WO2015165276A1 - Kit de réactifs utilisant le répresseur de transcription tale pour la construction modulaire d'une lignée de gènes synthétiques dans une cellule de mammifère - Google Patents

Kit de réactifs utilisant le répresseur de transcription tale pour la construction modulaire d'une lignée de gènes synthétiques dans une cellule de mammifère Download PDF

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WO2015165276A1
WO2015165276A1 PCT/CN2015/000140 CN2015000140W WO2015165276A1 WO 2015165276 A1 WO2015165276 A1 WO 2015165276A1 CN 2015000140 W CN2015000140 W CN 2015000140W WO 2015165276 A1 WO2015165276 A1 WO 2015165276A1
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target
promoter
protein
expression cassette
sequence
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PCT/CN2015/000140
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Chinese (zh)
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谢震
威斯
李寅青
蒋云
廖微曦
陈赫
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清华大学
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Priority claimed from CN201410342350.6A external-priority patent/CN104630267B/zh
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Publication of WO2015165276A1 publication Critical patent/WO2015165276A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the distance between the target of the TALER protein A upstream of the promoter B and the target of the TAREL protein A closest to the downstream of the promoter B is 72-100 bp.
  • kits comprising the recombinant vector A-I having the expression cassette A-I, the recombinant vector B-I having the expression cassette B-I, and the recombinant vector C having the expression cassette C;
  • the upstream and downstream of the promoter B each have at least one target of the TALER protein A, or the promoter upstream of the promoter B does not have the target of the TALER protein A But downstream thereof has at least one target of said TALER protein A;
  • the recombinant vector A-I having the expression cassette A-I, the recombinant vector B-I having the expression cassette B-I, and the recombinant vector C having the expression cassette C were introduced into a host cell by adding shRNA1-1 , ..., shRNA1-n, shRNA2-1, ... or shRNA2-n regulate the expression of the protein A and the expression of the protein B.
  • the upstream of the promoter A has one target of the TALER protein B and has 1-3 targets of the TALER protein B downstream thereof;
  • the distance between the target of the TALER protein B upstream of the promoter A and the target of the TAREL protein B closest to the promoter downstream of the promoter is 72-100 bp;
  • a third kit for the controllable expression of two proteins protected by the present invention is as follows (a) or (b):
  • the expression cassette B-I includes, in order from upstream to downstream, the following elements: the coding sequence of the feedback element, the promoter B, the coding gene of the protein B linked by the coding gene of the self-cleaving polypeptide, and the coding gene of the TALER protein B, the target Sequence B-I;
  • the target sequence B-I includes a target sequence of shRNA2-1, ..., a target sequence of shRNA2-n, and n is a natural number of 2 or more;
  • the expression cassette D-I includes, in order from upstream to downstream, the following elements: a coding sequence of a feedback element, a promoter, a gene encoding a protein B linked by a gene encoding a self-cleaving polypeptide, and a gene encoding a TALER protein B, a target a sequence D;
  • the target sequence D-I includes a target sequence of shRNA3-1, ..., a target sequence of shRNA3-n, and n is a natural number of 2 or more;
  • each of the upstream and downstream of the promoter has at least one target of the TALER protein A, or the promoter upstream of the promoter does not have the target of the TALER protein A But downstream thereof has at least one target of said TALER protein A;
  • the expression cassette C comprises, in sequence from upstream to downstream, a coding sequence for a constitutive promoter and an activation element; under the stimulation of the activation element, DNA is located downstream of the coding sequence of the feedback element.
  • a recombinant vector A-I having the expression cassette A-I a recombinant vector B-I having the expression cassette B-I, a recombinant vector having the expression cassette C, and having the expression cassette D-I
  • the recombinant vector D-I is introduced into a host cell, and the protein A is regulated by the addition of shRNA1-1, ..., shRNA1-n, shRNA2-1, ..., shRNA2-n, shRNA3-1, ... or shRNA3-n. Expression and protein B expression.
  • the upstream of the promoter A has one target of the TALER protein B and has 1-3 targets of the TALER protein B downstream thereof;
  • the upstream of the promoter has one target of the TALER protein A and has 1-3 targets of the TALER protein A downstream thereof.
  • the distance between the target of the TALER protein B upstream of the promoter A and the target of the TAREL protein B closest to the promoter downstream of the promoter is 72-100 bp;
  • the distance between the target of the TALER protein A upstream of the promoter B and the target of the TAREL protein A closest to the downstream of the promoter B is 72-100 bp;
  • the first kit for sorting cell A and/or cell B from mixed cells protected by the present invention is as follows (a) or (b):
  • the expression cassette B-II includes, in order from upstream to downstream, the following elements: the coding sequence of the feedback element, the promoter B, the coding gene of the fluorescent protein B linked by the coding gene of the self-cleaving polypeptide, and the coding gene of the TALER protein B, Target sequence B-II; the target sequence B-II comprises a target sequence of more than one (specifically 4) miRNA2;
  • each of the upstream and downstream of the promoter A has at least one target of the TALER protein B, or the promoter upstream of the promoter A does not have the target of the TALER protein B. But downstream thereof has at least one target of said TALER protein B;
  • kits comprising the recombinant vector A-II having the expression cassette A-II, the recombinant vector B-II having the expression cassette B-II, and the recombinant vector C having the expression cassette C;
  • the miRNAs 1-1, ..., miRNA1-n are specific miRNAs possessed by the cell A;
  • the miRNA 2-1, ..., miRNA2-n is a specific miRNA possessed by the cell B;
  • the upstream of the promoter A has one target of the TALER protein B and has 1-3 targets of the TALER protein B downstream thereof;
  • the expression cassette D-II comprises, in order from upstream to downstream, the following elements: the coding sequence of the feedback element, the promoter B, the coding gene of the fluorescent protein B linked by the coding gene of the self-cleaving polypeptide, and the coding of TALER protein B.
  • each of the upstream and downstream of the promoter A has at least one target of the TALER protein B, or the promoter upstream of the promoter A does not have the target of the TALER protein B. But downstream thereof has at least one target of said TALER protein B;
  • each of the upstream and downstream of the promoter B has at least one target of the TALER protein A, or the promoter upstream of the promoter B does not have the target of the TALER protein A But downstream thereof has at least one target of said TALER protein A;
  • the miRNA 2-1, ..., miRNA2-n is a specific miRNA possessed by the cell B;
  • the miRNAs 3-1, ..., miRNA3-n are specific miRNAs possessed by the cell B;
  • the 5 x UAS sequence can be any of the 5 x UAS sequences of any of the plasmids containing the 5 x UAS sequence involved in Examples 1-6.
  • the CMVmini promoter can be referred to in Examples 1-6 All of the CMVmini promoters in the plasmid containing the CMVmini promoter.
  • the CAG promoter may be any of the CAG promoters in the plasmids containing the CAG promoter involved in Examples 1-6.
  • the gene encoding Gal4/vp16 may be any one of the CAG promoters of the plasmid containing the gene encoding Gal4/vp16 involved in Examples 1-6.
  • the shRNA3 may specifically be RNA encoded by the coding gene of shRNA-FF3, RNA encoded by the coding gene of shRNA-FF4, RNA encoded by the coding gene of shRNA-FF5, or RNA encoded by the coding gene of shRNA-FF6.
  • the gene encoding the shRNA-FF3 may be the gene encoding shRNA-FF3 in the plasmid of any of Examples 1-6.
  • the gene encoding the shRNA-FF4 can be The gene encoding shRNA-FF4 in the plasmid of any of Examples 1-6.
  • the gene encoding the shRNA-FF5 may be the gene encoding shRNA-FF5 in the plasmid of any of Examples 1-6.
  • the gene encoding the shRNA-FF6 may be the gene encoding shRNA-FF6 in the plasmid of any of Examples 1-6.
  • the target sequence of the miRNA1 may specifically be a target sequence of miR21, a target sequence of miR18a, a target sequence of miR19ab or a target sequence of miR191.
  • the target sequence of the miRNA2 may specifically be a target sequence of miR21, a target sequence of miR18a, a target sequence of miR19ab or a target sequence of miR191.
  • the target sequence may specifically be a target sequence of miR21, a target sequence of miR18a, a target sequence of miR19ab or a target sequence of miR191.
  • the target sequence of the miR21 may specifically be the target sequence of miR21 in the plasmid of any of Examples 1-6.
  • the target sequence of the miR18a may specifically be the target sequence of miR18a in the plasmid of any of Examples 1-6.
  • the target sequence of the miR19ab may specifically be the target sequence of miR19ab in the plasmid of any of Examples 1-6.
  • the target sequence of the miR191 may specifically be the target sequence of miR191 in the plasmid of any of Examples 1-6.
  • the recombinant vector A-I may specifically be pT9+T9x3+72-mKate2-2A-TALER10-4xTarget ⁇ FF4 plasmid, pT9+T9x3+72-mKate2-2A-TALER12-4xTarget ⁇ FF6 plasmid, pT9+ T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF4 plasmid, pT9+T9x3+72-mKate2-2A-TALER21-4x Target ⁇ FF3 plasmid, pT10+T10x3+72-EYFP-2A-TALER9-4xTarget ⁇ FF5 plasmid, pT10 +T10x3+72-mKate2-2A-TALER12-4xTarget ⁇ FF6 plasmid, pT10+T10x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF4 plasmid, pT10+
  • the host cell may specifically be HEK293 cells, HeLa cells, HeLa:TagBFP or HEK293: iRFP_shRNA-FF4.
  • Engineered structurally complex gene lines are hampered by the lack of orthogonal and reversible transcriptional repressor elements.
  • the top 10 strongest TALERs effectively inhibited their corresponding promoters with little effect on the other nine promoters.
  • Synthetic biology aims to use modular principles to modularize the construction of well-defined synthetic gene lines.
  • gene routing in mammalian cells, synthesizing its components, quantitatively describing and predicting its function remains an important challenge.
  • the input and output levels of the TALER elements were simultaneously measured, and a color model was established which normalized the fluorescence levels of the input and output.
  • the inventors found through experiments-based input/output transfer functions that the properties of some TALERs can be used to construct TALER cascades and switches and to quantify their results.
  • more accurate predictions may require further feature testing and modeling. For example, a grading analysis may help eliminate copy number differences during transient transfection.
  • RNA interference provides a modular, modular and scalable interface between synthetic gene lines and endogenous molecular inputs in mammalian cells.
  • endogenous microRNA can be used to control the state of the TALER switch, and the sensitivity of the TALER switch to shRNA regulation can be adjusted by adjusting the ratio of the two components.
  • two cell type-specific microRNAs can tightly control the output of the TALER switch, enabling precise cell sorting in a mixed cell population.
  • the results of the invention of heat provide convenience for the selection of TALER switches that can sense cell-specific microRNAs, and can select the appropriate TALER directly from the TALER library to meet different expression levels. Demand.
  • TALER switches can be used to modularize more complex logic lines, such as using a logical design framework to more accurately detect cell type-specific microRNAs, or for programmable memory elements to track intracellular events and signals. process. As long as conditions such as efficient in vivo cell delivery and long-term stability of gene lines are resolved, future TALER switches will be widely used in biomedicine.
  • Figure 1 is a schematic diagram showing the mechanism of action of the TALER protein.
  • Figure 2 is a schematic representation of the mechanism of action of the pCMV-TALERx plasmid, pTx+Tx+ plasmid and pEF1a-TagBFP-2A plasmid.
  • Figure 6 is the result of step two of the second embodiment.
  • Figure 7 is the result of step three of the second embodiment.
  • Figure 10 is the result of step two of the third embodiment.
  • Figure 12 is the result of step two of the fourth embodiment.
  • Figure 13 is the result of the first step of the fifth embodiment.
  • Figure 16 is the result of the first step of the embodiment 6.
  • Figure 21 is a further expanded genetic line.
  • FIG. 2 A schematic diagram of the mechanism of action of pCMV-TALERx plasmid, pTx+Tx+ plasmid and pEF1a-TagBFP-2A plasmid is shown in Fig. 2.
  • TagBFP and Gal4/vp16 are expressed under the action of the pEF1a promoter (TagBFP and The 2A linker peptide between Gal4/vp16 is a self-splicing peptide, so TagBFP can represent the expression level of Gal4/vp16).
  • Gal4/vp16 activates the 5xUAS sequence, thereby activating the transcriptional initiation of the CMVmini promoter, and mKate2 is expressed.
  • EYFP and TALER1 proteins Under the action of the CMV promoter, EYFP and TALER1 proteins are expressed (the 2A linker peptide between EYFP and TALER1 proteins is a self-splicing peptide, so EYFP can represent the expression level of TALER1 protein).
  • the TALER1 protein binds to the T1 sequence and exerts transcriptional repression through steric hindrance.
  • the CMVmini promoter between the two T1 sequences is inactivated, and mKate2 is inhibited.
