WO2021139596A1

WO2021139596A1 - Gene expression switch based on red light and far-red light regulation, and construction method and application thereof

Info

Publication number: WO2021139596A1
Application number: PCT/CN2020/142005
Authority: WO
Inventors: 叶海峰; 周阳; 孔德强; 王欣怡
Original assignee: 华东师范大学
Priority date: 2020-01-08
Filing date: 2020-12-31
Publication date: 2021-07-15
Also published as: CN113088532B; CN113088532A

Abstract

Provided are a gene expression switch/system (REDMAP system) based on red light and far-red light regulation for mammalian cells, comprising a photoreceptor and an effector. The REDMAP system has the capabilities of simple design, small modules, high-efficiency induction, and rapid and sensitive transcription expression regulation. The REDMAP system can efficiently and accurately regulate transgenic expression of mammalian cells and a signaling pathway based on protein-protein interaction by means of red light and far-red light, and has the characteristics of non-toxicity, high efficiency, spatio-temporal specificity, reversible induction of gene expression and the like. The REDMAP system can accurately regulate expression of a target gene in vivo, facilitates long-term drug delivery of adeno-associated viruses (AAV), can be used for regulating insulin expression, can be used for various biological and pre-transformation application research, such as regulation of a cell signaling pathway, epigenetic engineering, and cell therapy based on drug delivery, and has potential application value in the fields of basic biological research, mammalian gene engineering, and regenerative medicine.

Description

Gene expression switch regulated by red light and far-red light, and construction method and application thereof

Technical field

The present invention relates to synthetic biology, optogenetics and other multidisciplinary cross-fields, in particular to a method for constructing a gene expression switch regulated by red light and far-red light in mammalian cells, which efficiently induces the transcription and expression of mammalian genes, and its application in Applications in mammalian genetic engineering.

Background technique

In the field of synthetic biology, molecular switches that precisely regulate gene expression artificially have become an indispensable means. At present, there are a variety of artificial regulation and induction gene expression systems. These regulation systems mainly induce and regulate gene expression through chemical inducers or physical methods. However, there are few systems that regulate gene expression through light induction.

Light is an ideal inducer of gene expression. It is ubiquitous in nature, is easy to obtain, has temporal and spatial specificity, and has low toxicity. Therefore, the use of light as an inducer to regulate gene expression and research in the field of genetic engineering has great application value.

At present, some optogenetic regulation tools have been developed and applied in various fields, including genetic engineering, industrial production, etc. However, the existing light-controlled transcription system is not perfect, and there are still a lot of optogenetic transcription regulation tools. Room for improvement. These existing light-controlled transcription activation systems, one type uses short-wave light such as blue or ultraviolet light as an inducer, such as CRY2/CIBN, LOV, and Magnet systems, but they have certain limitations, such as certain phototoxicity And low tissue penetration, it is not easy to regulate the target gene through the skin or abdominal cavity, which greatly limits the in-depth development and clinical application of the existing light control system.

The other type uses far red light or red light as the excitation switch. Although these transcription systems have low phototoxicity and better skin permeability, they still have some shortcomings, such as large protein modules in the BphP1/PpsR2 system, low activation efficiency, and background The leakage is serious, the induction multiple is poor (only 5 times), the BphS system activation conditions are harsh and the light response speed is insufficient, and it takes a long time for several hours a day to activate the light stimulation. Although the PhyB system can quickly activate transcription, its activation multiple is not high , And the shutdown of transcription needs to be placed under far red light for a long time, which is inconvenient for system shutdown. These shortcomings greatly limit the further application of these light control systems in the field of in vivo and translational medicine. In order to overcome these limitations, it is urgent to invent a gene expression switch that is simple to operate, safe and efficient, high induction efficiency, rapid response, small modular system, can be delivered by AAV virus, and can be precisely regulated in time and space, so that it can be used for basic research, Applied research on drug delivery and clinical translation.

Summary of the invention

In view of the above-mentioned shortcomings of the prior art, the present invention proposes a mammalian cell red light and far-red light regulation gene expression switch/system (hereinafter referred to as the REDMAP system) to regulate the transcription of mammalian cell genes. The present invention is non-toxic , High efficiency, high sensitivity, good induction multiples, strong temporal and spatial specificity, strong tissue penetration and reversible induction of gene expression, etc., which well solve the shortcomings of the current optical system. The present invention can respond to light in milliseconds. Stimulus, quick opening, no need for long time irradiation, convenient operation, strong practicability; at the same time, the present invention can quickly close, stop transcription activation, overcome the shortcomings of the existing optical system that cannot be closed in time, and has stronger controllability; in addition, the present invention also The dual regulation of chemical molecules and photons can be achieved, which solves the problem of serious background leakage. In summary, the present invention has great potential application value in the research of precise temporal and spatial specific regulation of cell behavior in the field of mammalian genetic engineering biomedicine.

The present invention proposes a mammalian cell REDMAP system for the first time. In the present invention, the photon energy of the red light and far-red light is much lower than that of the blue-violet light, and has less cytotoxicity and stronger skin permeability. The system is delivered to the deep tissues through AAV, which can achieve long-term It seamlessly regulates the expression and inhibition of the target gene in cells infected by AAV in the deep tissues, regulates the transcriptional expression of specific tissues and organs in the body, and serves as a controllable drug delivery vehicle.

The present invention also provides a mammalian cell REDMAP system. The invention optimizes the photoreceptors in the system to achieve the optimal induction effect, enables the system to have the maximum response ability to red light/far-red light, and provides technical support for the in-depth development and clinical application of the light control system. The REDMAP system of the present invention can regulate gene expression or protein interaction control system through the red light/far-red light delivered by the adeno-associated virus. The READMAP system has a small module and can be used for efficient packaging of AAV virus and delivery of organs in the body, realizing long-term light Control the expression of transgenes in organs in the body. The REDMAP system of the present invention can quickly and reversibly regulate gene transcription expression levels through red light/far-red light, provides a new tool for genetic engineering, has great potential application value, and can be widely promoted in clinical applications.

Each nucleotide sequence or amino acid sequence described in the present invention can be prepared by artificial synthesis.

The mammalian cell REDMAP system proposed by the present invention includes: photoreceptors and effectors.

In the present invention, the photoreceptor includes the Arabidopsis thaliana light-sensitive protein PhyA and its interacting protein FHY1/FHL. The amino acid sequence of the Arabidopsis photosensitive protein PhyA is shown in SEQ ID NO. 1, the amino acid sequence of the interacting protein FHY1 is shown in SEQ ID NO. 2, and the amino acid sequence of FHL is shown in SEQ ID NO. 3 shown.

In the present invention, the effector (inducible promoter effector) includes an operon, an inducible weak promoter and a target gene, namely _PREDMAP- Reporter. Wherein, the inducible promoter P _REDMAP includes an operon and a weak promoter, and the operon and inducible weak promoter can bind to the photoreceptor and its fusion protein; the report Reporter, that is, the target gene is any meaningful protein.

