comprise a maintenance phase in pluripotency and a differentiation phase towards the cellular phenotype, e.g., hPSC-derived cardiac muscle cells (hPSC-CM). In aggregate, these costs levitate the market price for HTS with hPSC at least 10- 100 times higher. However, when going from 10,000 compounds to 10 in traditional HTS, potentially safe and effective compounds in humans can be discarded because they show signs of unsafety or ineffectiveness in pre-clinical models that we know (after the FDA modernization act) to be poorly predictive. In other words, a drug to prevent heart attack (1 in 2 men, 1 in 3 women) may already have been discarded simply because it did not give the right signals when tested on rodent or human cells of other tissue, since the only human heart muscle cells can be derived from hPSCs. As an illustration of the importance of this fact, mention may be made of the public -private partnership CiPA initiative, which is trying to introduce computationally accurate models of human pathophysiology to “interpret” pro-arrhythmic signals measured using low-predictive HTS (https : / / cipaproj ect. org / ) .

[0012] Description of the Invention

[0013] The main aim of the present invention is to make stem cell lines containing endogenous marked proteins with differential expression.

[0014] Another object of the invention is to use computational models to interpret potential errors in HTS. In detail, the invention in accordance with the present invention allows HTS to be made more predictive by introducing hPSCs in a convenient maimer.

[0015] A further object of the present invention is to genetically engineer a line of hPSCs so that they express fluorescent proteins which are sensitive to a variety of pathophysiological signals. In this way, the cells would independently manufacture the reagent and the fixed component of the marginal cost for imHTS with hPSCs would disappear.

[0016] In other words, given a sufficiently high volume of sales, it is presumable to absorb the higher cost of cells, making the cost of reagents disappear.

[0017] This is because the cells directly express the necessary sensors, hereafter referred to as “instrumented hPSCs” or hPSCsins. Moreover, by carefully choosing the sensors to be implemented in hPSCsins, innovative analyses can be unlocked.

[0018] For example, the combination of sensors, hereinafter referred to as TEMPO makes possible new and important analyses in the context of drug screening and regenerative medicine (hereinafter referred to as hPSCsins = hPSCsTEMPO). Brief Description of the Drawings

[0019] Other characteristics and advantages of the present invention will become more apparent from the description of a preferred, but not exclusive, embodiment of stem cell lines containing endogenous marked proteins with differential expression, production methods and use thereof for cell cycle identification, illustrated by way of an indicative, yet non-limiting example in the attached tables of drawings in which:

[0020] Figure 1 is a schematic representation of the FUCCIplex sensor in accordance with the present invention;

[0021] Figures 2 and 3 are schematic representations of the gene construct in accordance with the present invention;

[0022] Figure 4 is representative of a functional analysis of the FUCCIplex gene construct in accordance with the present invention;

[0023] Figure 5 is representative of a computational analysis aimed at eliminating the overlap in the emission spectra between the green and cyan channels;

[0024] Figure 6 is representative of a segmentation analysis of cell compartments using the gene construct in accordance with the present invention;

[0025] Figures 7 and 8 are representative of expression analysis of the gene construct in accordance with the present invention.

[0026] Embodiments of the Invention

[0027] In a first aspect, the invention according to the invention relates to a gene construct, comprising:

[0028] (i) at least a first gene (FUCCIplex) for or a partial fragment of a gene for at least a first expression product the quantity of which in a cell varies in a cell cycle-dependent maimer; and at least one of:

[0029] (ii) at least a second gene for or a partial fragment of a gene for at least a second expression product the quantity of which in a cell varies in a cell structure-dependent manner;

[0030] (iii) at least a third gene for or a partial fragment of a gene for at least a third expression product the quantity of which in a cell varies in a cell functiondependent manner.

[0031] Preferably, the first expression product is at least one of: a cyan fluorescent protein (CFP) or an infrared fluorescent protein (iRFP).

[0032] Advantageously, the second expression product is at least one of: a fluorescent protein (RFP-LifeAct) the quantity of which in a cell varies depending on the presence of actin, a fluorescent protein the quantity of which in a cell varies depending on the presence of tubulin.

[0033] In addition, the third expression product is a fluorescent protein (GCaMP6f) the quantity of which in a cell varies depending on the presence of intracellular calcium.

[0034] In addition, the construct in accordance with the present invention comprises at least one fluorescent marker (FLUO-4) adapted to detect the presence of intracellular calcium.

[0035] Furthermore, in a second aspect, the invention relates to a method for performing the phase identification of a cell cycle, the method comprising the phases of: visualizing, using at least a first marker, at least one or more gene expression products the quantity of which in a cell vary in a cell cycle-dependent manner; and detecting at least the first marker so as to distinguish a proliferation phase of the cell cycle from a resting phase of the cell cycle.

[0036] The method according to the invention comprises visualizing, using at least a second marker, at least one or more gene expression products the quantity of which in a cell vary in a cell structure-dependent maimer; and detecting at least the second marker so as to distinguish actin microfilaments.

[0037] Still, the method according to the invention comprises visualizing, by using at least a third marker, at least one or more gene expression products the quantity of which in a cell vary in a cell function-dependent manner; and detecting at least the third marker so as to distinguish intracellular calcium.

[0038] Furthermore, in accordance with a third aspect, the invention relates to a cell, comprising an exogenous polynucleotide integrated into a target genomic locus, the exogenous polynucleotide comprising polynucleotide sequences encoding:

[0039] (a) at least a first selectable marker;

[0040] (b) at least a regulatory building element which is operationally connected to the first selectable marker;

[0041] (d) at least a second selectable marker which is different from the first selectable marker, where the target genomic locus is a locus of a gene that encodes for a differentially expressed protein.

[0042] Finally, in accordance with a fourth aspect, the invention relates to a lentiviral expression vector comprising the previously described gene construct.

[0043] Preferably, such a vector consists of the sequence SEQ. ID. No: 1.