  • the pCMV-TALER1 plasmid is shown in SEQ ID NO: 1.
  • the 1-549th nucleotide from the 5' end is the CMV promoter
  • the nucleotides 6013-1319 are the coding gene of EYFP (enhanced yellow fluorescent protein)
  • the nucleotides 1326-1379 The gene encoding the 2A-linked peptide, the nucleotides 1389-4220 are the genes encoding the TALER1 protein, and the nucleotides 4227-4259 are the genes encoding the nuclear localization signal SV40NLS.
  • the pT1+T1+ plasmid is shown in SEQ ID NO:27.
  • the nucleotides 4275-4367 from the 5' end are 5 ⁇ UAS sequences
  • the nucleotides 4383-4396 are T1 sequences (target sequences of TALER1 protein)
  • nucleotides 4403-4462 For the CMVmini promoter
  • nucleotides 4469-4482 are T1 sequences
  • nucleotides 4532-5237 are genes encoding mKate2 (far red fluorescent protein).
  • the pCMV-TALER2 plasmid is shown in SEQ ID NO: 2.
  • nucleotides 1-549 are CMV promoters
  • nucleotides 603-1319 are EYFP encoding genes
  • nucleotides 1326-1379 are 2A connecting peptide encoding genes
  • 1389- The 4220 nucleotide is the gene encoding the TALER2 protein
  • nucleotides 4227-4259 are the genes encoding the nuclear localization signal SV40NLS.
  • the pCMV-TALER4 plasmid is shown in SEQ ID NO:3.
  • nucleotides 1713 to 2301 are CMV promoters
  • nucleotides 2315 to 3031 are EYFP encoding genes
  • nucleotides 3038 to 3091 are encoding genes of 2A linked peptides
  • the 5932 nucleotide is the coding gene of the TALER4 protein
  • nucleotide 5939-5971 is the coding gene of the nuclear localization signal SV40NLS.
  • the pCMV-TALER5 plasmid is shown in SEQ ID NO: 4.
  • nucleotides 5842-6430 are CMV promoters
  • nucleotides 6444-7160 are encoding genes of EYFP
  • nucleotides 7167-7220 are encoding genes of 2A linking peptides
  • 7230- The 2387 nucleotide is the coding gene of the TALER5 protein
  • nucleotide 2394-2426 is the coding gene of the nuclear localization signal SV40NLS.
  • the pCMV-TALER9 plasmid is shown in SEQ ID NO: 5.
  • nucleotides 6148-6736 are the CMV promoter
  • nucleotides 6750-7466 are the coding gene of EYFP
  • nucleotides 7473-7526 are the coding genes of 2A junction peptide
  • 7536- The 2693 nucleotide is the coding gene of the TALER9 protein
  • the 2700-2732 nucleotide is the coding gene of the nuclear localization signal SV40NLS.
  • the pCMV-TALER10 plasmid is shown in SEQ ID NO: 6.
  • the 1792-2380 nucleotide is the CMV promoter
  • the 2394-3110 nucleotide is the EYFP encoding gene
  • the 3117-3170 nucleotide is the 2A linkage.
  • the coding gene of the peptide, the nucleotides 3180-6623 are the genes encoding the TALER10 protein
  • the nucleotides 6630-6662 are the genes encoding the nuclear localization signal SV40NLS.
  • the pCMV-TALER11 plasmid is shown in SEQ ID NO: 7.
  • the 1766-2354 nucleotide is the CMV promoter
  • the 2368-3084 nucleotide is the EYFP coding gene
  • the 3091-3144 nucleotide is the 2A junction peptide coding gene
  • the 3154- 4 The 6597 nucleotide is the coding gene of the TALER11 protein
  • the nucleotide 6604-6636 is the coding gene of the nuclear localization signal SV40NLS.
  • the pCMV-TALER13 plasmid is shown in SEQ ID NO: 9.
  • the nucleotides 1687-2275 are the CMV promoter
  • the nucleotides 2289-3005 are the coding genes of EYFP
  • the nucleotides 301-32-365 are the coding genes of the 2A junction peptide, pp. 3075-
  • the nucleotide at position 6212 is the gene encoding the TALER13 protein
  • the nucleotide at positions 6219-6251 is the gene encoding the nuclear localization signal SV40NLS.
  • the pCMV-TALER14 plasmid is shown in SEQ ID NO: 10.
  • the 1764-2352 nucleotide is the CMV promoter
  • the 2366-3082 nucleotide is the EYFP coding gene
  • the 3089-3142 nucleotide is the 2A junction peptide coding gene
  • the 3152- The 6289 nucleotide is the coding gene of the TALER14 protein
  • the nucleotides 6296-6328 are the coding genes of the nuclear localization signal SV40NLS.
  • the pCMV-TALER15 plasmid is shown in SEQ ID NO: 11.
  • nucleotides 1-549 are CMV promoters
  • nucleotides 603-1319 are EYFP encoding genes
  • nucleotides 1326-1379 are 2A linking peptide encoding genes
  • 1394- The 4597 nucleotide is the coding gene of the TALER15 protein
  • nucleotide 4605-4637 is the coding gene of the nuclear localization signal SV40NLS.
  • the pCMV-TALER16 plasmid is shown in SEQ ID NO: 12.
  • nucleotides 1-549 are CMV promoters
  • nucleotides 603-1319 are EYFP encoding genes
  • nucleotides 1326-1379 are 2A linking peptide encoding genes
  • 1394- The 4597 nucleotide is the coding gene of the TALER16 protein
  • nucleotide 4605-4637 is the coding gene of the nuclear localization signal SV40NLS.
  • the pCMV-TALER18 plasmid is shown in SEQ ID NO: 14.
  • nucleotides 1-549 are CMV promoters
  • nucleotides 603-13-119 are EYFP encoding genes
  • nucleotides 1326-1379 are 2A linking peptide encoding genes
  • 1394- The 4597 nucleotide is the coding gene of the TALER18 protein
  • nucleotide 4605-4637 is the coding gene of the nuclear localization signal SV40NLS.
  • the pCMV-TALER19 plasmid is shown in SEQ ID NO: 15.
  • the nucleotides 1711-2299 are CMV promoters
  • the nucleotides 2313-3029 are EYFP encoding genes
  • the nucleotides 3036-3089 are 2A.
  • the gene encoding the linker peptide, the nucleotides 3099-6440 are the genes encoding the TALER19 protein
  • the nucleotides 6447-6479 are the genes encoding the nuclear localization signal SV40NLS.
  • the pCMV-TALER20 plasmid is shown in SEQ ID NO: 16.
  • nucleotides 1-549 are CMV promoters
  • nucleotides 603-1319 are EYFP encoding genes
  • nucleotides 1326-1379 are 2A linking peptide encoding genes
  • 1394- The 4597 nucleotide is the coding gene of the TALER20 protein
  • nucleotide 4605-4637 is the coding gene of the nuclear localization signal SV40NLS.
  • the pCMV-TALER21 plasmid is shown in SEQ ID NO: 17.
  • nucleotides 1616-2204 are CMV promoters
  • nucleotides 2218-2934 are EYFP encoding genes
  • nucleotides 2941-2994 are 2A linking peptide encoding genes
  • 3004- The 6345 nucleotide is the coding gene of the TALER21 protein
  • nucleotide number 6352-6384 is the coding gene of the nuclear localization signal SV40NLS.
  • the pCMV-TALER23 plasmid is shown in SEQ ID NO: 19.
  • nucleotides 1-549 are CMV promoters
  • nucleotides 603-13-1 are EVFP encoding genes
  • nucleotides 1326-1379 are 2A linking peptide encoding genes
  • 1394- The 3985 nucleotide is the coding gene of the TALER23 protein
  • nucleotides 3993-4025 are the genes encoding the nuclear localization signal SV40NLS.
  • the pCMV-TALER24 plasmid is shown in SEQ ID NO: 20.
  • nucleotides 1-549 are CMV promoters
  • nucleotides 603-13-119 are EYFP encoding genes
  • nucleotides 1326-1379 are 2A connecting peptide encoding genes
  • 1394- The 3985 nucleotide is the coding gene of the TALER24 protein
  • nucleotides 3993-4025 are the genes encoding the nuclear localization signal SV40NLS.
  • the pCMV-TALER26 plasmid is shown in SEQ ID NO:21.
  • nucleotides 1679-2267 are CMV promoters
  • nucleotides 2281-2997 are EYFP encoding genes
  • nucleotides 3004-3057 are 2A connecting peptide encoding genes
  • 3064- The nucleotide at position 6009 is the gene encoding the TALER26 protein
  • the nucleotide at position 6024-6045 is the gene encoding the nuclear localization signal SV40NLS.
  • the pCMV-TALER29 plasmid is shown in SEQ ID NO:22.
  • the 1638-2226 nucleotide is the CMV promoter
  • the 2240-2956 nucleotide is the EYFP coding gene
  • the 2963-3016 nucleotide is the 2A junction peptide coding gene
  • the 3303- The 5560 nucleotide is the coding gene of the TALER29 protein
  • the nucleotides 5575-5596 are the coding genes of the nuclear localization signal SV40NLS.
  • the pCMV-TALER30 plasmid is shown in SEQ ID NO:23.
  • the nucleotides 1838-2426 are the CMV promoter
  • the nucleotides 2440-3156 are the coding genes of EYFP
  • the nucleotides 3163-3216 are the coding genes of the 2A junction peptide
  • the 5760 nucleotide is the coding gene of the TALER30 protein
  • the nucleotides 5775-5796 are the coding genes of the nuclear localization signal SV40NLS.
  • the pCMV-TALER31 plasmid is shown in SEQ ID NO:24.
  • the nucleotides 3403-3991 are the CMV promoter
  • the nucleotides 4005-4721 are the coding genes of EYFP
  • the nucleotides 4728-4781 are 2A.
  • the coding gene of the ligation peptide, the nucleotide of 4788-7325 is the coding gene of TALER31 protein
  • the nucleotide of 7340-7361 is the coding gene of nuclear localization signal SV40NLS.
  • the pCMV-TALER32 plasmid is shown in SEQ ID NO:25.
  • the nucleotides 1691-2279 are the CMV promoter
  • the nucleotides 2293-3009 are the coding genes of EYFP
  • the nucleotides 3016-3069 are the coding genes of the 2A junction peptide, 3076-
  • the nucleotide at position 5613 is the gene encoding the TALER32 protein
  • the nucleotide at position 5628-5649 is the gene encoding the nuclear localization signal SV40NLS.
  • the pT2+T2+ plasmid is shown in SEQ ID NO: 28.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-190 are T2 sequences (target sequence of TALER2 protein)
  • nucleotides 197-256 For the CMVmini promoter
  • nucleotides 263-276 are the T2 sequence
  • nucleotides 355-1073 are the coding genes for mKate2.
  • the pT4+T4+ plasmid is shown in SEQ ID NO:29.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-190 are T4 sequences (target sequence of TALER4 protein)
  • nucleotides 197-256 For the CMVmini promoter nucleotides 263-276 are T4 sequences
  • nucleotides 355-1073 are genes encoding mKate2.
  • the pT5+T5+ plasmid is shown in SEQ ID NO:30.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-194 are T5 sequences (target sequence of TALER5 protein)
  • nucleotides 201-260 For the CMVmini promoter nucleotides 267-284 are T5 sequences
  • nucleotides 363-1081 are genes encoding mKate2.
  • sequence 32 The pT10+T10+ plasmid is shown in sequence 32.
  • sequence 32 the nucleotides from the 5' end of the 7069-7161 nucleotide are 5 ⁇ UAS sequences, and the nucleotides from the 7177-7196 nucleotides are the T10 sequence (target sequence of the TALER10 protein), and nucleotides 7203-7262 For the CMVmini promoter, nucleotides 7269-7288 are T10 sequences, and nucleotides 78-796 are genes encoding mKate2.
  • the pT11+T11+ plasmid is shown in SEQ ID NO:33.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-196 are T11 sequences (target sequences of TALER11 protein)
  • nucleotides 203-262 For the CMVmini promoter the nucleotides 269-288 are the T11 sequence
  • nucleotides 367-1085 are the coding genes of mKate2.
  • the pT14+T14+ plasmid is shown in SEQ ID NO:36.
  • sequence 36 the nucleotides from the 5' end of the 7069-7161 nucleotides are 5 ⁇ UAS sequences, and the nucleotides at positions 7177-7193 are the T14 sequences (target sequences of the TALER14 protein), and nucleotides 7200-7259
  • nucleotides 7266-7282 are T14 sequences
  • nucleotides 78-796 are genes encoding mKate2.
  • the pT15+T15+ plasmid is shown in SEQ ID NO:37.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-201 are T15 sequences (target sequences of TALER15 protein)
  • nucleotides 208-267 For the CMVmini promoter
  • nucleotides 274-298 are the T15 sequence
  • nucleotides 377-1095 are the coding genes of mKate2.