The mechanism of action of the mammalian cell REDMAP system of the present invention is, as shown in Figure 1, PhyA and FHY1/FHL in the photoreceptor are fused and expressed with DNA binding protein (DBP) and transcription activation domain (AD), respectively. In the presence of PCB, once the photoreceptor is irradiated with red light, PhyA will specifically bind to FHL/FHY1, and then be transported into the nucleus. The fusion transcription activation domain is specifically recruited to the inducible promoter through the DNA binding protein. Thereby inducing transcriptional expression. When irradiated by far-red light, the photoreceptor quickly dissociates and exits the nucleus, terminating the transcriptional expression of the gene.

The components of the REDMAP system provided by the present invention can be constructed in a eukaryotic expression vector by genetic engineering technology, so as to realize the regulation and control of the transcription expression of the target gene. The REDMAP system provided by the present invention can use red light/far-red light that hardly damages cells or the body to specifically induce gene expression in eukaryotic host cells in time and space. The host cells can be of any type. Mammalian cells, such as hMSC-TERT, HEK-293, Hela, etc.

Wherein, the light intensity of the red light is 0-2 mW/cm ² ; the irradiation time is 0-10 s; the light intensity of the far-red light is 0-2 mW/cm ² , and the irradiation time is 0-10 min. Irradiation methods include pulsed irradiation, continuous irradiation or direct irradiation in space to control the gene expression levels of cells in different locations. By controlling the light source to produce different illumination times, different degrees of induction of endogenous genes can be achieved. The red light source may be an LED, a laser light, or the like. The wavelength of the red light is 660nm±10nm, and the wavelength of the far-red light is 720-900nm.

The present invention also proposes a method for constructing the REDMAP system, which includes the following steps:

(1) Building a photoreceptor

The fusion protein of PhyA and DNA binding protein, the fusion protein of FHY1/FHL and transcription activation domain, the nuclear localization/nuclear export signal of the aforementioned two fusion proteins, and the connecting peptide between the aforementioned two fusion proteins were constructed as the REDMAP. The photoreceptor of the system.

Wherein, the PhyA may be a PhyA protein with different lengths truncated, including but not limited to PhyA (1-598aa), ΔPhyA (1-617aa) (ΔPhyA), the amino acid sequence of which is shown in SEQ ID NO. 5-6, respectively ; Among them, preferably, ΔPhyA (1-617aa).

Wherein, the DNA binding protein is a protein that can bind to a specific DNA sequence-an operon (such as (UAS) ₅ , (TetO) ₇ DNA sequence), including GAL4, TetR, etc., the PhyA and DNA binding protein The fusion proteins include but are not limited to PhyA-GAL4 and PhyA-TetR, the amino acid sequences of which are shown in SEQ ID NOs. 7-8, respectively.

Wherein, the transcription activation domain is VP64, VPR, VP16 and other transcription factors that have the function of recruiting RNA polymerase, and the fusion protein of FHY1/FHL and the transcription activation domain includes but not limited to FHY1-VP64, FHL-VP64 , FHY1-VPR, FHL-VPR, FHY1-VP16, FHL-VP16, the amino acid sequences of which are shown in SEQ ID NO. 9-14, respectively.

Wherein, the nuclear localization/nuclear export signals of the two fusion proteins are NLS (Nuclear Import Signal Peptide) and NES (Nuclear Export Signal Peptide), and their amino acid sequences are shown in SEQ ID NO. 15-16. That is, the nuclear localization signal and nuclear export signal of the fusion protein of PhyA and DNA binding protein are NLS and NES, respectively; the nuclear localization signal and nuclear export signal of the fusion protein of FHY1/FHL and the transcription activation domain are NLS and NES, respectively.

Wherein, the connecting peptide between the two fusion proteins is a peptide chain of 0-30 amino acids, and its amino acid sequence includes, but is not limited to, as shown in SEQ ID NO. 17-18. That is, the connecting peptide of the fusion protein of PhyA and the DNA binding protein is shown in SEQ ID NO.17; the connecting peptide of the fusion protein of FHY1/FHL and the transcription activation domain is shown in SEQ ID NO.18.

(2) Construct effectors.

Construct an inducible promoter effector that responds to DNA binding proteins, including an operon, an inducible weak promoter, and a target gene, namely _PREDMAP- Reporter; wherein the inducible promoter _PREDMAP includes an operon, an inducible weak promoter The report Reporter is the target gene, which is any meaningful protein.

In the present invention, the operon is capable of interacting with DNA binding proteins, including but not limited to (UAS) ₅ , (TetO) ₇ , and preferably, its nucleotide sequence is as shown in SEQ ID NO. 19-20, respectively .

In the present invention, the inducible weak promoter includes but is not limited to hCMV _min and TATA. Preferably, the nucleotide sequences thereof are as shown in SEQ ID NOs. 21-22, respectively.

In the present invention, any meaningful protein includes insulin, etc.; preferably, its nucleotide sequence is shown in SEQ ID NO.4.

The REDMAP system of the present invention can respond to millisecond-long red light stimulation to initiate gene transcription and expression; the REDMAP system can respond to microwatt-level red light stimulation to initiate gene transcription and expression; the REDMAP system can quickly respond to remote Red light stimulation turns off gene transcription and expression.

The REDMAP system protein module of the present invention is small and can be packaged with adeno-associated virus for efficient delivery in vivo. The REDMAP system can regulate gene expression in different mammalian cell lines.

The REDMAP system of the present invention is used in regulating or inducing cell gene expression (such as insulin, etc.), regulating the interaction of proteins and proteins (such as the regulation of Ras signaling pathway). Wherein, the product that regulates or induces cell gene expression includes but is not limited to insulin, and the amino acid sequence of the insulin is shown in SEQ ID NO.4. Ras signaling pathway is an application case of REDMAP system regulating protein-protein interaction.

The present invention also provides a eukaryotic expression vector, host cell and/or engineered cell and/or engineered cell transplantation vector of the REDMAP system; wherein the host cell and/or engineered cell is transfected with the genuine Nuclear expression vector; wherein the engineered cell transplantation vector is a vector containing engineered cells, such as microcapsules and/or hydrogels. Wherein, the eukaryotic expression vector, host cell and/or engineered cell and/or engineered cell transplantation vector comprise the REDMAP system.

The eukaryotic expression vector includes a mammalian cell expression vector containing the REDMAP system. The expression vector may be a vector containing a gene encoding a photoreceptor alone or a vector containing a gene encoding an effector alone. Alternatively, the expression vector includes both a vector encoding a photoreceptor gene and a vector encoding an effector gene. See Table 1 for the construction methods of all the aforementioned mammalian cell expression vectors.

The present invention also proposes a method for preparing eukaryotic expression vectors, engineered cells or engineered cell transplantation vectors containing the REDMAP system.

The preparation method of the eukaryotic expression vector is shown in Table 1; the method of the engineered cell includes calcium phosphate transfection, PEI transfection, liposome transfection, electroporation transfection, or viral infection; the engineered cell The preparation method of the transplant carrier includes: preparing microcapsules, preparing sodium alginate rubber block skins, and preparing hollow fiber membrane transplantation tubes.

The present invention also proposes a method for regulating gene expression in host cells (mammalian cells, engineered cells) using the REDMAP system, which includes the steps:

a) Constructing the REDMAP system in a host cell eukaryotic plasmid expression vector;

b) introducing the expression vector (e.g., after transfection) into the host cell;

c) Inducing or regulating the expression of effector-encoding reporter genes (such as SEAP, Luciferase, insulin, etc.) in the host cell through red light and far-red light, so as to realize the expression of the REDMAP system encoding genes in the host cell;

d) Detect the expression of the target gene.