[0044] Such a vector is a lentiviral vector having the following nucleotide sequence:

[0045] SEQ. ID. No: 1

[0046] AATGTAGTCT TATGCAATAC TCTTGTAGTC TTGCAACATG

[0047] GTAACGATGA GTTAGCAACA TGCCTTACAA GGAGAGAAAA

[0048] AGCACCGTGC ATGCCGATTG GTGGAAGTAA GGTGGTACGA

[0049] TCGTGCCTTA TTAGGAAGGC AACAGACGGG TCTGACATGG

[0050] ATTGGACGAA CCACTGAATT GCCGCATTGC AGAGATATTG TATTTAAGTG CCTAGCTCGA TACATAAACG GGTCTCTCTG

[0051] GTTAGACCAG ATCTGAGCCT GGGAGCTCTC TGGCTAACTA

[0052] GGGAACCCAC TGCTTAAGCC TCAATAAAGC TTGCCTTGAG

[0053] TGCTTCAAGT AGTGTGTGCC CGTCTGTTGT GTGACTCTGG

[0054] TAACTAGAGA TCCCTCAGAC CCTTTTAGTC AGTGTGGAAA

[0055] ATCTCTAGCA GTGGCGCCCG AACAGGGACT TGAAAGCGAA

[0056] AGGGAAACCA GAGGAGCTCT CTCGACGCAG GACTCGGCTT

[0057] GCTGAAGCGC GCACGGCAAG AGGCGAGGGG CGGCGACTGG

[0058] TGAGTACGCC AAAAATTTTG ACTAGCGGAG GCTAGAAGGA

[0059] GAGAGATGGG TGCGAGAGCG TCAGTATTAA GCGGGGGAGA

[0060] ATTAGATCGC GATGGGAAAA AATTCGGTTA AGGCCAGGGG

[0061] GAAAGAAAAA ATATAAATTA AAACATATAG TATGGGCAAG

[0062] CAGGGAGCTA GAACGATTCG CAGTTAATCC TGGCCTGTTA

[0063] GAAACATCAG AAGGCTGTAG ACAAATACTG GGACAGCTAC

[0064] AACCATCCCT TCAGACAGGA TCAGAAGAAC TTAGATCATT

[0065] ATATAATACA GTAGCAACCC TCTATTGTGT GCATCAAAGG

[0066] ATAGAGATAA AAGACACCAA GGAAGCTTTA GACAAGATAG

[0067] AGGAAGAGCA AAACAAAAGT AAGACCACCG CACAGCAAGC

[0068] GGCCGCTGAT CTTCAGACCT GGAGGAGGAG ATATGAGGGA

[0069] CAATTGGAGA AGTGAATTAT ATAAATATAA AGTAGTAAAA

[0070] ATTGAACCAT TAGGAGTAGC ACCCACCAAG GCAAAGAGAA

[0071] GAGTGGTGCA GAGAGAAAAA AGAGCAGTGG GAATAGGAGC

[0072] TTTGTTCCTT GGGTTCTTGG GAGCAGCAGG AAGCACTATG

[0073] GGCGCAGCGT CAATGACGCT GACGGTACAG GCCAGACAAT

[0074] TATTGTCTGG TATAGTGCAG CAGCAGAACA ATTTGCTGAG

[0075] GGCTATTGAG GCGCAACAGC ATCTGTTGCA ACTCACAGTC

[0076] TGGGGCATCA AGCAGCTCCA GGCAAGAATC CTGGCTGTGG

[0077] AAAGATACCT AAAGGATCAA CAGCTCCTGG GGATTTGGGG

[0078] TTGCTCTGGA AAACTCATTT GCACCACTGC TGTGCCTTGG

[0079] AATGCTAGTT GGAGTAATAA ATCTCTGGAA CAGATTTGGA

[0080] ATCACACGAC CTGGATGGAG TGGGACAGAG AAATTAACAA TTACACAAGC TTAATACACT CCTTAATTGA AGAATCGCAA

[0081] AACCAGCAAG AAAAGAATGA ACAAGAATTA TTGGAATTAG

[0082] ATAAATGGGC AAGTTTGTGG AATTGGTTTA ACATAACAAA

[0083] TTGGCTGTGG TATATAAAAT TATTCATAAT GATAGTAGGA

[0084] GGCTTGGTAG GTTTAAGAAT AGTTTTTGCT GTACTTTCTA

[0085] TAGTGAATAG AGTTAGGCAG GGATATTCAC CATTATCGTT

[0086] TCAGACCCAC CTCCCAACCC CGAGGGGACC CGACAGGCCC

[0087] GAAGGAATAG AAGAAGAAGG TGGAGAGAGA GACAGAGACA

[0088] GATCCATTCG ATTAGTGAAC GGATCTCGAC GGTATCGCTA

[0089] GCTTTTAAAA GAAAAGGGGG GATTGGGGGG TACAGTGCAG

[0090] GGGAAAGAAT AGTAGACATA ATAGCAACAG ACATACAAAC

[0091] TAAAGAATTA CAAAAACAAA TTACAAAAAT TCAAAATTTT