  • sequence 38 The pT16+T16+ plasmid is shown in SEQ ID NO:38.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-201 are T16 sequences (target sequences of TALER16 protein)
  • nucleotides 274-298 are T16 sequences
  • nucleotides 377-1095 are genes encoding mKate2.
  • the pT17+T17+ plasmid is shown in SEQ ID NO:39.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-201 are T17 sequences (target sequences of TALER17 protein)
  • nucleotides 208-267 For the CMVmini promoter
  • the nucleotides 274-298 are the T17 sequence
  • the nucleotides 377-1095 are the coding genes of mKate2.
  • the pT19+T19+ plasmid is shown in sequence 41.
  • the nucleotides from the 5' end of the 7069-7161 nucleotide are 5 ⁇ UAS sequences
  • the nucleotides at the 7177-7195 nucleotides are the T19 sequence (the target sequence of the TALER19 protein)
  • the nucleotides of the 7th to 7261th nucleotides are the T19 sequence (the target sequence of the TALER19 protein)
  • nucleotides 7268-7286 are T19 sequences
  • nucleotides 78-796 are genes encoding mKate2.
  • sequence 42 The pT20+T20+ plasmid is shown as sequence 42.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-201 are T20 sequences (target sequences of TALER20 protein)
  • nucleotides 274-298 are T20 sequences
  • nucleotides 377-1095 are genes encoding mKate2.
  • sequence 43 The pT21+T21+ plasmid is shown in sequence 43.
  • nucleotides 7177-7195 are T21 sequences (target sequences for TALER21 protein)
  • nucleotides 7268-7286 are T21 sequences
  • nucleotides 78-796 are genes encoding mKate2.
  • sequence 44 The pT22+T22+ plasmid is shown as sequence 44.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-195 are T22 sequences (target sequences of TALER22 protein)
  • nucleotides 202-261 For the CMVmini promoter nucleotides 268-286 are T22 sequences
  • nucleotides 365-1083 are genes encoding mKate2.
  • sequence 45 The pT23+T23+ plasmid is shown in sequence 45.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-195 are T23 sequences (target sequences of TALER23 protein)
  • nucleotides 202-261 For the CMVmini promoter nucleotides 268-286 are T23 sequences
  • nucleotides 365-1083 are genes encoding mKate2.
  • sequence 46 The pT24+T24+ plasmid is shown in SEQ ID NO:46.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-195 are T24 sequences (target sequences of TALER24 protein)
  • nucleotides 202-261 For the CMVmini promoter nucleotides 268-286 are T24 sequences
  • nucleotides 365-1083 are genes encoding mKate2.
  • sequence 47 nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-194 are T26 sequences (target sequences of TALER26 protein), nucleotides 201-260 For the CMVmini promoter, nucleotides 267-284 are T26 sequences, and nucleotides 363-1081 are genes encoding mKate2.
  • the pT29+T29+ plasmid is shown in SEQ ID NO:48.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-190 are T29 sequences (target sequences of TALER29 protein)
  • nucleotides 197-256 For the CMVmini promoter the nucleotides 263-276 are the T29 sequence
  • nucleotides 355-1073 are the coding genes of mKate2.
  • the pT30+T30+ plasmid is shown in SEQ ID NO:49.
  • sequence 49 nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-190 are T30 sequences (target sequence of TALER30 protein), nucleotides 197-256 For the CMVmini promoter, the nucleotides 263-276 are the T30 sequence, and the nucleotides 355-1073 are the coding genes of mKate2.
  • sequence 50 The pT31+T31+ plasmid is shown as sequence 50.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-189 are T31 sequences (target sequences of TALER31 protein)
  • nucleotides 196-255 For the CMVmini promoter nucleotides 262-274 are T31 sequences
  • nucleotides 353-1071 are genes encoding mKate2.
  • sequence 51 The pT32+T32+ plasmid is shown in sequence 51.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-190 are T32 sequences (target sequence of TALER32 protein)
  • nucleotides 197-256 For the CMVmini promoter nucleotides 263-276 are T32 sequences
  • nucleotides 355-1073 are the coding genes for mKate2.
  • sequence 52 The pT35+T35+ plasmid is shown in sequence 52.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-190 are T35 sequences (target sequences of TALER35 protein)
  • nucleotides 197-256 For the CMVmini promoter nucleotides 263-276 are T35 sequences
  • nucleotides 355-1073 are genes encoding mKate2.
  • the pCMV-TALER1 plasmid, pT1+T1+ plasmid and pEF1a-TagBFP-2A plasmid were co-transfected into HEK293 cells (transfected with 200 ng pCMV-TALER1 plasmid, 50 ng pT1+T1+ plasmid and 30 ng pEF1a-TagBFP-2A plasmid per well), transfected.
  • Flow cytometry analysis was performed after 48 hours to measure the fluorescence intensity of EYFP, the fluorescence intensity of mKate2, and the fluorescence intensity of TagBFP.
  • a control treatment without the addition of the pCMV-TALER1 plasmid was set.
  • Inhibition ratio control group mKate2 fluorescence intensity correction value ⁇ experimental group mKate2 fluorescence intensity correction value.
  • Percent inhibition (control group mKate2 fluorescence intensity correction value - experimental group mKate2 fluorescence intensity correction value) ⁇ control group mKate2 fluorescence intensity correction value.
  • mKate2 fluorescence intensity correction value fluorescence intensity of mKate2 / fluorescence intensity of TagBFP.
  • the pCMV-TALER1 plasmid was replaced with the pCMV-TALER2 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with the pT2+T2+ plasmid.
  • the pCMV-TALER1 plasmid was replaced with the pCMV-TALER4 plasmid, and the above procedure was carried out by substituting the pT4+T4+ plasmid for the pT1+T1+ plasmid.
  • the pCMV-TALER1 plasmid was replaced with the pCMV-TALER5 plasmid, and the above procedure was carried out by substituting the pT5+T5+ plasmid for the pT1+T1+ plasmid.
  • the pCMV-TALER1 plasmid was replaced with the pCMV-TALER9 plasmid, and the above procedure was carried out by substituting the pT9+T9+ plasmid for the pT1+T1+ plasmid.
  • the pCMV-TALER1 plasmid was replaced with pCMV-TALER14 plasmid, and the above procedure was carried out using pT14+T14+ plasmid instead of pT1+T1+ plasmid.
  • the pCMV-TALER1 plasmid was replaced with pCMV-TALER15 plasmid, and the above procedure was carried out by substituting pT15+T15+ plasmid for pT1+T1+ plasmid.
  • the pCMV-TALER1 plasmid was replaced with pCMV-TALER16 plasmid, and the above procedure was carried out using pT16+T16+ plasmid instead of pT1+T1+ plasmid.
  • the pCMV-TALER1 plasmid was replaced with the pCMV-TALER17 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with pT17+T17+ plasmid.
  • the pCMV-TALER1 plasmid was replaced with pCMV-TALER20 plasmid, and the above procedure was carried out by substituting pT20+T20+ plasmid for pT1+T1+ plasmid.
  • the pCMV-TALER1 plasmid was replaced with the pCMV-TALER21 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with pT21+T21+ plasmid.
  • the pCMV-TALER1 plasmid was replaced with pCMV-TALER22 plasmid, and the above procedure was carried out using pT22+T22+ plasmid instead of pT1+T1+ plasmid.
  • the pCMV-TALER1 plasmid was replaced with pCMV-TALER23 plasmid, and the above procedure was carried out by substituting pT23+T23+ plasmid for pT1+T1+ plasmid.
  • the pCMV-TALER1 plasmid was replaced with the pCMV-TALER29 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with pT29+T29+ plasmid.
  • the pCMV-TALER1 plasmid was replaced with pCMV-TALER30 plasmid, and the above procedure was carried out by substituting pT30+T30+ plasmid for pT1+T1+ plasmid.
  • the pCMV-TALER1 plasmid was replaced with pCMV-TALER35 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with pT35+T35+ plasmid.
  • the pCMV-TALER1 plasmid, pT35+T35+ plasmid and pEF1a-TagBFP-2A plasmid were co-transfected into HEK293 cells (200 ng pCMV-TALER1 plasmid, 50 ng pT1+T1+ plasmid and 30 ng pEF1a-TagBFP-2A plasmid per well). After 48 hours of transfection, flow cytometry analysis was performed to detect the fluorescence intensity of EYFP, the fluorescence intensity of mKate2, and the fluorescence intensity of TagBFP. A control treatment without the addition of the pCMV-TALER1 plasmid was set.
  • the pCMV-TALER1 plasmid was the pCMV-TALER1 plasmid of Example 1.
  • sequence 54 The pT1+T1+72-DsRed plasmid is shown in sequence 54.
  • the nucleotides 2441-2533 from the 5' end are 5 x UAS sequences
  • the nucleotides 2549-2562 are T1 sequences (target sequences of TALER1 protein)
  • nucleotides 2569-2628 For the CMVmini promoter the nucleotides at positions 2635-2648 are T1 sequences
  • the nucleotides at positions 2668-3345 are genes encoding DsRed (red fluorescent protein).
  • sequence 56 The pT2+T1+72-DsRed plasmid is shown as sequence 56.
  • nucleotides 2441-2533 from the 5' end are 5 x UAS sequences
  • nucleotides 2549-2562 are T2 sequences
  • nucleotides 2569-2628 are CMVmini promoters
  • 2635- The 2648 nucleotide is the T1 sequence
  • the 2668-3345 nucleotide is the DsRed coding gene.
  • sequence 57 The pT2+T2+72-DsRed plasmid is shown in sequence 57.
  • nucleotides 2441-2533 from the 5' end are 5 x UAS sequences
  • nucleotides 2549-2562 are T2 sequences
  • nucleotides 2569-2628 are CMVmini promoters
  • 2635- The 2648 nucleotide is the T2 sequence
  • the 2668-3345 nucleotide is the DsRed coding gene.
  • the pT1+T1+72-mKate2 plasmid is shown in SEQ ID NO:58.
  • sequence 58 the nucleotides 4275-4367 from the 5' end are 5 ⁇ UAS sequences, the nucleotides 4383-4396 are T1 sequences, and the nucleotides 4403-4462 are CMVmini promoters, 4469- The 4482 nucleotide is the T1 sequence, and the nucleotides 4532-5237 are the coding genes of mKate2.
  • the pT1+T1+78-mKate2 plasmid is shown in SEQ ID NO:59.
  • nucleotides 7161-7253 from the 5' end are 5 ⁇ UAS sequences
  • nucleotides 7269-7282 are T1 sequences
  • nucleotides 6-65 are CMVmini promoters
  • 78- The nucleotide at position 91 is the T1 sequence
  • the nucleotide at positions 170-888 is the coding gene for mKate2.
  • the pT1+T1+83-mKate2 plasmid is shown in SEQ ID NO:60.
  • nucleotides 7166-7258 from the 5' end are 5 x UAS sequences
  • nucleotides 7274-7287 are T1 sequences
  • nucleotides 6-65 are CMVmini promoters
  • 83- The 96th nucleotide is the T1 sequence
  • nucleotides 175-893 are the coding genes of mKate2.
  • sequence 61 The pT1+T1+89-mKate2 plasmid is shown in sequence 61.
  • nucleotides 7172-7264 from the 5' end are 5 ⁇ UAS sequences
  • nucleotides 7280-7293 are T1 sequences
  • nucleotides 6-65 are CMVmini promoters
  • 89- The 102 nucleotide is the T1 sequence
  • nucleotides 181-899 are the coding genes of mKate2.
  • sequence 62 The pT1+T1+94-mKate2 plasmid is shown as sequence 62.
  • nucleotides 7177-7269 from the 5' end are 5 x UAS sequences
  • nucleotides 7285-7298 are T1 sequences
  • nucleotides 6-65 are CMVmini promoters
  • 94- The 107th nucleotide is the T1 sequence
  • nucleotides 186-904 are the coding genes of mKate2.
  • sequence 63 The pT1+T1+100-mKate2 plasmid is shown as sequence 63.
  • nucleotides 7203-7295 from the 5' end are 5 ⁇ UAS sequences
  • nucleotides 6-19 are T1 sequences
  • nucleotides 26-85 are CMVmini promoters
  • 120- The nucleotide at position 133 is the T1 sequence
  • the nucleotides at positions 212-930 are the coding genes for mKate2.
  • sequence 64 The pT2+T2+72-mKate2 plasmid is shown as sequence 64.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-190 are T2 sequences
  • nucleotides 197-256 are nucleosides.
  • the acid is the CMVmini promoter
  • the nucleotides 263-276 are the T2 sequence
  • nucleotides 355-1073 are the coding genes of mKate2.