The present invention also proposes a method for regulating and expressing transgenes in engineered cell transplantation vectors by using the REDMAP system, which includes the steps:

a) preparing a eukaryotic plasmid expression vector containing the REDMAP system;

b) using the eukaryotic plasmid expression vector of step a) to prepare engineered cells containing the REDMAP system;

c) using the engineered cells of step b) to prepare an engineered cell transplantation vector containing the REDMAP system;

d) Inducing expression of engineered cell transplantation vector by red light and far-red light, so that the effector encoding reporter gene (such as SEAP, Luciferase, insulin, etc.) in the transplantation vector is expressed, that is, realizing the expression of the REDMAP system in the Expression of encoding genes in the transplantation vector;

e) Detect the expression of the target gene.

The present invention also provides that the REDMAP system or the eukaryotic expression vector and/or host cell and/or engineered cell and/or engineered cell transplantation vector is used to regulate or induce (preferably mammalian) cell gene expression (insulin). Etc.) and/or control (preferably mammalian) protein-protein interaction/application in the kit.

Wherein, when the gene is expressed as insulin, preferably the amino acid sequence of insulin is shown in SEQ ID NO.4.

The present invention also provides a kit that contains the REDMAP system, or the drug or kit contains the above-mentioned eukaryotic expression vector, host cell and/or engineered cell and/or engineered cell transplantation vector .

In the present invention, the kit includes a kit for regulating the plasmids of each component of the REDMAP system, a kit containing mammalian cells for regulating the REDMAP system, and corresponding instructions.

The present invention also proposes a method for regulating protein-protein interaction using the REDMAP system, taking the regulation of the Ras-MAPK signal pathway as an example, the method includes:

a) Fusion of PhyA protein and cell membrane anchoring signal peptide CAAX sequence into PhyA-CAAX, and then anchored on the cell membrane after expression, the nucleotide sequence of PhyA-CAAX is shown in SEQ ID NO.23;

b) FHY1 protein and Ras protein guanine nucleotide exchange domain SOScat protein are fused and expressed as FHY1-SOScat, and the nucleotide sequence of FHY1-SOScat is shown in SEQ ID NO.24;

c) Under the irradiation of red light/far-red light, FHY1 protein will bind/dissociate with PhyA protein, and SOScat will be recruited/detached from the cell membrane, thereby activating/inhibiting the Ras-MAPK signaling pathway.

The present invention also provides a method of artificially constructed Ras-MAPK signal pathway regulated by the REDMAP system, the method comprising:

a) PhyA-CAAX, FHY1-SOScat, and the recombinant transcription factor pTetR-ElK1, TetR-responsive reporter pMF111 are transfected into the host cell;

b) Under the irradiation of red light/far-red light, the Ras-MAPK signaling pathway is activated/inhibited, thereby initiating the expression of the reporter gene (Figure 22c, d).

The invention also proposes an artificially constructed Ras-MAPK signal pathway.

The present invention also proposes the application of the REDMAP system in the application of epigenetic engineering. Specifically, the present invention proposes a method for regulating endogenous gene expression based on CRISPR-dCas9 in a host cell mediated by the REDMAP system, and the method includes:

a) Construction of the REDMAP system effector of the CRISPR-dCas9 transcriptional regulatory element MS2-p65-HSF1, namely P _REDMAP- MS2-p65-HSF1;

b) Co-transform the REDMAP system with the CRISPR-dCas9 protein element and introduce it into the host cell;

c) Inducing and regulating the host cell by irradiation with red light and far-red light, realizing the expression of the transcriptional regulatory element MS2-p65-HSF1 encoded by the effector, and finally realizing the regulation of the endogenous target gene.

The present invention also provides a method for treating diabetes by using the REDMAP system, the diabetes is preferably type I diabetes, and the method includes:

a) Construct the effector of REDMAP to regulate insulin expression, that is, the eukaryotic vector for insulin therapy, see Table 1;

b) Preparation of a transplantation vector for regulating insulin expression by the REDMAP system; wherein the preparation method of the transplantation vector includes:

Preparing a eukaryotic plasmid expression vector containing the REDMAP to regulate insulin expression;

Using the eukaryotic plasmid expression vector to prepare engineered cells containing the REDMAP system to regulate insulin expression;

Using the engineered cells to prepare a microcapsule-encapsulated cell transplantation carrier;

c) Through the irradiation of red light/far-red light, the expression level of insulin is controlled, so as to reduce blood sugar.

The present invention also provides a red light/far-red light control system (REDMAP) that can be delivered by adeno-associated virus (AAV) to regulate gene expression or protein interaction. The READMAP system has a small module and can be used for high-efficiency packaging and packaging of AAV viruses. In vivo organ delivery, so as to achieve light control of transgene expression control in in vivo organs.

In the present invention, the red light has a wavelength of 660±10 nm, and the far-red light has a wavelength of 720-900 nm.

The beneficial effect of the present invention is that the REDMAP system of the present invention can regulate gene transcription and expression through red light (start) and far-red light (off) efficiently, accurately, and ultra-fast reversibly through red light/far-red light (such as for regulating Transgene expression in mammalian cells and control of signal pathways based on protein-protein interaction), with fast response, high fold of induced gene expression (hundreds of times), rapid and sensitive regulation (less than 1 second), high temporal and spatial specificity It has the characteristics of flexibility, strong tissue penetration, non-toxic side effects, high efficiency, spatiotemporal specificity, and reversible induction of gene expression. The REDMAP is simple in design, small in system modules, and easy to deliver adeno-associated virus (AAV) to realize light-controlled gene expression in animals. The REDMAP system can be used to regulate the expression of insulin to control blood glucose homeostasis in mice. The REDMAP system disclosed in the present invention can be used in a variety of biological and pre-transformation application research, including regulating cell signal pathways, epigenetic engineering, and cell therapy based on drug delivery. The new light control system has great potential application value in the field of basic biology research, mammalian genetic engineering and regenerative medicine to achieve precise temporal and spatial specific regulation of cell behavior.

Description of the drawings

Figure 1: a is a schematic diagram of the photoprotein interaction and principle of the REDMAP system. b is a schematic diagram of the optical protein interaction of the REDMAP system and the regulation of gene transcription in host cells. In the presence of the cofactor PCB, FHY1-VP64 can be recruited to the effector (P _REDMAP- Reporter) through PhyA-Gal4 under 660nm light irradiation, thereby initiating transgene expression. When 730nm light is irradiated, the protein dissociates, thereby terminating the transgene expression.

Figure 2 shows the results of different truncation and optimization of PhyA elements in the REDMAP system.

Figure 3 shows the optimized results of PhyA's different chaperone proteins in the REDMAP system.

Figure 4 is a diagram of the optimization result of the nuclear localization signal of PhyA in the REDMAP system.

Figure 5 shows the optimized result of the nuclear localization signal of FHY1 in the REDMAP system.

Figure 6 is a diagram showing the results of optimizing different transcription activation domains in the REDMAP system.

Figure 7 is a graph showing the effect of different red light irradiation time on the activation efficiency of the REDMAP system.