[0092] ACTAGTGATT ATCGGATCAA CTTTGTATAG AAAAGTTGGG

[0093] CTCCGGTGCC CGTCAGTGGG CAGAGCGCAC ATCGCCCACA

[0094] GTCCCCGAGA AGTTGGGGGG AGGGGTCGGC AATTGAACCG

[0095] GTGCCTAGAG AAGGTGGCGC GGGGTAAACT GGGAAAGTGA

[0096] TGTCGTGTAC TGGCTCCGCC TTTTTCCCGA GGGTGGGGGA

[0097] GAACCGTATA TAAGTGCAGT AGTCGCCGTG AACGTTCTTT

[0098] TTCGCAACGG GTTTGCCGCC AGAACACAGG TAAGTGCCGT

[0099] GTGTGGTTCC CGCGGGCCTG GCCTCTTTAC GGGTTATGGC

[0100] CCTTGCGTGC CTTGAATTAC TTCCACCTGG CTGCAGTACG

[0101] TGATTCTTGA TCCCGAGCTT CGGGTTGGAA GTGGGTGGGA

[0102] GAGTTCGAGG CCTTGCGCTT AAGGAGCCCC TTCGCCTCGT

[0103] GCTTGAGTTG AGGCCTGGCC TGGGCGCTGG GGCCGCCGCG

[0104] TGCGAATCTG GTGGCACCTT CGCGCCTGTC TCGCTGCTTT

[0105] CGATAAGTCT CTAGCCATTT AAAATTTTTG ATGACCTGCT

[0106] GCGACGCTTT TTTTCTGGCA AGATAGTCTT GTAAATGCGG

[0107] GCCAAGATCT GCACACTGGT ATTTCGGTTT TTGGGGCCGC

[0108] GGGCGGCGAC GGGGCCCGTG CGTCCCAGCG CACATGTTCG

[0109] GCGAGGCGGG GCCTGCGAGC GCGGCCACCG AGAATCGGAC

[0110] GGGGGTAGTC TCAAGCTGGC CGGCCTGCTC TGGTGCCTGG TCTCGCGCCG CCGTGTATCG CCCCGCCCTG GGCGGCAAGG

[0111] CTGGCCCGGT CGGCACCAGT TGCGTGAGCG GAAAGATGGC

[0112] CGCTTCCCGG CCCTGCTGCA GGGAGCTCAA AATGGAGGAC

[0113] GCGGCGCTCG GGAGAGCGGG CGGGTGAGTC ACCCACACAA

[0114] AGGAAAAGGG CCTTTCCGTC CTCAGCCGTC GCTTCATGTG

[0115] ACTCCACGGA GTACCGGGCG CCGTCCAGGC ACCTCGATTA

[0116] GTTCTCGAGC TTTTGGAGTA CGTCGTCTTT AGGTTGGGGG

[0117] GAGGGGTTTT ATGCGATGGA GTTTCCCCAC ACTGAGTGGG

[0118] TGGAGACTGA AGTTAGGCCA GCTTGGCACT TGATGTAATT

[0119] CTCCTTGGAA TTTGCCCTTT TTGAGTTTGG ATCTTGGTTC

[0120] ATTCTCAAGC CTCAGACAGT GGTTCAAAGT TTTTTTCTTC

[0121] CATTTCAGGT GTCGTGACAA GTTTGTACAA AAAAGCAGGC

[0122] TGCCACCATG GTGAGCAAGG GCGAGGAGCT GTTCACCGGG

[0123] GTGGTGCCCA TCCTGGTCGA GCTGGACGGC GACGTAAACG

[0124] GCCACAAGTT CAGCGTGTCC GGCGAGGGCG AGGGCGATGC

[0125] CACCTACGGC AAGCTGACCC TGAAGTTCAT CTGCACCACC

[0126] GGCAAGCTGC CCGTGCCCTG GCCCACCCTC GTGACCACCC

[0127] TGTCCTGGGG CGTGCAGTGC TTCGCCCGCT ACCCCGACCA

[0128] CATGAAGCAG CACGACTTCT TCAAGTCCGC CATGCCCGAA

[0129] GGCTACGTCC AGGAGCGCAC CATCTTCTTC AAGGACGACG

[0130] GCAACTACAA GACCCGCGCC GAGGTGAAGT TCGAGGGCGA

[0131] CACCCTGGTG AACCGCATCG AGCTGAAGGG CATCGACTTC

[0132] AAGGAGGACG GCAACATCCT GGGGCACAAG CTGGAGTACA

[0133] ACTACTTTAG CGACAACGTC TATATCACCG CCGACAAGCA

[0134] GAAGAACGGC ATCAAGGCCA ACTTCAAGAT CCGCCACAAC

[0135] ATCGAGGACG GCGGCGTGCA GCTCGCCGAC CACTACCAGC

[0136] AGAACACCCC CATCGGCGAC GGCCCCGTGC TGCTGCCCGA

[0137] CAACCACTAC CTGAGCACCC AGTCCAAGCT GAGCAAAGAC

[0138] CCCAACGAGA AGCGCGATCA CATGGTCCTG CTGGAGTTCG TGACCGCCGC CGGGATCACT CTCGGCATGG ACGAGCTGTA CAA TACAGC ATGCTGCCTA GCCTCGGCGG CTACCCTAGC CATTGGAGGC CCCTGGAACC CTCTCCTGCC AGACCTGCCC TGAGAGCCCC