  • sequence 65 The pT2+T2x3+72-mKate2 plasmid is shown as sequence 65.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-190 are T2 sequences
  • nucleotides 197-256 are CMVmini promoters
  • 263- The nucleotide 276 is the T2 sequence
  • the nucleotides 279-292 are the T2 sequence
  • the nucleotides 295-308 are the T2 sequence
  • nucleotides 384-1106 are the coding genes of mKate2.
  • sequence 66 The pT4+T4+72-mKate2 plasmid is shown as sequence 66.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-190 are T4 sequences (target sequence of TALER4 protein)
  • nucleotides 197-256 For the CMVmini promoter nucleotides 263-276 are T4 sequences
  • nucleotides 355-1073 are genes encoding mKate2.
  • the pT4+T4x3+72-mKate2 plasmid is shown in SEQ ID NO:67.
  • sequence 67 nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-190 are T4 sequences, and nucleotides 197-256 are CMVmini promoters, 263- 276 nucleotides are T4 sequences, nucleotides 277-290 are T4 sequences, nucleotides 291-304 are T4 sequences, and nucleotides 383-1101 are mKate2 encoding genes.
  • sequence 71 nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-190 are T32 sequences, and nucleotides 197-256 are CMVmini promoters, 263- 276 nucleotides are T32 sequences, nucleotides 277-290 are T32 sequences, nucleotides 291-304 are T32 sequences, and nucleotides 383-1101 are mKate2 encoding genes.
  • the pCMV-TALER1 plasmid, pT1+T1+72-DsRed plasmid and pEF1a-TagBFP-2A plasmid were co-transfected into HEK293 cells (transfected with 200 ng pCMV-TALER1 plasmid per well, 50 ng pT1+T1+72-DsRed) Granules and 30 ng of pEF1a-TagBFP-2A plasmid) were analyzed by flow cytometry 48 hours after transfection to detect the fluorescence intensity of EYFP, the fluorescence intensity of DsRed and the fluorescence intensity of TagBFP. A control treatment without the addition of the pCMV-TALER1 plasmid was set.
  • the inhibition multiples and percent inhibition results are shown in Figures 6 and 4.
  • the TALER protein has no periodic inhibitory behavior, and the closer the TALER binding site is to the miniCMV promoter, the stronger the inhibitory effect.
  • the pCMV-TALER2 plasmid, pT2+T2+72-mKate2 plasmid (or T2+T2x3+72-mKate2 plasmid) and pEF1a-TagBFP-2A plasmid were co-transfected into HEK293 cells (transfected with 200 ng pCMV-TALER2 plasmid per well, 50 ng pT2 per well).
  • the pCMV-TALER2 plasmid was replaced with pCMV-TALER4 plasmid, and the pT2+T2+72-mKate2 plasmid was replaced with pT4+T4+72-mKate2 plasmid. ) Carry out the above steps.
  • the plasmid shown in SEQ ID NO: 72 of the Sequence Listing was synthesized.
  • sequence 72 the 4766-5033 nucleotide from the 5' end is the doxycycline response element TRE (wherein the 4766-4961 nucleotide is tetO, the first The 4976-5033 nucleotide is the CMVmini promoter), the 5113-8250 nucleotide is the coding gene of TALER14 protein, the nucleotides 9306-9549 are cHS4core, and the nucleotides 9625-9868 are cHS4core.
  • TRE doxycycline response element
  • the nucleotides 9998-10079 are 5 ⁇ UAS sequences
  • the nucleotides 10095-10111 are T14 sequences (target sequences of TALER14 protein)
  • the nucleotides 10118-10177 are CMVmini promoters
  • 10184- 10200 nucleotides are T14 sequences
  • 10201-10217 nucleotides are T14 sequences
  • 10218-10234 nucleotides are T14 sequences
  • 1013-11031 nucleotides are mKate2 encoding genes, 11979-
  • the nucleotide 12222 is cHS4core
  • the nucleotide at 12298-12541 is cHS4core
  • the nucleotide at 12658-12925 is the doxycycline response element TRE (wherein nucleotides 112658-12853 are tetO, 12868-
  • the 12925 nucleotide is the CMVmini promoter
  • doxycycline After the addition of doxycycline, doxycycline binds to rtTA, activates the doxycycline response element TRE, TALER14 protein and EYFP are expressed, TALER14 protein binds to the T14 sequence, and transcriptional inhibition is achieved by steric hindrance, CMVmini between T14 sequences
  • the promoter is inactivated, whereby mKate2 is inhibited from expression.
  • the expression level of TALFP14 was estimated to be under Dox-induced expression, and the change in the expression level of mKate2 reflects the inhibition of the CMVmini promoter between two T14 sequences by TALER14.
  • Example 3 modular construction of the gene line of the TALER protein cascade
  • the pCAG-rtTA-2A-Gal4/vp16 plasmid is shown in sequence 73.
  • sequence 73 the nucleotides 4253-4930 from the 5' end are the CAG promoter, the nucleotides 6004-6747 are the coding genes of rtTA, and the nucleotides 6748-6813 are the coding genes of the 2A junction peptide.
  • the nucleotide at position 6820-7503 is a gene encoding Gal4/vp16.
  • the pT14+T14+72-mKate2 plasmid is shown as sequence 74.
  • sequence 74 nucleotides 4275-4367 from the 5' end are 5 x UAS sequences, nucleotides 4383-4399 are T14 sequences (target sequence of TALER14 protein), nucleotides 4406-4465 For the CMVmini promoter, 4472-4488
  • the nucleotide is the T14 sequence, and the nucleotides 4538-5243 are the coding genes of mKate2.
  • the pT14+T14+72-mKate2 plasmid has a binding site for two TALER14 proteins (pT14BS2).
  • the pT14+T14x3+72-mKate2 plasmid is shown in sequence 75.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-193 are T14 sequences
  • nucleotides 200-259 are CMVmini promoters
  • 266- 282 nucleotides are T14 sequences
  • 283-299 nucleotides are T14 sequences
  • nucleotides 300-316 are T14 sequences
  • nucleotides 395-1113 are mKate2 encoding genes.
  • the pT14+T14x3+72-mKate2 plasmid has four binding sites for the TALER14 protein (pT14BS4).
  • sequence 76 The pT21+T21+72-mKate2 plasmid is shown as sequence 76.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4401 are T21 sequences (target sequences of TALER21 protein)
  • nucleotides 4408-4467 For the CMVmini promoter the nucleotide number 4447-4492 is the T21 sequence
  • the nucleotide number 4542-5247 is the coding gene of mKate2.
  • the pT21+T21+72-mKate2 plasmid has a binding site for two TALER21 proteins (pT21BS2).
  • the pTRE-EBFP2 plasmid is shown in SEQ ID NO: 78.
  • the nucleotides 4250-4555 from the 5' end are the doxycycline response element TRE (wherein the 4250-4482 nucleotide is tetO and the 4496-4555 nucleotide is the CMVmini promoter),
  • the nucleotide 4661-5380 is the coding gene for EBFP2 (enhanced blue luciferin).
  • the pTRE-TALER14-4xT plasmid is shown in sequence 79.
  • nucleotides 67-334 from the 5' end are the doxycycline response element TRE (wherein nucleotides 67-262 are tetO and nucleotides 277-334 are CMVmini promoters),
  • the nucleotide at positions 423-3560 is the gene encoding the TALER14 protein.
  • the pTRE-TALER21-4xT plasmid is shown in SEQ ID NO: 80.
  • nucleotides 67-334 from the 5' end are the doxycycline response element TRE (wherein nucleotides 67-262 are tetO and nucleotides 277-334 are CMVmini promoters),
  • the nucleotide at positions 423-3764 is the gene encoding the TALER21 protein.
  • sequence 81 The pT14+T14+72_TALER21 plasmid is shown in sequence 81.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4399 are T14 sequences
  • nucleotides 4406-4465 are CMVmini promoters
  • 4472- The 4488 nucleotide is the T14 sequence
  • nucleotide 4574-7915 is the gene encoding the TALER21 protein.
  • sequence 82 The pT14+T14x3+72_TALER21 plasmid is shown in sequence 82.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4399 are T14 sequences
  • the 4406-4465 nucleotide is the CMVmini promoter
  • the 4472-4488 nucleotide is the T14 sequence
  • the 4489-4505 nucleotide is the T14 sequence
  • the 4506-4522 nucleotide is the T14 sequence, the 4608th.
  • the nucleotide -7949 is the gene encoding the TALER21 protein.
  • sequence 83 The pT21+T21+72_TALER14 plasmid is shown in sequence 83.
  • nucleotides 4275-4367 from the 5' end are 5 ⁇ UAS sequences
  • nucleotides 4383-4401 are T21 sequences
  • nucleotides 4408-4467 are CMVmini promoters
  • 4474- The 4492 nucleotide is the T21 sequence
  • nucleotides 4578-7715 are the coding genes of the TALER14 protein.
  • sequence 84 The pT21+T21x3+72_TALER14 plasmid is shown as sequence 84.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4401 are T21 sequences
  • nucleotides 4408-4467 are CMVmini promoters
  • 4474- The 4492 nucleotide is the T21 sequence
  • the 4493-4511 nucleotide is the T21 sequence
  • the 4512-4530 nucleotide is the T21 sequence
  • the 4616-7753 nucleotide is the TALER14 protein coding gene.
  • the pCAG-EYFP plasmid is shown in SEQ ID NO:85.
  • the nucleotides 3320-3997 from the 5' end are the CAG promoter, and the nucleotides 5064-5783 are the coding genes of EYFP (enhanced yellow luciferin).
  • doxycycline After the addition of doxycycline, doxycycline binds to rtTA, activates the doxycycline response element TRE, TALER protein (TALER14 protein or TALER21 protein is exemplified) and EBFP2 are expressed, TALER protein binds to Tx sequence, sterically hindered The transcriptional repression is exerted, and the CMVmini promoter between the Tx sequences is inactivated, whereby mKate2 is inhibited from expressing.
  • the expression level of TALFP protein was estimated by EBFP2 expression level, and the expression level of mKate2 reflected the inhibition of CMVmini promoter between Tx sequences by TALER protein.
  • HEC293 cells were transfected with pCAG-rtTA-2A-Gal4/vp16 plasmid, pTRE-EBFP2 plasmid, pTRE-TALER14-4xT plasmid, pT14+T14+72-mKate2 plasmid and pCAG-EYFP plasmid (100 ng pCAG per well) rtTA-2A-Gal4/vp16 plasmid, 50 ng pTRE-EBFP2 plasmid, 50 ng pTRE-TALER14-4xT plasmid, 100 ng pT14+T14+72-mKate2 plasmid and 50 ng pCAG-EYFP plasmid), transfected simultaneously with DOX in cell culture system (Making a DOX concentration of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500 or 1000 ng/mL; setting a blank control without DOX, expressed as
  • a 24-well plate was prepared, and 0.5 mL of HEK293 cell suspension (containing 6 ⁇ 10 4 HEK293 cells) was inoculated into each well. After 24 hours of culture, the new DMEM medium was replaced and then subjected to plasmid transfection.
  • Fluorescence intensity of mKate2 / fluorescence intensity of EYFP fluorescence intensity of corrected mKate2.
  • Fluorescence intensity of EBFP2 / fluorescence intensity of EYFP fluorescence intensity of corrected EBFP2.
  • n(pTxBS2) or n(pTxBS4) represents the Hill coefficient.
  • the thick line area represents the input range for experimental observations.
  • the thin line region represents the predicted transfer equation curve for the fitted Hill equation.
  • the Hill coefficient ranges from 0.67 to 1.15, indicating that there is no strong synergistic effect of the binding of the TALER protein to the corresponding promoter.
  • the inventors developed and studied a composite gene line using the TALER protein as a building block.
  • the two TALER protein/promoter pairs are ligated in tandem to form a TALER cascade (see Figure 10A for a schematic representation), and the input of the next TALER protein promoter corresponds to the output of the previous TALER protein.
  • the second TALER protein inhibits the output of the reporter gene (mKate2 gene).
  • DOX induces the expression of the first TALER protein, it can inhibit the expression of the second TALER protein, thereby releasing its inhibition of mKate2 and increasing the expression level of mKate2.
  • the pCAG-rtTA-2A-Gal4/vp16 plasmid, pTRE-EBFP2 plasmid, pTRE-TALER14-4xT plasmid, pT14+T14+72_TALER21 plasmid, pT21+T21+72-mKate2 plasmid and pCAG-EYFP plasmid were co-transfected into HEK293 cells ( Each well was transfected with 100 ng of pCAG-rtTA-2A-Gal4/vp16 plasmid, 50 ng of pTRE-EBFP2 plasmid, 50 ng of pTRE-TALER14-4xT plasmid, 50 ng of T14+T14+72_TALER21 plasmid, 100 ng of pT21+T21+72-mKate2 plasmid and 50 ng of pCAG -EYFP plasmid), transfection while adding DOX to the cell culture
  • Plasmid transfection of cells 24-well plates were prepared, and 0.5 mL of HEK293 cell suspension (containing 6 ⁇ 10 4 HEK293 cells) was inoculated into each well. After 24 hours of culture, the new DMEM medium was replaced, followed by plasmid transfection.