Figure 8 is a graph showing the effect of different red light irradiation intensities on the activation efficiency of the REDMAP system.

Figure 9 is a graph showing the effect of different PCB concentrations on the activation efficiency of the REDMAP system.

Figure 10 is a graph showing the changes in the transcriptional activation ability of the REDMAP system over time.

Figure 11 shows the spatial specificity verification of the REDMAP system.

Figure 12 shows the reversibility verification of the REDMAP system.

Figure 13 shows how the REDMAP system works in different cells.

Figure 14 shows the comparison result of the REDMAP system and other red light/far-red light systems.

Figure 15 is a graph showing the effect of far-red light (730 nm) irradiation for different times on the shutdown efficiency of the REDMAP system.

Figure 16 is a graph showing the effect of different intensities of far-red light (730 nm) on the shutdown efficiency of the REDMAP system.

Figure 17 shows the effect of far-red light (780nm) irradiation for different time and intensity on the shutdown efficiency of the REDMAP system.

Figure 18 shows the result of the verification of the replaceability of the PCB in the REDMAP system.

Figure 19 is a diagram showing the application result of the REDMAP system for epigenetic engineering of CRISPR-dCas9.

Among them, Figure 19a is a schematic diagram of the epigenetic engineering application of the REDMAP system for CRISPR-dCas9; under red light irradiation, the sgRNA-dCas9 complex further recruits the hybrid transcription activator (MS2-p65-HSF1) through the MS2 box , The formation of a transcription complex initiates transgene expression, and the system can be shut down after far-red light is irradiated.

Figures 19b-c are diagrams of the illumination time and intensity dependence verification results of the REDMAP system used in the epigenetic engineering application of CRISPR-dCas9;

Figure 19d is a graph showing the reversibility results of the REDMAP system used in the epigenetic engineering application of CRISPR-dCas9;

Figures 19e-h are graphs showing the universal performance verification results of the REDMAP system used in the epigenetic engineering application of CRISPR-dCas9.

Figure 20 is a graph showing the effect of different red light irradiation time on the activation efficiency of the REDMAP system in mice;

Among them, the left picture is the luminous intensity statistical result of the in vivo imaging photos of the activation efficiency of the REDMAP system in mice with different red light irradiation time;

The image on the right is an example of in vivo imaging photos of the activation efficiency of the REDMAP system in mice with different red light irradiation times, which are the dark group and the 660nm light group.

Figure 21 is a graph showing the effect of different red light irradiation intensities on the activation efficiency of the REDMAP system in mice.

Among them, the left picture is the luminous intensity statistical result of the in vivo imaging photos of the activation efficiency of the REDMAP system in mice with different red light irradiation intensities;

The image on the right is an example of in vivo imaging photos of the activation efficiency of the REDMAP system in mice with different red light irradiation intensities, which are the dark group and the 660nm light group.

Figure 22 shows the results of the REDMAP system delivered by adeno-associated virus (AAV) in mice;

Among them, the left picture is the luminous intensity statistical result of the in vivo imaging photos regulated by the REDMAP system delivered by the adeno-associated virus (AAV) in mice;

The picture on the right is an example of in vivo imaging photos of the REDMAP system delivered by adeno-associated virus (AAV) in mice, which are the dark group and the 660nm light group.

Figure 23 is a schematic diagram of the treatment of the REDMAP system in a diabetic mouse model and the results; where a is a schematic diagram of mice irradiated with red light in vitro; b is a schematic diagram of the eukaryotic expression vector of the REDMAP system-insulin expression module; c is a small The result graph of the change of mouse blood glucose before and after treatment; d is the result graph of the change of mouse insulin content before and after treatment.

Figure 24 is a schematic diagram and results of the REDMAP system regulating the Ras-MAPK signaling pathway, and a schematic diagram and effects of the artificially constructed Ras-MAPK signaling pathway; where a is the schematic diagram of the REDMAP system regulating the Ras-MAPK signaling pathway; b is the Western Blot The result diagram of detecting the REDMAP system regulating the Ras-MAPK signal pathway; c is the principle diagram of the REDMAP system regulating the artificial Ras-MAPK signal pathway; d is the result diagram of the REDMAP system inducing the artificial Ras-MAPK signal pathway.

Detailed ways

With reference to the following specific embodiments and drawings, the present invention will be further described in detail. These examples are only used to illustrate the invention, and do not constitute any limitation on the scope of the invention. The process, conditions, and experimental methods for implementing the present invention, except for the content specifically mentioned below, are all common knowledge and common knowledge in the field, and the present invention has no special limitations. The reagents, instruments, etc. used in the following examples, as well as the experimental methods without specific conditions, are carried out in accordance with the conditions recommended by conventional or commercial suppliers.

Materials and Methods

The construction reagents, specific construction systems and steps of all expression plasmids of the present invention are as follows.

All primers used for PCR are synthesized by Jinweizhi Biotechnology Co., Ltd. The expression plasmids constructed in the examples of the present invention have all undergone sequence determination, which was completed by Jinweizhi Biotechnology Co., Ltd. The PhantaMax Super-Fidelity DNA polymerase used in the embodiment of the present invention was purchased from Nanjing Novazan Biotechnology Co., Ltd. Both endonuclease and T4DNA ligase were purchased from TaKaRa Company. The homologous recombinase was purchased from Heyuan Biotechnology (Shanghai) Co., Ltd. PhantaMax Super-Fidelity DNA polymerase is purchased with corresponding polymerase buffer and dNTP. Endonuclease, T4 DNA ligase, and homologous recombinase are purchased with corresponding buffers. Yeast Extract, Trypton, Agar Powder, and Ampicillin (Amp) were purchased from Shanghai Shenggong Biological Engineering Technology Co., Ltd. DNA Marker DL5000, DNA Marker DL2000 (Bao Biological Engineering Co., Ltd.); Nucleic Acid Dye EB (Guangdong Guoao Biotechnology Company); Plasmid Mini Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.); DNA gel recovery reagent The kit and PCR product purification kit were all purchased from Kangwei Century Biotechnology Co., Ltd.; the other reagents such as absolute ethanol and NaCl mentioned in the examples are all domestically produced analytical pure products.

(1) Polymerase chain reaction (PCR)

(2) Endonuclease digestion reaction

1. A system for double-enzyme digestion of plasmid vectors (x represents the volume when the mass of the plasmid is 1 μg; n represents the amount of sterilized ultrapure water μL that needs to be added to make the system reach the total volume)

2. A system for double digestion of PCR product fragments (x represents the volume when the plasmid mass is 1 μg; n represents the amount of sterilized ultrapure water μL that needs to be added to make the system reach the total volume)

3. Connect the PCR product fragments after double restriction digestion into the plasmid vector after double restriction digestion to form a circular plasmid system:

Note: The mass ratio of the PCR product fragment to the vector double enzyme digestion product is roughly between 2:1-6:1.

(3) Homologous recombination connection reaction

According to the instructions of Heyuan Biotechnology (Shanghai) Co., Ltd. Seamless Cloning (Assembly) Kit: When PCR product fragments are amplified, a 15bp nucleotide sequence homologous to the 15bp nucleic acid sequence on both sides of the linearized vector is added on both sides The 15bp nucleic acid sequence on both sides of the amplified PCR product is homologous to the nucleotide sequence on both sides of the linearized vector sequence. Under the action of the homologous recombinase, the PCR product fragments are homologously recombined with the linearized vector to form a circle.