[0139] TGCCTCCGCC ACCTCCGGCA GCAGAAAGAG AGCCAGACCT

[0140] CCTGCCGCCC CTGGCAGAGA TCAGGCCAGG CCCCCTGCCC

[0141] GCAGACGGCT CCGGCTGTCC GTGGATGAGG TGTCCAGCCC

[0142] TAGCACCCCC GAGGCCCCCG ATATCCCTGC CTGTCCCAGC

[0143] CCTGGCCAGA AAATCAAGAA GAGCACACCT GCCGCCGGAC

[0144] AGCCTCCTCA CCTGACCAGC GCCCAGGACC AGGACACCAT

[0145] CGTCGACGAG GGCAGGGGCA GCCTGCTGAC CTGCGGCGAC

[0146] GTGGAGGAGA ACCCCGGCCC CAGATCCATG GTAGCAGGTC

[0147] ATGCCTCTGG CAGCCCCGCA TTCGGGACCG CCTCTCATTC

[0148] GAATTGCGAA CATGAAGAGA TCCACCTCGC CGGCTCGATC

[0149] CAGCCGCATG GCGCGCTTCT GGTCGTCAGC GAACATGATC

[0150] ATCGCGTCAT CCAGGCCAGC GCCAACGCCG CGGAATTTCT

[0151] GAATCTCGGA AGCGTACTCG GCGTTCCGCT CGCCGAGATC

[0152] GACGGCGATC TGTTGATCAA GATCCTGCCG CATCTCGATC

[0153] CCACCGCCGA AGGCATGCCG GTCGCGGTGC GCTGCCGGAT

[0154] CGGCAATCCC TCTACGGAGT ACTGCGGTCT GATGCATCGG

[0155] CCTCCGGAAG GCGGGCTGAT CATCGAACTC GAACGTGCCG

[0156] GCCCGTCGAT CGATCTGTCA GGCACGCTGG CGCCGGCGCT

[0157] GGAGCGGATC CGCACGGCGG GTTCACTGCG CGCGCTGTGC

[0158] GATGACACCG TGCTGCTGTT TCAGCAGTGC ACCGGCTACG

[0159] ACCGGGTGAT GGTGTATCGT TTCGATGAGC AAGGCCACGG

[0160] CCTGGTATTC TCCGAGTGCC ATGTGCCTGG GCTCGAATCC

[0161] TATTTCGGCA ACCGCTATCC GTCGTCGACT GTCCCGCAGA

[0162] TGGCGCGGCA GCTGTACGTG CGGCAGCGCG TCCGCGTGCT

[0163] GGTCGACGTC ACCTATCAGC CGGTGCCGCT GGAGCCGCGG

[0164] CTGTCGCCGC TGACCGGGCG CGATCTCGAC ATGTCGGGCT

[0165] GCTTCCTGCG CTCGATGTCG CCGTGCCATC TGCAGTTCCT

[0166] GAAGGACATG GGCGTGCGCG CCACCCTGGC GGTGTCGCTG

[0167] GTGGTCGGCG GCAAGCTGTG GGGCCTGGTT GTCTGTCACC

[0168] ATTATCTGCC GCGCTTCATC CGTTTCGAGC TGCGGGCGAT CTGCAAACGG CTCGCCGAAA GGATCGCGAC GCGGATCACC

[0169] GCGCTTGAGA G GGCTACCC CAGCCACTGG CGGCCTCTGG

[0170] AAATGAACCC CAGCATGAAG CAGAAGCAGG AAGAGATCAA

[0171] AGAGAACATC AAGAACAGCA GCGTGCCCAG GCGGACCCTG

[0172] AAGATGATCC AGCCCAGCGC CAGCGGATCT CTGGTGGGCC

[0173] GGGAGAATGA GCTGTCTGCC GGCCTGAGCA AGCGGAAGCA

[0174] CCGGAACGAC CACCTGACCA GCACCACCTC TAGCCCAGGC

[0175] GTGATCGTGC CCGAGAGCAG CGAGAACAAG AACCTGGGCG

[0176] GCGTGACCCA AGAAAGCTTC GACCTGATGA TCAAAGAAAA

[0177] CCCCAGCAGC CAGTACTGGA AAGAGGTGGC CGAGAAGCGG

[0178] CGGAAGGCCC TGTAAACCCA GCTTTCTTGT ACAAAGTGGT

[0179] GATAATCGAA TTCCGATAAT CAACCTCTGG ATTACAAAAT

[0180] TTGTGAAAGA TTGACTGGTA TTCTTAACTA TGTTGCTCCT

[0181] TTTACGCTAT GTGGATACGC TGCTTTAATG CCTTTGTATC

[0182] ATGCTATTGC TTCCCGTATG GCTTTCATTT TCTCCTCCTT

[0183] GTATAAATCC TGGTTGCTGT CTCTTTATGA GGAGTTGTGG

[0184] CCCGTTGTCA GGCAACGTGG CGTGGTGTGC ACTGTGTTTG

[0185] CTGACGCAAC CCCCACTGGT TGGGGCATTG CCACCACCTG

[0186] TCAGCTCCTT TCCGGGACTT TCGCTTTCCC CCTCCCTATT

[0187] GCCACGGCGG AACTCATCGC CGCCTGCCTT GCCCGCTGCT

[0188] GGACAGGGGC TCGGCTGTTG GGCACTGACA ATTCCGTGGT

[0189] GTTGTCGGGG AAGCTGACGT CCTTTCCATG GCTGCTCGCC

[0190] TGTGTTGCCA CCTGGATTCT GCGCGGGACG TCCTTCTGCT

[0191] ACGTCCCTTC GGCCCTCAAT CCAGCGGACC TTCCTTCCCG

[0192] CGGCCTGCTG CCGGCTCTGC GGCCTCTTCC GCGTCTTCGC

[0193] CTTCGCCCTC AGACGAGTCG GATCTCCCTT TGGGCCGCCT

[0194] CCCCGCATCG GGAATTCCCG CGGTTCGAAT TCTACCGGGT

[0195] AGGGGAGGCG CTTTTCCCAA GGCAGTCTGG AGCATGCGCT

[0196] TTAGCAGCCC CGCTGGGCAC TTGGCGCTAC ACAAGTGGCC

[0197] TCTGGCCTCG CACACATTCC ACATCCACCG GTAGGCGCCA

[0198] ACCGGCTCCG TTCTTTGGTG GCCCCTTCGC GCCACCTTCT ACTCCTCCCC TAGTCAGGAA GTTCCCCCCC GCCCCGCAGC

[0199] TCGCGTCGTG CAGGACGTGA CAAATGGAAG TAGCACGTCT

[0200] CACTAGTCTC GTGCAGATGG ACAGCACCGC TGAGCAATGG

[0201] AAGCGGGTAG GCCTTTGGGG CAGCGGCCAA TAGCAGCTTT

[0202] GCTCCTTCGC TTTCTGGGCT CAGAGGCTGG GAAGGGGTGG

[0203] GTCCGGGGGC GGGCTCAGGG GCGGGCTCAG GGGCGGGGCG

[0204] GGCGCCCGAA GGTCCTCCGG AGGCCCGGCA TTCTGCACGC

[0205] TTCAAAAGCG CACGTCTGCC GCGCTGTTCT CCTCTTCCTC

[0206] ATCTCCGGGC CTTTCGACCT CACGTGGCCA CCATGAAAAA

[0207] GCCTGAACTC ACCGCGACGT CTGTCGAGAA GTTTCTGATC

[0208] GAAAAGTTCG ACAGCGTCTC CGACCTGATG CAGCTCTCGG

[0209] AGGGCGAAGA ATCTCGTGCT TTCAGCTTCG ATGTAGGAGG

[0210] GCGTGGATAT GTCCTGCGGG TAAATAGCTG CGCCGATGGT

[0211] TTCTACAAAG ATCGTTATGT TTATCGGCAC TTTGCATCGG

[0212] CCGCGCTCCC GATTCCGGAA GTGCTTGACA TTGGGGAATT

[0213] TAGCGAGAGC CTGACCTATT GCATCTCCCG CCGTGCACAG

[0214] GGTGTCACGT TGCAAGACCT GCCTGAAACC GAACTGCCCG

[0215] CTGTTCTGCA GCCGGTCGCG GAGGCCATGG ATGCGATCGC

[0216] TGCGGCCGAT CTTAGCCAGA CGAGCGGGTT CGGCCCATTC

[0217] GGACCGCAAG GAATCGGTCA ATACACTACA TGGCGTGATT

[0218] TCATATGCGC GATTGCTGAT CCCCATGTGT ATCACTGGCA

[0219] AACTGTGATG GACGACACCG TCAGTGCGTC CGTCGCGCAG

[0220] GCTCTCGATG AGCTGATGCT TTGGGCCGAG GACTGCCCCG

[0221] AAGTCCGGCA CCTCGTGCAC GCGGATTTCG GCTCCAACAA

[0222] TGTCCTGACG GACAATGGCC GCATAACAGC GGTCATTGAC

[0223] TGGAGCGAGG CGATGTTCGG GGATTCCCAA TACGAGGTCG

[0224] CCAACATCTT CTTCTGGAGG CCGTGGTTGG CTTGTATGGA

[0225] GCAGCAGACG CGCTACTTCG AGCGGAGGCA TCCGGAGCTT

[0226] GCAGGATCGC CGCGGCTCCG GGCGTATATG CTCCGCATTG

[0227] GTCTTGACCA ACTCTATCAG AGCTTGGTTG ACGGCAATTT

[0228] CGATGATGCA GCTTGGGCGC AGGGTCGATG CGACGCAATC GTCCGATCCG GAGCCGGGAC TGTCGGGCGT ACACAAATCG

[0229] CCCGCAGAAG CGCGGCCGTC TGGACCGATG GCTGTGTAGA

[0230] AGTACTCGCC GATAGTGGAA ACCGACGCCC CAGCACTCGT

[0231] CCGAGGGCAA AGGAATAGGG TACCTTTAAG ACCAATGACT

[0232] TACAAGGCAG CTGTAGATCT TAGCCACTTT TTAAAAGAAA

[0233] AGGGGGGACT GGAAGGGCTA ATTCACTCCC AACGAAGACA