  • Fluorescence intensity of mKate2 / fluorescence intensity of EYFP fluorescence intensity of corrected mKate2.
  • the inventors used Rainbow Calibration Particles to convert the corresponding amount of fluorescent units into a standardized unit amount.
  • the peak of the EYFP fluorescence mean using the microparticles and its absolute MEFL unit amount establish a linear relationship between MEFL and EYFP in the log domain.
  • a linear relationship is also established between MEBFP and TagBFP or EBFP2, MECY and mKate2 or DsRed, MEAPCY7 and iRFP in a similar manner.
  • the scatter plots were drawn using the corrected data using Matlab (MathWorks) improved flow cell data reading and visualization tools.
  • mean (FL) indicates the mean value of MECY, MEFL or MEBFP in the test cell population.
  • Mean (Control) represents the mean of the reported fluorescence of the constitutive expression in the cell population. The inventors then calculated the percent inhibition and the inhibition multiple using the following formula:
  • the pCAG-EYFP plasmid was the pCAG-EYFP plasmid of Example 3.
  • the pTRE-EBFP2 plasmid was the pTRE-EBFP2 plasmid of Example 3.
  • the pTRE-mKate2 plasmid is shown in sequence 87.
  • sequence 87 the nucleotides 4250-4555 from the 5' end are the doxycycline response element TRE (wherein the 4250-4482 nucleotide is tetO and the 4496-4555 nucleotide is the CMVmini promoter),
  • the nucleotide at positions 4466-5369 is the coding gene for mKate2.
  • sequence 88 The pEF1a-rtTA-2A-Hyg plasmid is shown in sequence 88.
  • nucleotides 6207-7380 from the 5' end are pEF1a (promoter)
  • nucleotides 7441-8184 are rtTA encoding genes
  • nucleotides 8185-8250 are 2A linked peptides.
  • the coding gene, nucleotides 8263-9288, is the Hyg gene (hygromycin resistance gene).
  • the pTRE-EYFP-2A-TALER14 plasmid is shown in SEQ ID NO: 89.
  • the nucleotides 4250-4555 from the 5' end are the doxycycline response element TRE (wherein the 4250-4482 nucleotide is tetO and the 4496-4555 nucleotide is the CMVmini promoter),
  • the nucleotides of 4676-5392 are the coding genes of EYFP
  • the nucleotides of nucleotides 5399-5452 are the genes encoding the 2A junction peptide
  • the nucleotides of positions 5462-8599 are the genes encoding the TALER14 protein.
  • sequence 93 The pT10+T10x3+72-mKate2 plasmid is shown in Sequence 93.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-196 are T10 sequences (target sequences of TALER10 protein)
  • nucleotides 203-262 For the CMVmini promoter, the nucleotides 269-288 are T10 sequences, the nucleotides 289-308 are T10 sequences, the nucleotides 309-328 are T10 sequences, and the nucleotides 407-1152 are The coding gene of mKate2.
  • sequence 94 The pT12+T12x3+72-mKate2 plasmid is shown as sequence 94.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4400 are T12 sequences (target sequences of TALER12 protein)
  • nucleotides 4491-4508 are T12 sequences
  • the nucleotides of 4509-4526 are T12 sequences
  • nucleotides of 4576-5281 are The coding gene of mKate2.
  • the pTRE-EYFP-2A-TALER9 plasmid is shown in SEQ ID NO:95.
  • the nucleotides 4250-4555 from the 5' end are the doxycycline response element TRE (wherein the 4250-4482 nucleotide is tetO and the 4496-4555 nucleotide is the CMVmini promoter),
  • the nucleotides 4676-5392 are the coding genes of EYFP
  • the nucleotides 5399-5452 are the coding genes of the 2A junction peptide
  • the nucleotides 5426-9007 are the coding genes of the TALER9 protein.
  • the pTRE-EYFP-2A-TALER10 plasmid is shown as sequence 96.
  • the nucleotides 4250-4555 from the 5' end are the doxycycline response element TRE (wherein the 4250-4482 nucleotide is tetO and the 4496-4555 nucleotide is the CMVmini promoter),
  • the nucleotides of 4676-5392 are the coding genes of EYFP
  • the nucleotides of nucleotides 5399-5452 are the genes encoding the 2A junction peptide
  • the nucleotides of positions 5462-8905 are the genes encoding the TALER10 protein.
  • the pTRE-EYFP-2A-TALER12 plasmid is shown in Sequence 97.
  • the nucleotides 4250-4555 from the 5' end are the doxycycline response element TRE (wherein the 4250-4482 nucleotide is tetO and the 4496-4555 nucleotide is the CMVmini promoter),
  • the nucleotides 4676-5392 are the coding genes of EYFP
  • the nucleotides 5399-5452 are the coding genes of the 2A junction peptide
  • the nucleotides 5426-5871 are the coding genes of the TALER12 protein.
  • the pTRE-EYFP-2A-TALER21 plasmid is shown in sequence 98.
  • the nucleotides 4250-4555 from the 5' end are the doxycycline response element TRE (wherein the 4250-4482 nucleotide is tetO and the 4496-4555 nucleotide is the CMVmini promoter),
  • the nucleotides 4676-5392 are the coding genes of EYFP
  • the nucleotides 5399-5452 are the coding genes of the 2A junction peptide
  • the nucleotides 5468-8803 are the coding genes of the TALER21 protein.
  • HEK293 cells were co-transfected with pEF1a-rtTA-2A-Hyg plasmid, pCAG-EYFP plasmid, pTRE-EBFP2 plasmid and pTRE-mKate2 plasmid (100 ng pEF1a-rtTA-2A-Hyg plasmid, 100 ng pCAG-EYFP plasmid per well, 100 ng of pTRE-EBFP2 plasmid and 100 ng of pTRE-mKate2 plasmid), transfection and addition of DOX to the cell culture system (to a DOX concentration of 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500 or 1000 ng/ mL; set blank control without DOX, expressed as 0 ng/mL), flow cytometry analysis 48 hours after transfection, detection of fluorescence intensity of mKate2, fluorescence intensity of EBFP2, and fluorescence intensity of EYFP.
  • DOX DOX
  • Plasmid transfection of cells 24-well plates were prepared, and 0.5 mL of HEK293 cell suspension (containing 6 ⁇ 10 4 HEK293 cells) was inoculated into each well. After 24 hours of culture, the new DMEM medium was replaced, followed by plasmid transfection.
  • the gene lines used to construct the EBFP2 and mKate2 transformation models are shown in Figure 11A.
  • Figure 11C shows the relationship between the corrected EBFP2 and mKate2, and the linear regression equation is plotted above the graph.
  • HEK293 cells were co-transfected with pEF1a-rtTA-2A-Hyg plasmid, pCAG-TagBFP plasmid, pTRE-EYFP-2A-TALER14 plasmid and pTRE-mKate2-2A-TALER14 plasmid (transfection of 100 ng pEF1a-rtTA-2A-Hyg per well) Plasmid, 100 ng pCAG-TagBFP plasmid, 100 ng pTRE-EYFP-2A-TALER14 plasmid and 100 ng pTRE-mKate2-2A-TALER14 plasmid), transfected and added DOX to the cell culture system (to a DOX concentration of 0.5, 1, 2) 5, 10, 20, 50, 100, 200, 500 or 1000 ng/mL; set blank control without DOX, expressed as 0 ng/mL), flow cytometry analysis 48 hours after transfection, detection of fluorescence intensity of mKate2 , Flu
  • Plasmid transfection of cells 24-well plates were prepared, and 0.5 mL of HEK293 cell suspension (containing 6 ⁇ 10 4 HEK293 cells) was inoculated into each well. After 24 hours of culture, the new DMEM medium was replaced, followed by plasmid transfection.
  • the gene lines used to construct the EYFP and mKate2 normalization models are shown in Figure 11B.
  • Figure 11D shows the relationship between the corrected EYFP and mKate2, and the linear regression equation is plotted above the graph.
  • the pEF1a-rtTA-2A-Hyg plasmid, pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pTRE-EYFP-2A-TALER9 plasmid and pT9+T9x3+72-mKate2 plasmid were co-transfected into HEK293 cells (per well) Transfection of 100 ng of pEF1a-rtTA-2A-Hyg plasmid, 100 ng of pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, 100 ng of pTRE-EYFP-2A-TALER9 plasmid and 100 ng of p9+T9x3+72-mKate2 plasmid, transfection At the same time, DOX was added to the cell culture system (the DOX concentration was 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500 or 1000 ng/mL;
  • Plasmid transfection of cells 24-well plates were prepared, and 0.5 mL of HEK293 cell suspension (containing 6 ⁇ 10 4 HEK293 cells) was inoculated into each well. After 24 hours of culture, the new DMEM medium was replaced, followed by plasmid transfection.
  • the pTRE-EYFP-2A-TALER9 plasmid was replaced with the pTRE-EYFP-2A-TALER9 plasmid, and the pT9+T10x3+72-mKate2 plasmid was used instead of the pT9+T9x3+72-mKate2 plasmid.
  • the pTRE-EYFP-2A-TALER12 plasmid was replaced with the pTRE-EYFP-2A-TALER9 plasmid, and the pT9+T12x3+72-mKate2 plasmid was replaced with the pT12+T12x3+72-mKate2 plasmid, and the above procedure was carried out.
  • the pTRE-EYFP-2A-TALER9 plasmid was replaced with the pTRE-EYFP-2A-TALER9 plasmid, and the pT9+T14x3+72-mKate2 plasmid was replaced with the pT14+T14x3+72-mKate2 plasmid, and the above procedure was carried out.
  • the pTRE-EYFP-2A-TALER21 plasmid was replaced with the pTRE-EYFP-2A-TALER9 plasmid, and the pT9+T21x3+72-mKate2 plasmid was replaced with the pT21+T21x3+72-mKate2 plasmid, and the above procedure was carried out.
  • the gene line for the transfer function curve analysis is shown in Fig. 12A (the indicated line with an arrow at the end indicates positive regulation, and the indicated line with short end indicates negative regulation).
  • n (TALERx) represents the Hill coefficient, which ranges from 0.51 to 1.56.
  • the thick line area represents the input range for experimental observations.
  • the thin line region represents the predicted transfer function curve for the fitted Hill equation.
  • B represents the fluorescence intensity of the output
  • A represents the fluorescence intensity of the input representing the TALER concentration
  • ⁇ 2 represents the maximum production rate of the TALER promoter
  • ⁇ 2 represents the leakage production rate of the TALER promoter
  • k represents the inhibition ratio of 50%. Enter the concentration, n for the Hill coefficient and ⁇ for the decay rate.
  • each TALER For cascading action multiple prediction, the input and output of each TALER are first normalized to MEBFP by linear interpolation, and then the output of the first level TALER is used as the input of the second level TALER. Then the experimentally detected first-stage TALER input range enters the simulation of the gene line and passes the second-level TALER The output calculates the maximum change factor.
  • Sequence Target ⁇ FF4 nucleotides 8909-8930 are target sequence of shRNA-FF4 Target ⁇ FF4, nucleotides 8913-8952 are target sequences of shRNA-FF4 Target ⁇ FF4, nucleotides 8959-8980 The acid is the target sequence of shRNA-FF4 Target ⁇ FF4.
  • sequence 100 The pT9+T9x3+72-mKate2-2A-TALER12-4xTarget ⁇ FF6 plasmid is shown as sequence 100.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4403 are T9 sequences (target sequences of TALER9 protein)
  • nucleotides 4410-4469 For the CMVmini promoter, the 4476-4496 nucleotide is the T9 sequence, the 4497-4517 nucleotide is the T9 sequence, the 4518-4538 nucleotide is the T9 sequence, and the 4600-5295 nucleotide is The coding gene of mKate2, the nucleotide number 5302-5355 is the coding gene of 2A-ligation peptide, the nucleotide of 5365-8604 is the coding gene of TALER12 protein, and the nucleotide of 8689-8710
  • sequence 101 The pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF4 plasmid is shown in sequence 101.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4403 are T9 sequences (target sequences of TALER9 protein)
  • nucleotides 4476-4496 are T9 sequences
  • nucleotides 4497-4517 are T9 sequences
  • nucleotides 4518-4538 are T9 sequences
  • nucleotides 4603-5295 are The coding gene of mKate2, the nucleotide number 5302-5355 is the coding gene of 2A-ligation peptide, the nucleotide of 5365-8502 is the coding gene of TALER14 protein, and the nucleotide of 8593-8614 is the target of shRNA-FF4
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4403 are T9 sequences (target sequences of TALER9 protein)
  • the 4476-4496 nucleotide is the T9 sequence
  • the 4497-4517 nucleotide is the T9 sequence
  • the 4518-4538 nucleotide is the T9 sequence
  • the 4600-5295 nucleotide is The coding gene of mKate2
  • nucleotide number 5302-5355 is the coding gene of 2A-ligation peptide
  • nucleotide of nucleotides 5365-8706 is the coding gene of TALER21 protein
  • sequence 103 The pT10+T10x3+72-EYFP-2A-TALER9-4xTarget ⁇ FF5 plasmid is shown in Sequence 103.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4402 are T10 sequences (target sequences of TALER10 protein)
  • nucleotides 4409-4468 For the CMVmini promoter
  • the nucleotides 4475-4494 are T10 sequences
  • the nucleotides 4495-4514 are T10 sequences
  • the nucleotides 4515-4534 are T10 sequences
  • nucleotides 4596-5312 are The coding gene of EYFP
  • nucleotides 5319-5372 are the coding genes of the 2A-ligation peptide
  • nucleotides 5382-8927 are the genes encoding the TALER9 protein
  • the sequence Target ⁇ FF5 is the target sequence of shRNA-FF5 Target ⁇ FF5
  • the nucleotide number 9061-9082 is the target sequence of shRNA-FF5 Target ⁇ FF5
  • the nucleoside at position 9083-9104 The acid is the target sequence of shRNA5 Target ⁇ FF5.