Note: The value of n depends on the size and concentration of PCR product fragments.

(4) Preparation of E. coli competent cells

All solutions and consumables used for the preparation of competent cells are sterilized by high temperature and high pressure.

1. Streak the Escherichia coli DH5α strain on a plate without antibiotics and invert it for 12-16 hours at 37°C; pick a single colony into a 2mL LB shake tube without any antibiotics, shake at 37°C at 180 rpm Cultivate overnight

2. Transfer 1 mL of bacterial liquid to 250 mL Erlenmeyer flask LB medium for expansion culture (expand culture at a ratio of 1:100), shake culture on a shaker at 180 rpm at 37°C for 2 to 3 hours until OD600 is between 0.4 and 0.5;

3. Transfer the culture solution to a centrifuge tube, place it on ice for 10 minutes, and then centrifuge at 3000 rpm at 4°C for 10 minutes (the centrifuge needs to be pre-cooled in advance), and carefully discard the supernatant;

4. Add pre-cooled 0.1M CaCl2 4mL (0.1M CaCl2 should be pre-cooled in advance), after fully pre-cooling, use a vortex mixer to quickly suspend the bacteria, and then ice bath for 10 minutes, combine the two tubes into one tube; 5.4℃, 4000rpm Centrifuge for 6min;

6. Discard the supernatant, add 4mL ice-cold 0.1M CaCl2 and 0.1mL pre-cooled sterilized pure glycerin, and suspend the precipitate;

7. Dispense the above suspension into PCR tubes at 100 μL/tube (PCR tubes are best placed on ice in advance), and store in liquid nitrogen for later use;

(5) Transformation of ligation products into E. coli

1. Thaw the prepared competent cells (thaw on ice), add an appropriate volume of ligation product and mix well, and then ice bath for 30 minutes. Usually the volume of the ligation product added is less than 1/10 of the volume of the competent cell; heat shock in a 2.42℃ water bath for 90s, and then quickly place it on ice for 5 minutes;

3. Add the bacterial solution to 800μL LB liquid medium (without antibiotics), mix well and culture with shaking at 37°C for 40-60min;

4. Transfer the bacterial solution to a 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 5 minutes, discard part of the supernatant, leave about 100 μL of the supernatant, and then blow the cells into a cell suspension;

5. Spread the above suspension on the LB solid medium containing Amp, and place it upside down in a 37°C incubator for overnight culture; other experimental operations, such as DNA fragment gel recovery, purification and recovery, and the steps are based on the DNA gel recovery kit , PCR product purification kit (Kangwei Century Biotechnology Co., Ltd.) operating instructions; plasmid extraction steps according to the plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) extraction kit

Production and purification of adeno-associated virus

The preparation and purification of the adeno-associated virus involved in the present invention are obtained according to the following steps

1. Prepare 6 dishes of HEK-293 cells and pass them to 24 dishes and 10 cm culture dishes according to the ratio of 1:4;

2. When the cell density grows to 80% density, use PEI for transfection. Add 10μg AAV8 plasmid+10μg Helper plasmid+10μg AAV target gene plasmid+DMEM to each 10cm culture dish to 910μL+90μL PEI;

3. Shake and mix well, and let stand at room temperature for 15 minutes;

4. Add 1ml of mixed solution to each dish of cells, and replace with fresh complete medium after 9-10 hours;

5. After 60 hours of transfection, collect the cell culture supernatant into a 50ml centrifuge tube, centrifuge at 4000rpm and 4°C for 20 minutes, and concentrate all the supernatants to 10-15ml by centrifugation;

6. Blow down and collect the cells into a 50ml centrifuge tube, centrifuge at 1500rpm for 10 minutes, discard the supernatant and add 3ml of cell lysis buffer (150mM sodium chloride, 20mM tris, pH 8.0) to resuspend;

7. The cells were repeatedly frozen and thawed three times in an alcohol bath at -80°C and a water bath at 37°C;

8. Mix the concentrated supernatant and cell suspension, add 1M magnesium chloride to a final concentration of 1mM;

9. Add Benzonase (Merk 70746-10kU) to a final concentration of 25U/ml. 37°C water bath for 40 minutes after mixing;

10. Centrifuge at 4000 rpm for 30 minutes at 4°C, and take the supernatant;

11. Calculate the volume of the supernatant to prepare the Beckman finger seal tube for loading;

12. Use a sterile 15 cm long gavage needle to add the sample, add different samples with different gavage needles, each gradient of iodixanol can choose to wash with PBS. The corresponding sample volume should be the same. Before sealing the virus supernatant, add virus lysate and then 17% iodixanol 6ml, then 25% iodixanol 6ml, then 40% iodixanol 5ml, and then 60% iodixanol. 5ml of Salanol, then the virus supernatant until it fills the finger-seal tube, and finally seal it with a lighter;

13. Use an ultracentrifuge at 60,000 rpm for 2 hours at a temperature of 16°C;

14. After centrifugation, the virus is stored in 40% Iodixanol solution;

15. Add 1XPBS to 50ml to the collected virus liquid, take 15ml amount and transfer it to the ultrafiltration tube of Milipore, centrifuge at 3500rpm for 30 minutes, the temperature is 4℃, remove the liquid in the collection tube, and then add 15ml virus liquid Centrifuge under the same conditions until the filtration of 50ml virus solution is completed;

16. Finally, collect the virus in the ultrafiltration tube, and store the sterile EP tube in a refrigerator at -80°C for later use.

Hydrodynamic transfection of mouse liver

1. Plasmid large extraction, according to Tiangen removal endotoxin plasmid large extraction kit to obtain high-concentration and high-quality REDMAP system plasmid.

2. Use Ringers' solution to supplement the target system plasmid to about 2ml (8-10% of the mass of the mouse), and inject it through the tail vein. The injection time is controlled within 3-5s.

3. Press the wound for 5 seconds to stop bleeding.

Selection and production of light source

In the embodiments of the present invention, LEDs with wavelengths of 660 nm and 730/780 nm are used as examples to illustrate the regulation method of the gene loop remote regulation system of the present invention, but the protection scope of the present invention is not limited.

The LEDs used in the experiment were purchased from Shenzhen Best Optoelectronics Technology Co., Ltd. The rest of the accessories are all domestic conventional consumables. The 660nm wavelength LED is to provide a low-power red light emitter; the 730/780nm wavelength LED is to provide a low-power far-red light emitter. The connection method of the 4×6 LED board is as follows: according to the arrangement of the cell culture plate (24-well plate), each LED corresponds to the center position of each hole, and each LED is connected in parallel. Adjust the light time and light intensity according to the needs of the experiment.

Cell culture and transfection

In the examples of the present invention, the following cell lines and PEI transfection are used as examples to illustrate the working conditions of the REDMAP system in cells, but the protection scope of the present invention is not limited.

The 10cm cell culture dishes, cell culture plates (24 wells), 15mL and 50mL centrifuge tubes used for cell culture were all purchased from Thermo Fisher Scientific (Labserv) in the United States; used modified Eagle medium, fetal bovine serum, and penicillin The streptomycin solution was purchased from Gibico, USA; the PEI used for transfection was purchased from Polysciences; the cell incubator was purchased from Thermo Fisher Scientific, USA; the rest of the consumables were ordinary domestic consumables.