[0234] AGATCTGCTT TTTGCTTGTA CTGGGTCTCT CTGGTTAGAC

[0235] CAGATCTGAG CCTGGGAGCT CTCTGGCTAA CTAGGGAACC

[0236] CACTGCTTAA GCCTCAATAA AGCTTGCCTT GAGTGCTTCA

[0237] AGTAGTGTGT GCCCGTCTGT TGTGTGACTC TGGTAACTAG

[0238] AGATCCCTCA GACCCTTTTA GTCAGTGTGG AAAATCTCTA

[0239] GCAGTAGTAG TTCATGTCAT CTTATTATTC AGTATTTATA

[0240] ACTTGCAAAG AAATGAATAT CAGAGAGTGA GAGGAACTTG

[0241] TTTATTGCAG CTTATAATGG TTACAAATAA AGCAATAGCA

[0242] TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC

[0243] TAGTTGTGGT TTGTCCAAAC TCATCAATGT ATCTTATCAT

[0244] GTCTGGCTCT AGCTATCCCG CCCCTAACTC CGCCCATCCC

[0245] GCCCCTAACT CCGCCCAGTT CCGCCCATTC TCCGCCCCAT

[0246] GGCTGACTAA TTTTTTTTAT TTATGCAGAG GCCGAGGCCG

[0247] CCTCGGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT

[0248] TTTTGGAGGC CTAGGGACGT ACCCAATTCG CCCTATAGTG

[0249] AGTCGTATTA CGCGCGCTCA CTGGCCGTCG TTTTACAACG

[0250] TCGTGACTGG GAAAACCCTG GCGTTACCCA ACTTAATCGC

[0251] CTTGCAGCAC ATCCCCCTTT CGCCAGCTGG CGTAATAGCG

[0252] AAGAGGCCCG CACCGATCGC CCTTCCCAAC AGTTGCGCAG

[0253] CCTGAATGGC GAATGGGACG CGCCCTGTAG CGGCGCATTA

[0254] AGCGCGGCGG GTGTGGTGGT TACGCGCAGC GTGACCGCTA

[0255] CACTTGCCAG CGCCCTAGCG CCCGCTCCTT TCGCTTTCTT

[0256] CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TCCCCGTCAA

[0257] GCTCTAAATC GGGGGCTCCC TTTAGGGTTC CGATTTAGTG

[0258] CTTTACGGCA CCTCGACCCC AAAAAACTTG ATTAGGGTGA TGGTTCACGT AGTGGGCCAT CGCCCTGATA GACGGTTTTT

[0259] CGCCCTTTGA CGTTGGAGTC CACGTTCTTT AATAGTGGAC

[0260] TCTTGTTCCA AACTGGAACA ACACTCAACC CTATCTCGGT

[0261] CTATTCTTTT GATTTATAAG GGATTTTGCC GATTTCGGCC

[0262] TATTGGTTAA AAAATGAGCT GATTTAACAA AAATTTAACG

[0263] CGAATTTTAA CAAAATATTA ACGCTTACAA TTTAGGTGGC

[0264] ACTTTTCGGG GAAATGTGCG CGGAACCCCT ATTTGTTTAT

[0265] TTTTCTAAAT ACATTCAAAT ATGTATCCGC TCATGAGACA

[0266] ATAACCCTGA TAAATGCTTC AATAATATTG AAAAAGGAAG

[0267] AGTATGAGTA TTCAACATTT CCGTGTCGCC CTTATTCCCT

[0268] TTTTTGCGGC ATTTTGCCTT CCTGTTTTTG CTCACCCAGA

[0269] AACGCTGGTG AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT

[0270] GCACGAGTGG GTTACATCGA ACTGGATCTC AACAGCGGTA

[0271] AGATCCTTGA GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT

[0272] GATGAGCACT TTTAAAGTTC TGCTATGTGG CGCGGTATTA

[0273] TCCCGTATTG ACGCCGGGCA AGAGCAACTC GGTCGCCGCA

[0274] TACACTATTC TCAGAATGAC TTGGTTGAGT ACTCACCAGT

[0275] CACAGAAAAG CATCTTACGG ATGGCATGAC AGTAAGAGAA

[0276] TTATGCAGTG CTGCCATAAC CATGAGTGAT AACACTGCGG

[0277] CCAACTTACT TCTGACAACG ATCGGAGGAC CGAAGGAGCT

[0278] AACCGCTTTT TTGCACAACA TGGGGGATCA TGTAACTCGC

[0279] CTTGATCGTT GGGAACCGGA GCTGAATGAA GCCATACCAA

[0280] ACGACGAGCG TGACACCACG ATGCCTGTAG CAATGGCAAC

[0281] AACGTTGCGC AAACTATTAA CTGGCGAACT ACTTACTCTA

[0282] GCTTCCCGGC AACAATTAAT AGACTGGATG GAGGCGGATA

[0283] AAGTTGCAGG ACCACTTCTG CGCTCGGCCC TTCCGGCTGG

[0284] CTGGTTTATT GCTGATAAAT CTGGAGCCGG TGAGCGTGGG

[0285] TCTCGCGGTA TCATTGCAGC ACTGGGGCCA GATGGTAAGC

[0286] CCTCCCGTAT CGTAGTTATC TACACGACGG GGAGTCAGGC

[0287] AACTATGGAT GAACGAAATA GACAGATCGC TGAGATAGGT

[0288] GCCTCACTGA TTAAGCATTG GTAACTGTCA GACCAAGTTT ACTCATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA

[0289] ATTTAAAAGG ATCTAGGTGA AGATCCTTTT TGATAATCTC

[0290] ATGACCAAAA TCCCTTAACG TGAGTTTTCG TTCCACTGAG

[0291] CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA

[0292] TCCTTTTTTT CTGCGCGTAA TCTGCTGCTT GCAAACAAAA

[0293] AAACCACCGC TACCAGCGGT GGTTTGTTTG CCGGATCAAG

[0294] AGCTACCAAC TCTTTTTCCG AAGGTAACTG GCTTCAGCAG

[0295] AGCGCAGATA CCAAATACTG TTCTTCTAGT GTAGCCGTAG

[0296] TTAGGCCACC ACTTCAAGAA CTCTGTAGCA CCGCCTACAT

[0297] ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG

[0298] TGGCGATAAG TCGTGTCTTA CCGGGTTGGA CTCAAGACGA

[0299] TAGTTACCGG ATAAGGCGCA GCGGTCGGGC TGAACGGGGG

[0300] GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC

[0301] CGAACTGAGA TACCTACAGC GTGAGCTATG AGAAAGCGCC

[0302] ACGCTTCCCG AAGAGAGAAA GGCGGACAGG TATCCGGTAA

[0303] GCGGCAGGGT CGGAACAGGA GAGCGCACGA GGGAGCTTCC

[0304] AGGGGGAAAC GCCTGGTATC TTTATAGTCC TGTCGGGTTT

[0305] CGCCACCTCT GACTTGAGCG TCGATTTTTG TGATGCTCGT

[0306] CAGGGGGGCG GAGCCTATGG AAAAACGCCA GCAACGCGGC

[0307] CTTTTTACGG TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC

[0308] ATGTTCTTTC CTGCGTTATC CCCTGATTCT GTGGATAACC

[0309] GTATTACCGC CTTTGAGTGA GCTGATACCG CTCGCCGCAG

[0310] CCGAACGACC GAGCGCAGCG AGTCAGTGAG CGAGGAAGCG

[0311] GAAGAGCGCC CAATACGCAA ACCGCCTCTC CCCGCGCGTT

[0312] GGCCGATTCA TTAATGCAGC TGGCACGACA GGTTTCCCGA

[0313] CTGGAAAGCG GGCAGTGAGC GCAACGCAAT TAATGTGAGT

[0314] TAGCTCACTC ATTAGGCACC CCAGGCTTTA CACTTTATGC

[0315] TTCCGGCTCG TATGTTGTGT GGAATTGTGA GCGGATAACA

[0316] ATTTCACACA GGAAACAGCT ATGACCATGA TTACGCCAAG

[0317] CGCGCAATTA ACCCTCACTA AAGGGAACAA AAGCTGGAGC

[0318] TGCAAGCTT. The insertion of the vector within the cells can be done in various ways; these must be inserted into the cells. Such insertion can be done in various ways known to the technician in the field, including preferably:

[0319] • transfection: by means of the application of a short electrical discharge that creates temporary holes in the cell membrane, the insertion of external material into the cell is allowed. For example, this allows the CAS9 protein and RNA guides to enter to add the GFP tag specifically to the tubulin gene so that protein is expressed with the sensor attached;