  • sequence 104 The pT10+T10x3+72-mKate2-2A-TALER12-4xTarget ⁇ FF6 plasmid is shown as sequence 104.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4402 are T10 sequences (target sequences of TALER10 protein)
  • nucleotides 4409-4468 For the CMVmini promoter, the nucleotides 4475-4494 are T10 sequences
  • the nucleotides 4495-4514 are T10 sequences
  • nucleotide of nucleotides 5298-5351 is the coding gene of 2A-ligation peptide
  • nucleotide of 5361-8600 is the coding gene of TALER12 protein
  • sequence 105 The pT10+T10x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF4 plasmid is shown as sequence 105.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4402 are T10 sequences (target sequence of TALER10 protein)
  • nucleotides 4409-4468 For The CMVmini promoter has a T10 sequence at nucleotides 4475-4494, a T10 sequence at nucleotides 4495-4514, a T10 sequence at nucleotides 4515-4534, and a mKate2 at nucleotides 4599-5291.
  • the coding gene, the 5298-5351 nucleotide is the coding gene of the 2A junction peptide
  • the nucleotides 5361-8498 are the coding genes of the TALER14 protein
  • the nucleotides 8889-8610 are the target sequences of shRNA-FF4.
  • Target ⁇ FF4 nucleotides 8611-8632 are target sequences of shRNA-FF4 Target ⁇ FF4
  • nucleotides 8633-8654 are target sequences of shRNA-FF4 Target ⁇ FF4
  • sequence 106 The pT10+T10x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF5 plasmid is shown as sequence 106.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4402 are T10 sequences (target sequences of TALER10 protein)
  • nucleotides 4409-4468 For the CMVmini promoter, the nucleotides of 4475-4494 are T10 sequences, the nucleotides of 4495-4514 are T10 sequences, the nucleotides of 4515-4534 are T10 sequences, and the nucleotides of 4599-5291 are The coding gene of mKate2, the nucleotide of nucleotides 5298-5351 is the coding gene of 2A-ligation peptide, the nucleotide of 5361-8498 is the coding gene of TALER14 protein, and the nu
  • sequence 107 The pT10+T10x3+72-mKate2-2A-TALER21-4xTarget ⁇ FF3 plasmid is shown as sequence 107.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4402 are T10 sequences (target sequences of TALER10 protein)
  • nucleotides 4409-4468 For the CMVmini promoter, the nucleotides of 4475-4494 are T10 sequences, the nucleotides of 4495-4514 are T10 sequences, the nucleotides of 4515-4534 are T10 sequences, and the nucleotides of 4599-5291 are The coding gene of mKate2, the nucleotide of nucleotides 5298-5351 is the coding gene of 2A-linked peptide, the nucleotide of 5361-8702 is the coding gene of TALER21 protein, and the nucle
  • sequence 108 The pT12+T12x3+72-EYFP-2A-TALER9-4xTarget ⁇ FF5 plasmid is shown in sequence 108.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4400 are T12 sequences (target sequences of TALER12 protein)
  • the nucleotides of 4473-4490 are T12 sequences
  • the nucleotides of 4491-4508 are T12 sequences
  • the nucleotides of 4509-4526 are T12 sequences
  • nucleotides of 4588-5304 are The coding gene of EYFP
  • nucleotides of 5311-5364 are the genes encoding the 2A-ligation peptide
  • nucleotides of nucleotides 5374-8919 are the genes encoding the TALER9 protein
  • the sequence Target ⁇ FF5 the nucleotides 9031-9052 are the target sequence of shRNA-FF5 Target ⁇ FF5
  • the nucleotides at 0903-9074 are the target sequence of shRNA-FF5 Target ⁇ FF5
  • the nucleosides 9075-9096 The acid is the target sequence of shRNA-FF5 Target ⁇ FF5.
  • Sequence Target ⁇ FF4 nucleotides 8918-8939 are the target sequence of shRNA-FF4 Target ⁇ FF4, nucleotides 8940-8961 are target sequences of shRNA-FF4 Target ⁇ FF4, nucleosides 8968-8989 The acid is the target sequence of shRNA-FF4 Target ⁇ FF4.
  • sequence 110 The pT12+T12x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF4 plasmid is shown as sequence 110.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4400 are T12 sequences (target sequences of TALER12 protein)
  • the nucleotides of 4473-4490 are T12 sequences
  • the nucleotides of 4491-4508 are T12 sequences
  • the nucleotides of 4509-4526 are T12 sequences
  • nucleotides of 4591-5283 are The coding gene of mKate2
  • the 5290-5343 nucleotide is the coding gene of 2A-ligation peptide
  • the 5353-8490 nucleotide is the coding gene of TALER14 protein
  • nucleotides 8581-8602 are the target of sh
  • the sequence Target ⁇ FF3, the nucleotide number 8808-8828 is the target sequence of shRNA-FF3 Target ⁇ FF3
  • the nucleotide number 8834-8854 is the target sequence of shRNA-FF3 Target ⁇ FF3
  • the acid is the target sequence of shRNA-FF3 Target ⁇ FF3.
  • sequence 112 The pT14+T14x3+72-EYFP-2A-TALER9-4xTarget ⁇ FF5 plasmid is shown as sequence 112.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4399 are T14 sequences (target sequence of TALER14 protein)
  • nucleotides 4406-4465 For the CMVmini promoter, the nucleotides 4472-4488 are T14 sequences, the nucleotides 4489-4505 are T14 sequences, the nucleotides 4506-4522 are T14 sequences, and the nucleotides 4584-5300 are The coding gene of EYFP, the 5307-5360 nucleotide is the coding gene of 2A junction peptide, the 5370-8915 nucleotide is the coding gene of TALER9 protein, and the nucleotide of 9005-9026 is the target of shRNA-FF
  • sequence 113 The pT14+T14x3+72-EYFP-2A-TALER12-4xTarget ⁇ FF5 plasmid is shown as sequence 113.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4399 are T14 sequences (target sequence of TALER14 protein)
  • nucleotides 4406-4465 For the CMVmini promoter, the nucleotides 4472-4488 are T14 sequences, the nucleotides 4489-4505 are T14 sequences, the nucleotides 4506-4522 are T14 sequences, and the nucleotides 4584-5300 are The coding gene of EYFP, the 5307-5360 nucleotide is the coding gene of 2A-ligation peptide, the nucleotide of 5370-8609 is the coding gene of TALER12 protein, and the nucleotide of 8699-8720 is the target of
  • sequence 114 The pT14+T14x3+72-mKate2-2A-TALER21-4xTarget ⁇ FF3 plasmid is shown as sequence 114.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4399 are T14 sequences (target sequence of TALER14 protein)
  • nucleotides 4406-4465 For the CMVmini promoter, the nucleotides 4472-4488 are T14 sequences, the nucleotides 4489-4505 are T14 sequences, the nucleotides 4506-4522 are T14 sequences, and the nucleotides 4584-5279 are The coding gene of mKate2, the nucleotide of nucleotides 5286-5339 is the coding gene of 2A-ligation peptide, the nucleotide of nucleotides 5349-8690 is the coding gene of TALER21 protein, and the nu
  • sequence 115 The pT21+T21x3+72-EYFP-2A-TALER14-4xTarget ⁇ FF4 plasmid is shown as sequence 115.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4401 are T21 sequences (target sequences of TALER21 protein)
  • nucleotides 4408-4467 For the CMVmini promoter, the nucleotide number 4447-4492 is the T21 sequence, the nucleotides 4493-4511 are the T21 sequence, the nucleotides 4512-4530 are the T21 sequence, and the nucleotides 4925-5308 are The coding gene of EYFP, the nucleotides at positions 5315-5368 are the genes encoding the 2A-linker peptide, the nucleotides at positions 5378-8515 are the genes encoding the TALER14 protein, and the nucleotides at positions 80
  • the sequence Target ⁇ FF4 is the target sequence of shRNA-FF4 Target ⁇ FF4
  • the nucleotide number 8650-8671 is the target sequence of shRNA-FF4 Target ⁇ FF4
  • the acid is the target sequence of shRNA-FF4 Target ⁇ FF4.
  • sequence 116 The pT21+T21x3+72-EYFP-2A-TALER9-4xTarget ⁇ FF5 plasmid is shown as sequence 116.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4401 are T21 sequences (target sequences of TALER21 protein)
  • nucleotides 4408-4467 For the CMVmini promoter the nucleotides 4447-4490 are T21 sequences
  • the nucleotides 4491-4509 are T21 sequences.
  • nucleotides 4510-4530 are T21 sequences
  • nucleotides 4592-5308 are EYFP encoding genes
  • nucleotides 5315-5368 are 2A connecting peptide encoding genes
  • 5378-8923 nucleus The nucleotide is the gene encoding the TALER9 protein
  • the nucleotides of nucleotides 9013-9034 are the target sequence of shRNA-FF5 Target ⁇ FF5
  • nucleotides of nucleotides 9035-9056 are the target sequence of shRNA-FF5 Target ⁇ FF5
  • the nucleotide sequence of 9078 is the target sequence of shRNA-FF5 Target ⁇ FF5
  • nucleotide of nucleotides 9079-9100 is the target sequence of shRNA-FF5 Target ⁇ FF5.
  • sequence 117 The pT21+T21x3+72-EYFP-2A-TALER10-4xTarget ⁇ FF4 plasmid is shown as sequence 117.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4401 are T21 sequences (target sequences of TALER21 protein)
  • nucleotides 4408-4467 For the CMVmini promoter, the nucleotide number 4447-4492 is the T21 sequence, the nucleotides 4493-4511 are the T21 sequence, the nucleotides 4512-4530 are the T21 sequence, and the nucleotides 4925-5308 are The coding gene of EYFP, the nucleotides at positions 5315-5368 are the genes encoding the 2A linker peptide, the nucleotides at positions 5378-8821 are the genes encoding the TALER10 protein, and the nucleotides at nucleo
  • the pSIREN_U6-shRNA-FF4 plasmid is shown as sequence 121.
  • sequence 121 the nucleotides 229-477 from the 5' end are the U6 promoter, the nucleotides 484-536 are the coding genes of shRNA-FF4, and the nucleotides 741-1329 are the CMV IE promoter.
  • the nucleotides 1361-2311 are genes encoding iRFP (near-infrared luciferin).
  • the pSIREN_U6-shRNA-FF6 plasmid is shown as sequence 123.
  • sequence 123 the nucleotides 229-477 from the 5' end are the U6 promoter, the nucleotides 484-536 are the genes encoding shRNA-FF6, and the nucleotides 741-1329 are the CMV IE promoter.
  • the nucleotides 1361-2311 are genes encoding iRFP (near-infrared luciferin).
  • shRNA-FF3, shRNA-FF4, shRNA-FF5, and shRNA-FF6 are shRNAs.
  • a synthetic gene switch is composed of two elements that are mutually transcriptionally repressible, and the switch can suppress the transition of the state of one of the transcriptional repressing elements by an external signal molecule.
  • TAREL proteins with high transcriptional repression, the inventors used two mutually inhibited TALER proteins as modules to construct TALER switches, and used microRNA/shRNA as a signal to control the state of TALER switches (Fig. 13A, wavy lines represent shRNA, shRNA targets) The locus is represented by a small square).
  • the TALER9 protein, TALER10 protein, TALER12 protein, TALER14 protein and TALER21 protein which have strong inhibition and strong orthogonality in the previous analysis, were selected as modules for constructing the TALER protein switch.
  • a double gene expression vector is formed by ligating the TALER protein and the mKate2 or EYFP fluorescent reporter gene from the cleavage peptide 2A.