Cell culture: The cells involved in the patent include human embryonic kidney cells (HEK-293, ATCC: CRL-11268), which stably integrate a copy of the E1 gene (ThermoFisher, R70507), and telomere human mesenchymal cells (hMSC-TERT). ) And HEK-293-derived Hana3A cells, all cells were cultured in modified Eagle medium (DMEM) with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin added to the medium And streptomycin solution; cells are cultured in an incubator containing 5% carbon dioxide at 37°C. Transfection: There are two transfection methods for all cell lines. The first transfection method uses optimized PEI (Wieland M, Methods 56(3):351). To put it simply, the culture system is a 10mL 10cm cell culture dish inoculated with 6×10 ⁴ cells, after 18 hours of culture, the optimal ratio of DNA is mixed with PEI at a mass ratio of 3:1 (PEI:DNA) Dissolve in the culture medium and let stand for 6h. (Polyethyleneimine, molecular weight 40,000, stock solution 1mg/mL in ddH2O; Polysciences; Cat. no. 24765) The number of cells was counted with a Countess II automated cell counter. The second transfection method uses Lip8000 ^™ : a. The day before transfection, 200,000-700,000 cells per well are seeded into a six-well plate for culture, so that the cell density can reach 70%-80% the next day. b. Replace the six-well plate with fresh complete medium (containing serum and double antibodies). c. Take a clean and sterile centrifuge tube, add 125μl DMEM to each well, add 2.5μg plasmid DNA, mix gently, add 4μl Lipo8000 ^TM transfection reagent, mix gently, remember not to Vortex or centrifuge. d. Add 125μl of mixed solution evenly to the entire well and mix gently.

Reporter gene secreted alkaline phosphatase (SEAP) detection: Homoarginine, magnesium chloride, diethanolamine, and HCl used to configure the reporter gene reaction buffer were purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.; The color substrate (p-nitrophenol phosphate) was purchased from Shanghai Jingchun Biochemical Technology Co., Ltd. (Aladdin).

(1) Reagent configuration:

· 2x buffer:

·20mM Homoarginine Note: Its role is to inhibit endogenous alkaline phosphatase activity

·1mM Magnesium Chloride

·2% Diethanolamine

·Adjust the pH to 9.8 with HCl

Substrate solution:

·120mM chromogenic substrate (p-nitrophenol phosphate)

·In 2x assay buffer

(2) Experimental steps:

1. Aspirate the cell culture supernatant, 200μL into a centrifuge tube (Note: Generally more than 150μL, because subsequent heating will lose a part of the volume).

2. Water bath at 65°C for 30 minutes (Note: heating is mainly to inactivate endogenous alkaline phosphatase, while SEAP is resistant to high temperatures and will not be inactivated at this temperature).

3. Pipet 80μL (diluted according to the experimental situation) to a 96-well plate, and quickly add 100μl of pre-heated 2x buffer and 20μl of substrate solution.

4. The microplate reader measures 10 times at 405nm with an interval of 1 min (depending on the conditions of the experiment, additional conditions can be set).

(3) Calculation of enzyme activity

The enzyme activity of alkaline phosphatase (SEAP) is defined as: when 37℃, pH 9.8, it reacts with the substrate disodium p-nitrophenylphosphate (PNPP-Na2) within 1 min to generate 1mol/L p-nitrophenol alkalinity Phosphatase is defined as 1 activity unit (1U). The p-nitrophenol itself has a bright yellow color. At a wavelength of 405nm, different concentrations of p-nitrophenol correspond to different absorbance values. The calculation method is: the slope of the curve of the OD value measured at different time points during the reaction between the sample and the substrate *256.8 is the enzyme activity, in U/L.

Western blot experiment procedure

(1) Cells are planted in a 6-well plate. After the plate is full, discard the medium, wash it with PBS first, then place the plate on ice, discard the PBS, add 100～300μl RIPA, and scrape the cells with a cell scraper .

(2) Ultrasonic lysis of protein

The whole process needs to be completed on ice and water, the voltage is adjusted to the maximum, and the total time is 10min (over 0.25s, rest 0.25s).

(3) Collect the supernatant by centrifugation at 4℃

After the lysis is completed, centrifuge at 12000 rpm at 4°C for 15 min, and collect the supernatant.

(4) With glue, 10% separating glue

(5) BCA principle to measure protein concentration

Detect the absorbance value at 528nm, dilute the protein 10 times and add 25μl to (200μl A+4μl B), and put it at 37°C for 30 minutes after completion, and read the value.

(6) Protein denaturation

(7) Run electrophoresis

(8) Transfer film

(9) Washing film

(10) The milk is closed for 1h～1.5h

(11) Incubate the primary antibody in a shaker at 4°C overnight. After completion, wash the membrane three times, once for 10 minutes.

(12) Incubate the secondary antibody in a shaker at room temperature for 1 to 2 hours. After completion, wash the membrane three times, once for 10 minutes.

(13) The instrument sweeps the film.

Example 1 Different truncation and optimization of PhyA elements in REDMAP system

This example uses SEAP as the reporter gene to verify the different truncation and optimization of PhyA elements in the REDMAP system, but does not limit the protection scope of the present invention. Specific steps are as follows:

The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

The second step is to inoculate cells. The well-growing HEK-293 cells were digested with 0.25% trypsin and planted in two 24-well plates, each well with 6×10 ⁴ cells, and 500 μL containing 10% FBS, 1% (volume/ Volume) of penicillin and streptomycin in DMEM medium.

The third step is transfection. Divide two 24-well plates into dark group and light group, and each group is divided into 6 groups. Within 16 to 24 hours of seeding the cells, pDQ15, pDL6 and PhyA proteins of different lengths, pYZ180, pYZ179, pYZ181, pYZ182, pYZ183, and pYZ308 were added to each group at a ratio of 1:1:1 (w/w/w) and PEI Mix the staining reagent and serum-free DMEM, let it stand at room temperature for 15 minutes, and add it to the 24-well culture plate evenly. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 h of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin, streptomycin and 5 μM PCB were replaced for culture.

The fourth step is light. After changing the solution for 14-18 hours, divide them into 2 groups (numbered 1 and 2 respectively). Place the No. 1 24-well plate in the dark, and place the No. 2 24-well plate at a wavelength of 660nm and a light intensity of 2mW/cm ² Illuminate under LED (refer to experimental materials and methods for specific connection method) for 1 hour, and place it in the dark immediately after light treatment.

The fifth step is to detect the reporter gene. The cell culture supernatants of the dark group and the light group were taken 24 hours after the end of light to determine the expression of SEAP (refer to materials and methods for specific methods).

The results show that in the REDMAP system, when the length of PhyA is 1-617aa, the activation efficiency is the most significant. For details of the experimental data, please refer to Figure 2 of the specification.

Example 2 The activation efficiency of different PhyA chaperone proteins in the REDMAP system on the reporter gene

This example uses SEAP as the reporter gene to verify the activation efficiency of the different PhyA chaperone proteins in the REDMAP system on the reporter gene, but does not limit the protection scope of the present invention. Specific steps are as follows:

The second step is to inoculate cells (the specific steps are the same as in Example 1).