[0320] • infection: the genetic construct is integrated within an inactivated virus, preferably the lentivirus vector (LV). Cells are infected with the LV which inserts the genetic circuit into one or more random locations in the genome. This method is more suitable for large-sized sensors, such as FUCCIplex, but has the disadvantage that it is not possible to know in advance where the construct will be inserted.

[0321] In detail, one object in accordance with the present invention relates to genetically engineering a line of hPSCs so that they express fluorescent proteins sensitive to a variety of pathophysiological signals.

[0322] In the remainder of this disclosure, such engineered cells will be referred to by the expression hPSCsTEMPO.

[0323] Preferably, the selection of physiological variables in hPSCsTEMPO consists of:

[0324] • Actin as a structural signal. This signal makes the edge of all cells visible for analysis of morphology and dedicated structures of great interest for migration of all cell types (lamella, lamellopodia, phyllopodia, stress fibers), or specific to cardiac muscle (sarcomeres) and neurons (actin rings in axons).

[0325] • Intracellular calcium as a functional signal. This signal makes visible the change in calcium ion concentration within cells that are electrically (cardiac muscle) or chemically activated (neurons, smooth and cardiac muscle, pancreatic cells, etc.).

[0326] • Cell cycle as a coordination signal. This signal makes the phase of the cell cycle visible in which the cell is at the time of acquisition of the structural or functional signal (see also design). • Tubulin: as an alternative or in addition to actin, tubulin can also be considered in various structural applications. Alternatively, actin and tubulin can both be inserted as structural signals by removing the calcium sensor.

[0327] Finally, the expression assays of the construct in accordance with the present invention in human keratinocytes (HaCaT) are detailed below.

[0328] Culture and culture medium of the HaCaT cell line.

[0329] HaCaT cells were cultured in DMEM / F12 without phenol red (catalog no. 21041- 025, Gibco) with 10% fetal bovine serum (FBS) (catalog no. 10270-106, Gibco) and penicillin / streptomycin (P / S) (catalog no. A001, HiMedia). Cells were never allowed to reach confluence and were divided at 70% confluence.

[0330] Generation of the HaCaT FUCCIplex prototype clonal cell line.

[0331] Generation of the HaCaT FUCCIplex prototype clonal cell line consisted of 3 different and sequential steps. First of all, HaCaT cells were infected with FUCCIplex lentivirus and positively selected with antibiotics. A second lentiviral infection with a genetically encoded Lifeact-ACTB-RFP probe created a dual reporter cell line that has filamentous actin (F-actin) marked with a red fluorescent reporter in addition to the FUCCIplex cell cycle marker. After antibiotic selection, the cells were genomically modified to mark alpha-tubulin with an EGFP fluorophore at the N-terminus; this new 3 -reporter cell line was then clonally expanded and selected. In more detail, the HaCaT cells were plated at a density of 6x 104 / well in a 12-well plate in complete DMEM / F12. The next day, the medium was removed from the tissue culture plate by aspiration and replaced with fresh complete medium containing 8 pg / ml polybrene. Next, 10 pl of concentrated FUCCIplex lentivirus particles (>108 TU / ml, VectorBuilder) were resuspended in 500 pl of complete medium and directly added to the well. The cells were incubated overnight at 37 degrees, 5% CO2, and the next morning the transduction medium was aspirated and replaced with fresh complete medium. After 4 days, the positive transduced cells were selected with 20 pl / ml Hygromycin B (50 mg / ml in PBS), (Catalog No. 10687010, Invitrogen). HaCaT FUCCIplex cells were then clonally seeded and expanded. Positive cells were evaluated by means of PCR and confocal imaging to assess the correct expression of different reporters. For Lifeact-ACTB-RFP lentivirus infection (rLV Ubi- LifeAct-TagRFP, 1x107 TU / ml, 10 pl) cells were subjected to the same procedure with the only difference that 0.62 pl / ml Puromycin (1 pg / l) was used to select positive cells. Finally, HaCaT cells were subjected to genomic editing using the ThermoFisher Scientific True Tag system. Donor sequence and primers with homology arms specific for GFP tagging of the N terminus of alpha-tubulin (TUBA IB, NM006082.3) were designed online with the assistance of the ThermoFisher Scientific TrueDesign Genome Editor tool. Briefly, the True Tag dsDNA oligo donor linear with TUBA 1 A homology arms was amplified by PCR using Phusion Flash High-Fidelity PCR Master Mix, TrueTag Donor DNA Kit, GFP (Catalog No. A42992, Thermo). The forward sequence of the primer of the TrueTag homology arm is:

[0332] TCCTGTCGCCTTCGCCTCCTAATCCCTAGCCACTATGGGAGGTAAGC CCTTGCATTCG; the reverse is:

[0333] CCTGAAAGCAGCCGGGAGCCGCACGGCTTACTCACACCGCTTCCACT ACCTGAACC). Next, TrueGuide Synthetic gRNA (sgRNA) (ThermoFisher Scientific, sequence: GCACGGCTTACTCACCATAG) and True cut CAS9 Protein V2 (Catalog No. A36496, ThermoFisher Scientific) were combined to form the ribonucleoprotein (RNP) complex (CAS9+gRNA). True cut CAS9 V2 protein (2ug, 12 pmol), sgRNA (400 ng, 12 pmol), True Tag donor (500 ng) were resuspended in 10 pl of Resuspension Buffer R (catalog no. MPK1025, ThermoFisher Scientific) and then combined with 8 x 104 cells resuspended in 8 pl of Resuspension Buffer R for a final total volume of 18 pl. 10 pl of this mixture was used to electroporate the cells with the Neon transfection system (catalog No. MPK5000, ThermoFisher Scientific). The conditions for electroporation were as follows: 1200 V / 20 ms / 4 pulses. Electroporated cells were seeded in a 24-well plate filled with 0.5 ml DMEM / F12 medium containing 10% FBS without antibiotics. The plate was incubated at 37°C in a humidified CO2 incubator. The positive cells were then clonally plated and expanded.

[0334] Culture and culture medium of the HT-1080 LifeAct-TagGFP2 cell line.

[0335] HT-1080 LifeAct-TagGFP2 cells were purchased from iBidi (catalog no. 40101) and cultured in DMEM / F12 without phenol red (catalog no. 21041-025, Gibco) with 10% fetal bovine serum (FBS) (catalog no. 10270-106, Gibco) and penicillin / streptomycin (P / S) (catalog no. A001, HiMedia). Cells were never allowed to reach confluence and were divided at 70% confluence.

[0336] Generation of the ;x HT-1080 clonal cell line The strategy for the generation of the triple reporter and 4-color FUCCIplex HT-1080 prototype clonal cell line consisted of 2 separate steps and followed the same procedure used for the generation of the FUCCIplex HaCaT prototype cell line with minor modifications. First of all, LifeAct-TagGFP2 HT-1080 cells already expressing the genetically encoded LifeAct-TagGFP2 were genomically edited at the tubulin locus by using the ThermoFisher Scientific’s True Tag system. The sequence of donor and primers with homology arms specific for RFP tagging of the N terminus of alpha-tubulin (TUBA1 B, NM006082.3) were designed online with the assistance of the ThermoFisher Scientific TrueDesign Genome Editor tool as described above. Briefly, the generation of the linear dsDNA True Tag donor with TUBA 1 A homology arms was achieved by means of PCR using Phusion Flash High-Fidelity PCR Master Mix, TrueTag Donor DNA Kit, RFP (catalog No. A42993, Thermo). Next, the cells were electroporated (1200 V / 20 ms / 4 pulses) in the presence of ribonucleoprotein (RNP) complex (CAS9+gRNA). The electroporated cells were seeded in a 24-well plate filled with 0.5 ml DMEM / F12 medium containing 10% FBS without antibiotics. The plate was incubated at 37 °C in a humidified CO2 incubator. The positive cells were then clonally plated and expanded. Finally, FUCCIplex cell cycle indicator was introduced by infecting the cells with 10 pl of concentrated FUCCIplex lentivirus particles (>108 TU / ml, VectorBuilder). After antibiotic selection with 4 ul / ml Hygromycin B (50 mg / ml in PBS), FUCCIplex-positive cells were expanded. To clonally isolate HT-1080 FUCCIplex cells that have a clear and specific miRFP670-geminin nuclear signal, the following strategy was used. First of all, the frequency of miRFP670-geminin-positive cells was increased by seeding the cells loosely in a 6-well plate and letting them grow in small clusters. When a suitable cluster was detected at low magnification, the plate was marked and the cluster was harvested under sterile conditions and expanded. Then, the cells were diluted to 0.8 cells / well and seeded in a 96-well plate. Only clones that had defined signals of both mTurquoise2-cdtl and miRFP670-geminin were selected.

[0337] Maintenance ofhiPSC, differentiation and selection of cardiomyocytes.

[0338] The lines hiPSC WTC-11 (GM25256) and GCaMP6 (WTC11-AAV-CAG- GCaMP6) were kindly provided by Dr. Bruce Conklin (Gladstone. Institute of Cardiovascular Research). Cells were maintained in complete Essential 8 Flex Medium (catalog no. A2858501, ThermoFisher Scientific) supplemented with Penicillin / Streptomycin (P / S) (catalog no. A001, HiMedia) on Matrigel Growth Factor Reduced (GFR) 1: 100 (7-10 mg / ml, depending on the batch) without phenol red (catalog no. 356231, Coming). Once 70% confluence was reached (usually in 3-4 days), the cells were detached using 0.5 mM EDTA (ethylenediaminetetraacetic acid, Life Technologies) in Dulbecco’s PBS (DPBS) without Ca2+ or Mg2+ (catalog No. C-40232, PromoCell) for 5 min at 37 degrees 5%, CO2 environment. Next, cells were expanded in 6-well plates by means of 1: 10 passage in the presence of RevitaCell (catalog No. A26445-01, LifeTechnologies). Twenty-four hours after plating, the medium was replaced with complete Essential 8 Flex Medium (without RevitaCell) and changed every other day.

[0339] The hiPS cells were differentiated into cardiomyocytes (CMs) using a direct differentiation method adapted from the small molecule protocol described in Lian et al (PMID: 23257984). More specifically, hiPSCs were plated at a density of 8 x 104 / well in 12-well plates which were pre-coated with Matrigel (day -3 / 4) in the presence of RevitaCell. The next day, the medium was changed to complete Essential 8 Flex Medium (without RevitaCell). After 3-4 days, or when the cells reached 90% confluence (day 0), the medium was replaced with pre-warmed cardio differentiation medium (RPMI 1640+ Glutamax Medium (lx) (catalog no. 72400, Gibco)) supplemented with B-27 minus insulin (catalog no. A 1895601, Gibco) with the addition of 7.5 pM CHIR99021 (catalog no. 72054, StemCell). Exactly after 48 hours (day 2), the medium was replaced with pre-warmed cardio differentiation medium with the addition of 2pM Wnt-C59 (catalog No. 16644, Cayman Chemical Company). At day 4 (day 4), when exactly 48 hours had elapsed, the medium was aspirated and cardio differentiation medium was added; the cells were then allowed to differentiate for another 2 days. After 48 hours (day 6) the medium was changed and the cells were fed with cardio culture medium (RPMI 1640+ Glutamax Medium (lx) plus B-27 Supplement (50x), serum-free (catalog No. 17504044, Gibco)) and refreshed every 2 days. At day 12, cells were exposed to glucose-free Cardio Selection Medium DMEM (catalog no. 11966, Gibco) supplemented with 4 mM sodium DL-lactate (catalog no. 71720, SIGMA) to metabolically select only the differentiated cardiomyocytes. The medium was renewed every other day for four days; then, cardiomyocytes were cultured in the presence of cardio culture medium until experiments were performed following the specific time points of the assays.