  • Plasmid transfection of cells 24-well plates were prepared, and 0.5 mL of HEK293 cell suspension (containing 6 ⁇ 10 4 HEK293 cells) was inoculated into each well. After 24 hours of culture, the new DMEM medium was replaced, followed by plasmid transfection.
  • the pT10+T10x3+72-EYFP-2A-TALER9-4xTarget ⁇ FF5 plasmid was replaced with pT14+T14x3+72-EYFP-2A-TALER9-4xTarget ⁇ FF5 plasmid, and pT9+T9x3+72-mKate2-2A-TALER14-4xTarget was used.
  • the above procedure was carried out by substituting the FF4 plasmid for the pT9+T9x3+72-mKate2-2A-TALER10-4xTarget ⁇ FF4 plasmid.
  • the pT10+T10x3+72-EYFP-2A-TALER9-4xTarget ⁇ FF5 plasmid was replaced with pT14+T14x3+72-EYFP-2A-TALER10-4xTarget ⁇ FF4 plasmid, and pT10+T10x3+72-mKate2-2A-TALER14-4xTarget was used.
  • the above procedure was carried out by substituting the FF5 plasmid for the pT9+T9x3+72-mKate2-2A-TALER10-4xTarget ⁇ FF4 plasmid.
  • the pT10+T10x3+72-EYFP-2A-TALER9-4xTarget ⁇ FF5 plasmid was replaced with pT21+T21x3+72-EYFP-2A-TALER10-4xTarget ⁇ FF4 plasmid, and pT10+T10x3+72-mKate2-2A-TALER21-4xTarget was used.
  • the above procedure was carried out by substituting the FF3 plasmid for the pT9+T9x3+72-mKate2-2A-TALER10-4xTarget ⁇ FF4 plasmid.
  • the pT10+T10x3+72-EYFP-2A-TALER9-4xTarget ⁇ FF5 plasmid was replaced with pT14+T14x3+72-EYFP-2A-TALER12-4xTarget ⁇ FF5 plasmid, and pT12+T12x3+72-mKate2-2A-TALER14-4xTarget was used.
  • the above procedure was carried out by substituting the FF4 plasmid for the pT9+T9x3+72-mKate2-2A-TALER10-4xTarget ⁇ FF4 plasmid.
  • FIG. 13C The matrix shows a representative flow cytometry plot, where each row is labeled as the same TALER protein linked to mKate2 in the transfection experiment, and each column is labeled as the same EYFP-linked in the transfection experiment.
  • TALER protein The ratio of EYFP and mKate2 obtained by the experiment and predicted by the zero-displacement analysis has a good correlation, and the goodness of fit is 0.85. This indicates that the experimentally derived zero-displacement analysis can guide the design of the TALER switch.
  • the transfer equation curve based on the Hill equation is used to perform zero-displacement analysis on the TALER switch as shown in Fig. 13B (both solid and dashed lines represent the TALER transfer equation curve, where the horizontal axis of the dashed line corresponds to the output and the vertical axis corresponds to the input, thick line
  • the area represents the input range of the experimental observation
  • the thin line area represents the calculated transfer equation curve of the fitted Hill equation
  • the equilibrium state of the TALER switch represented by the intersection point.
  • the TALER switch being tested usually has two results.
  • Fluorescence intensity of mKate2 / fluorescence intensity of TagBFP fluorescence intensity of corrected mKate2.
  • Fluorescence intensity of EYFP / Fluorescence intensity of TagBFP fluorescence intensity of corrected EYFP.
  • the fluorescence intensity of the corrected mKate2 and the fluorescence intensity of the corrected EYFP were used in both Fig. 14 and Fig. 15.
  • the first group pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT9+T9x3+72-mKate2-2A-TALER12-4xTarget ⁇ FF6 plasmid and pT12+T12x3+72-EYFP-2A-TALER9 HEK293 cells were co-transfected with -4xTarget ⁇ FF5 plasmid (100ng pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, 100ng pT9+T9x3+72-mKate2-2A-TALER12-4xTarget ⁇ FF6 plasmid and 100ng per well) pT12+T12x3+72-EYFP-2A-TALER9-4xTarget ⁇ FF5 plasmid); the second group: pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT9
  • the first group pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF4 plasmid and pT14+T14x3+72-EYFP-2A-TALER9 HEK293 cells were co-transfected with -4xTarget ⁇ FF5 plasmid (100ng pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, 100ng pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF4 plasmid and 100ng per well) pT14+T14x3+72-EYFP-2A-TALER9-4xTarget ⁇ FF5 plasmid); the second group: pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT9
  • the first group pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT9+T9x3+72-mKate2-2A-TALER21-4xTarget ⁇ FF3 plasmid and pT21+T21x3+72-EYFP-2A-TALER9 HEK293 cells were co-transfected with -4xTarget ⁇ FF5 plasmid (100ng pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, 100ng per well) pT9+T9x3+72-mKate2-2A-TALER21-4xTarget ⁇ FF3 plasmid and 100ng pT21+T21x3+72-EYFP-2A-TALER9-4xTarget ⁇ FF5 plasmid); Group 2: pCAG-Gal4/vp16-2A-TagBFP -2A-Bla Plasmid, pT9
  • the first group pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT10+T10x3+72-mKate2-2A-TALER12-4xTarget ⁇ FF6 plasmid and pT12+T12x3+72-EYFP-2A-TALER10 HEK293 cells were co-transfected with -4xTarget ⁇ FF4 plasmid (100ng pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, 100ng pT10+T10x3+72-mKate2-2A-TALER12-4xTarget ⁇ FF6 plasmid and 100ng per well) pT12+T12x3+72-EYFP-2A-TALER10-4xTarget ⁇ FF4 plasmid); the second group: pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT10
  • the first group pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT10+T10x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF5 plasmid and pT14+T14x3+72-EYFP-2A-TALER10 HEK293 cells were co-transfected with -4xTarget ⁇ FF4 plasmid (transfected with 100 ng of pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, 100 ng of pT10+T10x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF 5 plasmid per well and 100ng pT14+T14x3+72-EYFP-2A-TALER10-4xTarget ⁇ FF4 plasmid); the second group: pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasm
  • the first group pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT10+T10x3+72-mKate2-2A-TALER21-4xTarget ⁇ FF3 plasmid and pT21+T21x3+72-EYFP-2A-TALER10 HEK293 cells were co-transfected with -4xTarget ⁇ FF4 plasmid (100ng pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, 100ng pT10+T10x3+72-mKate2-2A-TALER21-4xTarget ⁇ FF3 plasmid and 100ng per well) pT21+T21x3+72-EYFP-2A-TALER10-4xTarget ⁇ FF4 plasmid); the second group: pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT10
  • the first group pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT12+T12x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF4 plasmid and pT14+T14x3+72-EYFP-2A-TALER12 HEK293 cells were co-transfected with -4xTarget ⁇ FF5 plasmid (100ng pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, 100ng pT12+T12x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF4 plasmid and 100ng per well) pT14+T14x3+72-EYFP-2A-TALER12-4xTarget ⁇ FF5 plasmid); the second group: pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT12
  • the first group pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT12+T12x3+72-mKate2-2A-TALER21-4xTarget ⁇ FF3 plasmid and pT21+T21x3+72-EYFP-2A-TALER12 HEK293 cells were co-transfected with -4xTarget ⁇ FF5 plasmid (100ng pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, 100ng pT12+T12x3+72-mKate2-2A-TALER21-4xTarget ⁇ FF3 plasmid and 100ng per well) pT21+T21x3+72-EYFP-2A-TALER12-4xTarget ⁇ FF5 plasmid); the second group: pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT12
  • the first group pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT14+T14x3+72-mKate2-2A-TALER21-4xTarget ⁇ FF3 plasmid and pT21+T21x3+72-EYFP-2A-TALER14 HEK293 cells were co-transfected with -4xTarget ⁇ FF4 plasmid (100ng pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, 100ng pT14+T14x3+72-mKate2-2A-TALER21-4xTarget ⁇ FF3 plasmid and 100ng per well) pT21+T21x3+72-EYFP-2A-TALER14-4xTarget ⁇ FF4 plasmid); the second group: pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid, pT14
  • the histogram shows the mean value of the fluorescence of EYFP or mKate2 ⁇ standard deviation, and the graph under the histogram shows a representative flow cytogram obtained at 48 hours after the transfection.
  • the pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid was the pCAG-Gal4/vp16-2A-TagBFP-2A-Bla plasmid of Example 4.
  • the pT9+T9x3+72-mKate2-2A-TALER10-4xTarget ⁇ FF4 plasmid was the pT9+T9x3+72-mKate2-2A-TALER10-4xTarget ⁇ FF4 plasmid of Example 5.
  • the pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF4 plasmid was the pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF4 plasmid of Example 5.
  • the pT14+T14x3+72-EYFP-2A-TALER9-4xTarget ⁇ FF5 plasmid was the pT14+T14x3+72-EYFP-2A-TALER9-4xTarget ⁇ FF5 plasmid of Example 5.
  • the pSIREN_U6-shRNA-FF5 plasmid was the pSIREN_U6-shRNA-FF5 plasmid of Example 5.
  • the pT21+T21x3+72-EYFP-2A-TALER12-4xTarget ⁇ FF5 plasmid was the pT21+T21x3+72-EYFP-2A-TALER12-4xTarget ⁇ FF5 plasmid of Example 5.
  • the pCH150 plasmid is shown as sequence 125.
  • sequence 125 nucleotides 4781-4861 from the 5' end are pEF1a (promoter), and nucleotides 6750-7439 are genes encoding TagBFP.
  • TagBFP is a constitutive expression.
  • the pCAG-Gal4/vp16 plasmid is shown as sequence 127.
  • sequence 127 the nucleotides 4253-5962 from the 5' end are the CAG promoter, and the nucleotides 6019-6702 are the coding genes of Gal4/vp16.
  • sequence 128 The pT14+T14x3+72-EYFP-2A-TALER9-4xTarget ⁇ miR21 plasmid is shown as sequence 128.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4399 are T14 sequences (target sequence of TALER14 protein)
  • nucleotides 4406-4465 For the CMVmini promoter, the nucleotides 4472-4486 are T14 sequences, the nucleotides 4487-4503 are T14 sequences, the nucleotides 4450-4522 are T14 sequences, and the nucleotides 4584-5300 are The coding gene of EYFP, the 5307-5360 nucleotide is the coding gene of 2A-ligation peptide, the nucleotide of 5370-8915 is the coding gene of TALER9 protein, and the nucleotide of 8973-9018 is
  • nucleotides 9021 to 9042 are the target sequence of miR21 Target ⁇ miR21
  • nucleotides 9056-9077 are target sequences of miR21 Target ⁇ miR21
  • nucleotides 9080-9101 are target sequences of miR21.
  • nucleotides 4518-4538 are T9 sequences
  • nucleotides 4603-5295 are the coding genes of mKate2
  • nucleotides 5302-5355 are the coding genes of 2A junction peptide
  • nucleotides 5365-8502 The nucleotide is the gene encoding the TALER14 protein
  • the nucleotide number 8593-8614 is the target sequence of shRNA-FF5 Target ⁇ FF5
  • nucleotide number 8615-8636 is the target sequence of shRNA-FF5 Target ⁇ FF5
  • the 8658 nucleotide is the target sequence of shRNA-FF5 Target ⁇ FF5
  • nucleotide number 8659-8680 is the target sequence Target ⁇ FF5 of shRNA-FF5.
  • sequence 130 The pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ miR18a plasmid is shown as sequence 130.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4403 are T9 sequences (target sequence of TALER9 protein)
  • nucleotides 4476-4496 are T9 sequences
  • nucleotides 4497-4517 are T9 sequences
  • nucleotides 4518-4538 are T9 sequences
  • nucleotides 4603-5295 are The coding gene of mKate2, the nucleotides 5302-5355 are the coding genes of the 2A-ligation peptide, the nucleotides of the 5365-8502 nucleotides are the genes encoding the TALER14 protein, and the nucleotides of the 8575-85
  • the nucleotide number 8859-8620 is the target sequence of miR18a Target ⁇ miR18a
  • the nucleotide number 8621-8643 is the target sequence of miR18a Target ⁇ miR18a
  • the nucleotide number 8644-8666 is the target sequence of miR18a Target ⁇ miR18a.
  • sequence 131 The pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ miR19ab plasmid is shown as sequence 131.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4403 are T9 sequences (target sequence of TALER9 protein)
  • nucleotides 4476-4496 are T9 sequences
  • nucleotides 4497-4517 are T9 sequences
  • nucleotides 4518-4538 are T9 sequences
  • nucleotides 4603-5295 are The coding gene of mKate2, the nucleotides 5302-5355 are the coding genes of the 2A-ligation peptide, the nucleotides of the 5365-8502 nucleotides are the genes encoding the TALER14 protein, and the nucleotides of the 5575
  • the nucleotide number 8859-8620 is the target sequence of miR19ab Target ⁇ miR19ab
  • the nucleotide number 8621-8643 is the target sequence of miR19ab Target ⁇ miR19ab
  • the nucleotide number 8644-8666 is the target sequence of miR19ab Target ⁇ miR19ab.