The third step is transfection. Divide the two 24-well plates into a dark group and a light group, and each group is divided into two groups. Within 16 to 24 hours of cell inoculation, pYZ181, pDL6 and different PhyA chaperone proteins pDQ15, pDQ16 were added to each group at a ratio of 1:1:1 (w/w/w) mixed with PEI transfection reagent and serum-free DMEM. Evenly, let it stand at room temperature for 15 minutes and add it to the 24-well culture plate evenly. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin, streptomycin, and 5 μM PCB were replaced for culture.

The fourth step is lighting (the specific steps are the same as in Example 1).

The fifth step is to detect the reporter gene (the specific steps are the same as in Example 1).

The results show that in the REDMAP system, when FHY1 is used as a chaperone protein of PhyA, the system has a higher efficiency in activating the report. For details of the experimental data, please refer to Figure 3 of the specification.

Example 3 The activation efficiency of different nuclear localization signals of ΔPhyA in the REDMAP system on the reporter gene

This example uses SEAP as the reporter gene to verify the activation efficiency of the different nuclear localization signals of ΔPhyA in the REDMAP system on the reporter gene, but does not limit the protection scope of the present invention. Specific steps are as follows:

The third step is transfection. Divide the two 24-well plates into a dark group and a light group, and each group is divided into 3 groups. Within 16 to 24 hours of cell seeding, pDQ16, pDL6 and PhyA proteins pYZ181, pYZ216, and pYZ318 with different nuclear localization signals were added to each group at a ratio of 1:1:1 (w/w/w) with PEI transfection reagent Mix it with serum-free DMEM, let it stand at room temperature for 15 minutes, and add it to the 24-well culture plate evenly. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin, streptomycin, and 5 μM PCB were replaced for culture.

The fourth step is lighting (the specific steps are the same as in Example 1).

The results show that in the REDMAP system, when the ΔPhyA protein does not carry any nuclear localization signal, the activation report of the system has the highest efficiency. For details of the experimental data, please refer to Figure 4 of the specification.

Example 4 PhyA chaperone protein FHY1 in the REDMAP system has different nuclear localization signals to activate the reporter gene

This example uses SEAP as the reporter gene to verify that the chaperone protein FHY1 of PhyA in the REDMAP system has different nuclear localization signals to activate the reporter gene, but does not limit the protection scope of the present invention. Specific steps are as follows:

The third step is transfection. Divide the two 24-well plates into a dark group and a light group, and each group is divided into 3 groups. Within 16 to 24 hours of seeding the cells, pYZ181, pDL6 and FHY1 proteins pDQ16, pDQ18, pYZ217 with different nuclear localization signals were added to each group at a ratio of 1:1:1 (w/w/w) with PEI transfection reagent Mix it with serum-free DMEM, let it stand at room temperature for 15 minutes, and add it to the 24-well culture plate evenly. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin, streptomycin, and 5 μM PCB were replaced for culture.

The fourth step is lighting (the specific steps are the same as in Example 1).

The results show that in the REDMAP system, when the FHY1 protein does not carry any nuclear localization signal, the activation report of the system has the highest efficiency. For details of the experimental data, please refer to Figure 5 of the specification.

Example 5 The fusion of different transcriptional activation domains with the chaperone protein FHY1 of PhyA in the REDMAP system to activate the reporter gene

This example uses SEAP as the reporter gene to verify the activation efficiency of the fusion of different transcription activation domains of the PhyA chaperone protein FHY1 in the REDMAP system on the reporter gene, but does not limit the protection scope of the present invention. Specific steps are as follows:

The third step is transfection. Divide the two 24-well plates into a dark group and a light group, and each group is divided into 5 groups. Within 16 to 24 hours of seeding the cells, pYZ181, pDL6 and FHY1 proteins fused with different transcription activation domains pZQ53, pZQ54, pZQ56, pZQ57, and pDQ16 were added to each group at a ratio of 1:1:1 (w/w/w) Mix it with PEI transfection reagent and serum-free DMEM, let it stand at room temperature for 15 minutes, and add it to a 24-well culture plate evenly. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin, streptomycin, and 5 μM PCB were replaced for culture.

The fourth step is lighting (the specific steps are the same as in Example 1).

The results show that in the REDMAP system, taking into account the size of the transcription activation domain, when the FHY1 protein is fused with the smallest transcription activation domain VP64, the system has the highest activation report efficiency. For details of the experimental data, please refer to Figure 6 of the specification.

Example 6 The response of the REDMAP system to red light stimuli of different durations

This example uses SEAP as the reporter gene to verify the response of the REDMAP system to different long-term red light stimulations, but does not limit the protection scope of the present invention. Specific steps are as follows:

The third step is transfection. Divide 10 24-well plates into 10 groups evenly. Within 16 to 24 hours of cell inoculation, pYZ181, pDL6, and pDQ16 were added to each group at a ratio of 1:1:1 (w/w/w) and mixed with PEI transfection reagent and serum-free DMEM. After standing at room temperature for 15 minutes Evenly drop into the 24-well culture plate. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin, streptomycin, and 5 μM PCB were replaced for culture.

The fourth step is light. After 14-18 hours of fluid exchange, 10 groups were numbered (respectively numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), and the No. 1 24-well plate was placed in the dark, 2- No. 10 24-well plates were placed under LEDs with a wavelength of 660nm and a light intensity of 0.2mW/cm ² (refer to experimental materials and methods for specific connection) for different periods of time (0-10s), and immediately placed in the dark after the light treatment .

The results show that when the red light irradiation intensity is 0.2mW/cm ² , the activation ability of the REDMAP system increases with the irradiation time, and the switch is activated for 5 seconds at an intensity of ^{0.2mW/cm 2} Ability no longer increases with time. For details of the experimental data, please refer to Figure 7 of the specification.

Example 7 The response of the REDMAP system to red light stimuli of different intensities

This example uses SEAP as the reporter gene to verify the response of the REDMAP system to red light stimulations of different intensities, but does not limit the protection scope of the present invention. Specific steps are as follows:

The third step is transfection. Divide 8 24-well plates into 8 groups evenly. Within 16 to 24 hours of cell seeding, add pYZ181, pDL6, and pDQ16 to each group at a ratio of 1:1:1 (w/w/w), mix with PEI transfection reagent and serum-free DMEM, and let stand at room temperature for 15 minutes Then evenly drip into the 24-well culture plate. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin, streptomycin, and 5 μM PCB were replaced for culture.

The fourth step is light. 14-18 hours after changing the liquid, number 8 groups (numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 respectively), and place them at a wavelength of 660nm and different light intensity (0-2mW/cm ² ) LED (refer to experimental materials and methods for specific connection methods) for 1 second, and place in the dark immediately after light treatment.

The results show that when the REDMAP system is irradiated with red light for 1s, the activation ability of the report increases with the increase of the light intensity. When the light intensity is 1mW/cm ² under the condition of continuous stimulation for 1s, the activation ability of the switch is not Then increase with the increase of light intensity. For details of the experimental data, please refer to Figure 8 of the specification.