[0340] Generation of the hiPS TEMPO clonal cell line.

[0341] The strategy for the generation of the triple reporter and 4-color hiPS TEMPO clonal cell line followed the same scheme adopted for the creation of the HaCaT FUCCIplex prototype clonal cell line with some adaptations (see below). The FUCCIplex cell cycle indicator and the genetically encoded actin sensor RFP- LifeAct were introduced into the WTCl l-AAV-CAG-GCaMP6 cell line. After antibiotic selection (Hygromycin B (50 mg / ml in PBS) 50 ul / ml for FUCCIplex- positive cells and 2 pl / ml Puromycin (1 pg / pl) for LifeAct and nd-positive cells), TEMPO hiPSc were sorted and positive clones were expanded.

[0342] Generation of the hiPS R26-CAG-FUCCIplex clonal cell line

[0343] To generate the R26-CAG-FUCCIplex cell line, hiPS WTC-11 (GM25256) cells were genomically edited to specifically insert the FUCCIplex cassette into the ROSA26 THUMPD3-AS1 locus (PMID: 18037879) by means of CRISPR / Cas9n-based gene-trap strategy. Unless otherwise indicated, genomic editing was performed as described in Bertero et al (PMID: 27899508). First of all, the FUCCIplex cDNA fragment flanked by ROSA26 homology arms was amplified by PCR starting from the original FUCCIplex plasmid (pLV-Hygro- EFla-FUCCIplex) using primers fwd: CATCATTTTGGCAAAGAATTAATTCGGATCCgccaccatggtgagcaagg and rev: CGAGGCTGATCAGCGAGCTACGCGTttacagggccttccgccg. In parallel, the plasmid pR26-Bsd_CAG-EGFPd2 (kindly provided by Professor Bertero, Department of Molecular Biotechnology and Health Sciences, Turin, Italy) was digested with BamHI and Mlul to remove the EGFPd2 fragment and ligated by NEBuilder HiFi DNA assembly (catalog No. E5520S, New England Biolabs) with the FUCCIplex DNA fragment to generate the pR26-Bsd_C AG- FUCCIplex construct. Next, hiPSc were transfected using the Lipofectamine stem cell reagent (catalog No. STEM00015, Invitrogen) in combination with two other plasmids, pSpCas9n(BB)_R26-L and pSpCas9n(BB)_R26-R to induce a specific doublestrand break in the intron between exons 1 and 2 of THUMPDS3-AS1 on chromosome 3 (ROSA26 locus). Gene-targeted hPSC clonal lines were screened by means of genomic PCR to verify site-specific targeting using the following primers, fwd: AGCCTCTGCTAACCATGTTC, rev:

[0344] GCCCATAATCGAGAAGCGTC.

[0345] Finally, in accordance with a further aspect, the present invention relates to a method for the production of a cell comprising at least one endogenous marked protein, expressed in a differential maimer, the method comprising:

[0346] (a) providing a first nuclease specific to a target genomic locus of a differentially expressed protein;

[0347] (b) providing a donor plasmid comprising: a first polynucleotide encoding for a selection cassette, where the selection cassette comprises a first selectable marker; a second polynucleotide encoding for a second selectable marker that is different from the first selectable marker;

[0348] (c) introducing the first nuclease of (a) and the donor plasmid of (b) into a cell such that the first and second polynucleotides are inserted into the target genomic locus;

[0349] (d) selecting the cells expressing the first selectable marker; and

[0350] (e) introducing into the cells of (d): a second nuclease capable of removing the selection cassette to generate an endogenous protein marked with the second selectable marker, in which the endogenous marked protein is substantially devoid of a scar sequence; thus producing the cell comprising the at least one differentially expressed endogenous marked protein.

[0351] It has in practice been ascertained that the described invention achieves the intended objects.

[0352] In particular, it is emphasized that the fact of genetically engineering a line of hPSCs so that they express sensitive fluorescent proteins allows monitoring of pathophysiological signals.

Claims

CLAIMS1) A genic construct, comprising:(i) at least a first gene (FUCCIplex) for or a partial fragment of a gene for at least a first expression product the quantity of which in a cell varies in a cell cycle-dependent maimer; and at least one of:(ii) at least a second gene for or a partial fragment of a gene for at least a second expression product the quantity of which in a cell varies in a cell structure-dependent manner;(iii) at least a third gene for or a partial fragment of a gene for at least a third expression product the quantity of which in a cell varies in a cell functiondependent manner.2) Genic construct according to claim 1, wherein said at least a first expression product is at least one of: a cyan fluorescent protein (CFP) or an infrared fluorescent protein (iRFP).3) Genic construct according to one or more of the preceding claims, wherein said at least a second expression product is at least one of: a fluorescent protein (RFP-LifeAct) the quantity of which in a cell varies depending on the presence of actin, a fluorescent protein the quantity of which in a cell varies depending on the presence of tubulin.4) Genic construct according to one or more of the preceding claims, wherein said at least a third expression product is a fluorescent protein (GCaMP6f) the quantity of which in a cell varies depending on the presence of intracellular calcium.5) Genic construct according to one or more of the preceding claims, comprising at least one fluorescent marker (FLUO-4) adapted to detect the presence of intracellular calcium.6) A method for performing the phase identification of a cell cycle, the method comprising the phases of: visualizing, using at least a first marker, at least one or more gene expression products the quantity of which in a cell vary in a cell cycle-dependent manner;and detecting at least said first marker so as to distinguish a proliferation phase of the cell cycle from a resting phase of the cell cycle.7) Method according to claim 6, comprising at least visualizing, using at least a second marker, at least one or more gene expression products the quantity of which in a cell vary in a cell structure-dependent maimer; and detecting at least said second marker so as to distinguish actin microfilaments.8) Method according to claims 6 and 7, comprising at least visualizing, by using at least a third marker, at least one or more gene expression products the quantity of which in a cell vary in a cell function-dependent manner; and detecting at least said third marker so as to distinguish the intracellular calcium.9) A cell, comprising an exogenous polynucleotide integrated into a target genomic locus, the exogenous polynucleotide comprising polynucleotide sequences encoding:(a) at least a first selectable marker;(b) at least a regulatory building element which is operationally connected to the first selectable marker;(d) at least a second selectable marker which is different from the first selectable marker, wherein the target genomic locus is a locus of a gene that encodes for a differentially expressed protein.10) Lentiviral expression vector comprising the construct according to one or more of claims 1-5.11) Vector according to claim 10, consisting of the sequence SEQ. ID. No: 1.

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