  • sequence 132 The pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ miR191 plasmid is shown as sequence 132.
  • nucleotides 4275-4367 from the 5' end are 5 x UAS sequences
  • nucleotides 4383-4403 are T9 sequences (target sequence of TALER9 protein)
  • nucleotides 4410-4469 For the CMVmini promoter, nucleotides 4476-4496 are T9 sequences, nucleotides 4497-4517 are T9 sequences, nucleotides 4518-4538 are T9 sequences, and nucleotides 4603-5295 are The coding gene of mKate2, the nucleotides 5302-5355 are the coding genes of the 2A-ligation peptide, the nucleotides of the 5365-8502 nucleotides are the genes encoding the TALER14 protein, and the nucleotides
  • the nucleotide number 8859-8620 is the target sequence of miR191 Target ⁇ miR191
  • the nucleotide number 8621-8643 is the target sequence of miR191 Target ⁇ miR191
  • the acid is the target sequence of miR191 Target ⁇ miR191.
  • the inventors selected a pair of unbalanced TALER proteins (TALER9 protein and TALER14 protein), and constructed a closed loop structure switch that is inhibited by synthetic shRNA-FF5 (Fig. 16A), and one inhibited by TALER9 protein.
  • the promoter of the TALER10 protein, while the TALER10 protein does not inhibit the open-loop switch of the promoter of the TALER9 protein (Fig. 16B).
  • Plasmid transfection of cells 24-well plates were prepared, and 0.5 mL of HEK293 cell suspension (containing 6 ⁇ 10 4 HEK293 cells) was inoculated into each well. After 24 hours of culture, the new DMEM medium was replaced, followed by plasmid transfection.
  • Fluorescence intensity of mKate2 / fluorescence intensity of TagBFP fluorescence intensity of corrected mKate2.
  • Fluorescence intensity of EYFP / Fluorescence intensity of TagBFP fluorescence intensity of corrected EYFP.
  • the fluorescence intensity of the corrected mKate2 and the fluorescence intensity of the corrected EYFP were used.
  • microRNA expression database indicated that miR18a, miR191 and miR19ab (miR19a plus miR19b) are highly expressed in HEK293 cells and lowly expressed in HeLa cells.
  • the HeLa cell-specific microRNA is miR21. See Figure 17B.
  • the pCH150 plasmid was introduced into HeLa cells to obtain HeLa which expressed the blue fluorescent protein TagBFP.
  • Cell line (HeLa: TagBFP).
  • the pCH169 plasmid was introduced into HEK293 cells to obtain a HEK293 cell line (HEK293: iRFP_shRNA-FF4) expressing the near-infrared fluorescent proteins iRFP and shRNA-FF4.
  • the specific method is as follows:
  • a 24-well plate containing about 2 ⁇ 10 5 HeLa cells per well was added, and 1 mL of primary virus solution, 1 ml of medium, and 10 ⁇ g/mL of polybrene (Millipore) were added to each well, and cultured for 72 hours; then, Inoculation of blasticidin (InvivoGen) at a final concentration of 5 ⁇ g/mL for 6 days; then, the concentration of blasticidin was increased to 10 ⁇ g/mL, cultured for 2 days, confirmed by flow cytometry, about 95% HeLa: TagBFP cells are positive for TagBFP.
  • DMEM complete medium containing about 2 ⁇ 10 5 HEK293-FT cells was added to each well for 24 hours, and then pCH169 plasmid was packaged with Lipofectamine LTX plus Plus reagent, packaging vector pCMV-dR8.2 ( Addgene company was co-transfected with pCMV-VSV-G (Addgene), and after 24 hours of transfection, the culture supernatant was collected, which was a virus solution containing a lentivirus expressing iRFP_shRNA-FF4, abbreviated as a primary virus solution.
  • a 24-well plate containing about 2 ⁇ 10 5 HEK293 cells per well was added, and 1 mL of primary virus solution, 1 ml of medium and 10 ⁇ g/mL of polybrene (Millipore) were added to each well for 72 hours; then, Inoculation of blasticidin (InvivoGen) at a final concentration of 5 ⁇ g/mL for 6 days; then, the concentration of blasticidin was increased to 10 ⁇ g/mL, cultured for 2 days, confirmed by flow cytometry, about 40% HEK293: iRFP_shRNA-FF4 cells were iRFP positive; enriched with iRFP-positive HEK293:iRFP_shRNA-FF4 cells by trypsinization, centrifugation at 300g for 5 minutes, using 1xPBS containing 10% FBS (Invitrogen) and 1% sodium pyruvate (Invitrogen) Resuspend; cell sorting using BD AriaII.
  • the flow cytometry plots of HeLa cells (also known as wild-type cells) and HeLa:TagBFP (also known as engineered cells) are shown in Figure 17A, with the ellipse representing the HEK293:iRFP_shRNA-FF4 and HeLa:TagBFP regions.
  • the flow cytometry plot of HEK293 cells (also known as wild-type cells), HEK293:iRFP_shRNA-FF4 (also known as engineered cells) is shown in Figure 17A, and the ellipse indicates HEK293: iRFP_shRNA-FF4 and HeLa:TagBFP regions.
  • HEK293:iRFP_shRNA-FF4 cells were significantly different from HeLa:TagBFP cells.
  • HeLa:TagBFP cells and HEK293:iRFP_shRNA-FF4 cells were mixed to obtain a mixed cell population.
  • the pCAG-Gal4/vp16 plasmid, pT14+T14x3+72-EYFP-2A-TALER9-4xTarget ⁇ miR-21 plasmid and pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF4 plasmid were co-transfected into a mixed cell population ( Each well was transfected with 100 ng of pCAG-Gal4/vp16 plasmid, x ng pT14+T14x3+72-EYFP-2A-TALER9-4xTarget ⁇ miR-21 plasmid and y ng pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF4 Plasmid), transfection, DOX was added to the cell culture system
  • FIG. 18A The schematic diagram is shown in Figure 18A.
  • the results are shown in Figure 18B: the histogram shows the ratio of HeLa:TagBFP cells and HEK293:iRFP_shRNA-FF4 cells in the EYFP + or mKate2 + cell population, all bars represent the mean ⁇ standard deviation of three independent replicates; iRFP
  • the -TagBFP flow cytometry plot shows the EYFP + cell population distribution, the lower row shows the mKate2 + cell population distribution, and the ellipse indicates the modified HEK293 or HeLa cell population.
  • Figure 18C shows a representative EYFP-mKate2 flow cytometry plot.
  • HeLa:TagBFP cells were mixed with HEK293:iRFP_shRNA-FF4 cells to obtain a mixed cell population.
  • the pCAG-Gal4/vp16 plasmid, pT14+T14x3+72-EYFP-2A-TALER9-4xTarget ⁇ miR-21 plasmid and pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ miR18a plasmid were co-transfected into a mixed cell population ( Each well was transfected with 100 ng of pCAG-Gal4/vp16 plasmid, 100 ng of pT14+T14x3+72-EYFP-2A-TALER9-4xTarget ⁇ miR-21 plasmid and 100 ng of pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ miR18a plasmid)
  • DOX was
  • a 24-well plate was prepared, and 0.5 mL of a mixed cell suspension (containing 6 ⁇ 10 4 mixed cells) was inoculated into each well. After 24 hours of culture, a new DMEM medium was replaced, followed by plasmid transfection.
  • the pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ miR19ab plasmid was used in place of the pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ miR18a plasmid.
  • Figure 19A the histogram shows the ratio of HeLa:TagBFP cells and HEK293:iRFP_shRNA-FF4 cells in the EYFP + or mKate2 + cell population, and all bars represent the mean ⁇ standard deviation of three independent replicates; iRFP
  • the -TagBFP flow cytometry plot shows the EYFP + cell population distribution, the lower row shows the mKate2 + cell population distribution, and the ellipse indicates the modified HEK293 or HeLa cell population.
  • Figure 19B shows a representative EYFP-mKate2 flow cytometry plot.
  • HEK293-specific miR18a and HeLa-specific miR21 are the best inputs for endogenous microRNA classifier signals. This also indicates that the TALER switch can be controlled by endogenous microRNAs.
  • miRx represents miR18a, miR191 or miR19ab
  • Tx represents the quadruple repeat of the fully complementary target site of miR18a
  • FF5 represents the quadruple repeat of the fully complementary target site of shRNA-FF5 sequence.
  • HeLa:TagBFP cells were mixed with HEK293:iRFP_shRNA-FF4 cells to obtain a mixed cell population.
  • the pCAG-Gal4/vp16 plasmid, pT21+T21x3+72-EYFP-2A-TALER12-4xTarget ⁇ FF5 plasmid and pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF5 plasmid were co-transfected into mixed cell population (per well) Transfection of 100 ng of pCAG-Gal4/vp16 plasmid, 100 ng of pT21+T21x3+72-EYFP-2A-TALER12-4xTarget ⁇ FF5 plasmid and 100 ng of pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF5 plasmid)
  • DOX was added to the cell culture system to make the DOX concentration 1000
  • a 24-well plate was prepared, and 0.5 mL of a mixed cell suspension (containing 6 ⁇ 10 4 mixed cells) was inoculated into each well. After 24 hours of culture, a new DMEM medium was replaced, followed by plasmid transfection.
  • the pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ miR191 plasmid was used in place of the pT9+T9x3+72-mKate2-2A-TALER14-4xTarget ⁇ FF5 plasmid.
  • Figure 20B The left panel shows the EYFP-mKate2 flow cytometry plot of representative HEK293:iRFP_shRNA-FF4 cells and HeLa:TagBFP cells; the right panel shows the microRNA knockout efficiency in the corresponding cells, the histograms are The ratio of the mean value of mKate2 fluorescence values to the mean value of EYFP fluorescence values obtained from three independent replicate experiments.
  • Figure 20C Bar graph showing the proportion of HEK293 and HeLa cells after transformation in the EYFP + or mKate2 + cell population, both representing the mean ⁇ standard deviation of three independent replicates; iRFP-TagBFP flow cytometry Shown as EYFP + cell distribution, the lower row shows the mKate2 + cell distribution, and the ellipse indicates the modified HEK293 or HeLa region.
  • Fluorescent reporter gene assays in mixed cell populations indicated that miR18a has significant RNAi knockout effects, whereas miR191 and miR19ab do not, and open-loop TAREL switches that do not inhibit each other are not effective for cell sorting.
  • TALER a, b represents the TALER gene
  • 2A represents the self-cleaving amino acid sequence
  • Ta and Tb represent the binding sites of TALERa and TALERb
  • Activator represents the transcriptional activator
  • 5 ⁇ ARS represents the transcriptional activation response site
  • miniCMV represents the minimal CMV promoter
  • pCAG represents a constitutive promoter
  • OUTa and OUTb represent two different outputs.
  • shRNA or microRNA a1, a2 and b1, b2, etc. are input.
  • the corresponding logical expression is shown below.
  • TALE transcriptional activator-like factor

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Abstract

Cette invention concerne un kit de réactifs utilisant un répresseur de transcription TALE pour la construction modulaire d'une lignée de gènes synthétiques dans une cellule de mammifère. Plus spécifiquement, l'invention concerne un kit de réactifs permettant de réguler l'expression de deux protéines, comprenant un vecteur recombiné A contenant une cassette d'expression A, un vecteur recombiné B contenant une cassette d'expression B, et un vecteur recombiné C contenant une cassette d'expression C ; la cassette d'expression A comprenant : une séquence codant pour un élément de rétroaction, un promoteur A, une protéine A et un gène codant pour une protéine A du TALER, liés au moyen d'un polypeptide auto-clivant, et une séquence cible A comprenant une séquence cible shRNA1 ; la cassette d'expression B comprenant : une séquence codant pour un élément de rétroaction, un promoteur B, une protéine B et un gène codant pour une protéine A du TALER, liés au moyen d'un polypeptide auto-clivant, et une séquence B cible comprenant une séquence shRNA2 cible ; et la cassette d'expression C comprenant une séquence codante constituée d'un promoteur et d'un élément d'activation. Les vecteurs recombinés A, B et C sont introduits dans une cellule hôte, et shRNA1 ou shRNA2 est ajouté pour réguler l'expression des protéines A et B.
PCT/CN2015/000140 2014-04-30 2015-03-06 Kit de réactifs utilisant le répresseur de transcription tale pour la construction modulaire d'une lignée de gènes synthétiques dans une cellule de mammifère WO2015165276A1 (fr)

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