Example 8 The influence of different PCB concentrations on the activation reporting ability of the REDMAP system

This example uses SEAP as the reporter gene to verify the influence of different PCB concentrations on the activation reporting ability of the REDMAP system, but does not limit the protection scope of the present invention. Specific steps are as follows:

The third step is transfection. Divide the two 24-well plates into the dark group and the light group. Each group is divided into 5 groups. Within 16 to 24 hours of cell inoculation, pYZ181, pDL6, and pDQ16 are added to each group at a ratio of 1:1:1 (w/w/w) and mixed with PEI transfection reagent and serum-free DMEM. , After standing at room temperature for 15 minutes, add dropwise to the 24-well culture plate evenly. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin and streptomycin mixture was replaced for culture, and the corresponding amount of PCB (0-10 μM) was added to each group.

The fourth step is lighting (the specific steps are the same as in Example 1).

The results show that the activation reporting ability of the REDMAP system increases with the increase of PCB concentration, and approaches saturation when the PCB content is 10μM. For details of the experimental data, please refer to Figure 9 of the specification.

Example 9 Exploring the change in the amount of activation reported by the REDMAP system after receiving red light stimulation over time

In this example, SEAP is used as the reporter gene to explore the amount of activation of the report generated by the REDMAP system after receiving the red light stimulation for different periods of time, but it does not limit the protection scope of the present invention. Specific steps are as follows:

The third step is transfection. Divide the two 24-well plates into the dark group and the light group. Each group is divided into 8 groups. Within 16 to 24 hours of cell inoculation, pYZ181, pDL6, and pDQ16 are added to each group at a ratio of 1:1:1 (w/w/w) and mixed with PEI transfection reagent and serum-free DMEM. , After standing at room temperature for 15 minutes, add dropwise to the 24-well culture plate evenly. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin, streptomycin, and 5 μM PCB were replaced for culture.

The fourth step is lighting (the specific steps are the same as in Example 1).

The fifth step is to detect the reporter gene. The cell culture supernatants of the dark group and the light group were taken at different times (0-72h) after the end of light to determine the expression of SEAP (refer to materials and methods for specific methods).

The results showed that after the REDMAP system was stimulated by red light, the reported expression level increased with time. For details of the experimental data, please refer to Figure 10 of the specification.

Example 10 Research on the spatial specificity of the REDMAP system

This example uses EGFP as the reporter gene to explore the spatial specificity of the REDMAP system, but does not limit the protection scope of the present invention. Specific steps are as follows:

The second step is to inoculate cells. After digesting the well-growing HEK-293 cells with 0.25% trypsin, about 3×10 ⁶ HEK-293 cells were seeded in a culture dish with a diameter of 10 cm, and 10 mL containing 10% FBS, 1% (volume /Volume) of penicillin and streptomycin in DMEM medium.

The third step is transfection. Within 16 to 24 hours of cell inoculation, pYZ181, pDQ63, and pDQ16 were added to each group at a ratio of 1:1:1 (w/w/w) and mixed with PEI transfection reagent and serum-free DMEM. After standing at room temperature for 15 minutes Evenly drop into the petri dish. The total volume is 1000 μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 10 mL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin, streptomycin, and 5 μM PCB were replaced for culture.

The fourth step is light. After transfection for 18 hours, pass through a designed mask, ^{irradiate with red light (660nm, 1mW/cm 2} ) for 10s, and then immediately darken the treatment.

The fifth step is to detect the reporter gene. Clinx imaging equipment (ChemiScope 4300Pro, Clinx, Shanghai, China) was used to measure the fluorescence signal after 24 hours of light.

The results show that the REDMAP system has a high degree of spatial specificity. For details of the experimental data, please refer to Figure 11 of the specification.

Example 11 Research on the reversibility of REDMAP system

This example uses SEAP as the reporter gene to explore the reversibility of the REDMAP system, but does not limit the protection scope of the present invention. Specific steps are as follows:

The third step is transfection. Divide 4 24-well plates into OFF group and ON group. Choose 3 wells from each group as a group and divide them into 15 groups. Within 16 to 24 hours of cell inoculation, add pYZ181, pDQ16, and pDL6 to each group at a ratio of 1:1:1 (w/w/w) and mix with PEI transfection reagent and serum-free DMEM. After standing at room temperature for 15 minutes Evenly drop into the 24-well culture plate. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin, streptomycin, and 5 μM PCB were replaced for culture.

The fourth step is light. After changing the liquid for 14-18 hours, place the 24-well plate of the OFF group ^{under an LED with a wavelength of 730nm and a light intensity of 1mW/cm 2} (refer to experimental materials and methods for specific connection methods) for 1 min, and place the 24-well plate of the ON group in the The wavelength is 660nm, the light intensity is 1mW/cm ^{2 under} the LED (refer to the experimental materials and methods for specific connection) for 10s, and it is placed in the dark immediately after the light treatment. At the same time, the corresponding 24-well plate was replaced with light conditions (660nm/730nm) and fresh medium every 24h.

The fifth step is to detect the reporter gene. Take a small group of cell culture supernatants from the 24-well plate of the OFF group and the 24-well plate of the ON group for 6 hours each time, and determine the expression level of SEAP (refer to materials and methods for specific methods).

The results show that the REDMAP system has good reversibility and controllability. For details of the experimental data, please refer to Figure 12 of the specification.

Example 12 The working conditions of the REDMAP system in different cell lines

This example uses SEAP as the reporter gene to verify the working conditions of the REDMAP system in different cell lines, but does not limit the protection scope of the present invention. Specific steps are as follows:

The second step is to inoculate cells. The well-growing HEK-293 cells, Hana3A cells, hMSC-TERT cells, NIH 3T3 cells, and HeLa cells were digested with 0.25% trypsin and then planted in 2 blocks (each cell is planted in 4 wells on each plate) ) In different 24-well plates, plant 6×10 ⁴ cells per well, and add 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin and streptomycin.

The third step is transfection. Two 24-well plates were divided into dark group and light group, each group was divided into 5 groups (HEK-293 cell group, Hana3A cell group, hMSC-TERT cell group, NIH 3T3 cell group, HeLa cell group). Add pYZ181, pDQ16, and pDL6 to the PEI transfection reagent at a ratio of 1:1:1 (w/w/w) to HEK-293 cell group, Hana3A cell group, and hMSC-TERT cell group within 16 to 24 hours after cell seeding. Mix it with serum-free DMEM, let it stand at room temperature for 15 minutes, and add it to the 24-well culture plate evenly. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) of penicillin, streptomycin and 5 μM PCB was changed into DMEM medium for culture. Add pYZ181, pDQ16, and pDL6 to the NIH 3T3 cell group and HeLa cell group at a ratio of 1:1:1 (w/w/w) and Lip8000 ^TM transfection reagent and mix them, and follow the instructions in the materials and methods.

The fourth step is light. The dark group was placed in the dark, and the light group was placed ^{under an LED with a wavelength of 660nm and a light intensity of 1mW/cm 2} (refer to experimental materials and methods for specific connection methods) for 1 min, and immediately placed in the dark after the light treatment.

The results show that the REDMAP system can work well in a variety of different cell lines, exhibiting its broad-spectrum properties. For details of the experimental data, please refer to Figure 13 of the specification.

Example 13 Comparison results of REDMAP system and other red light/far-red light systems

This example uses SEAP as the reporter gene to verify the response of the REDMAP system to far-red light stimulation at different times, but does not limit the protection scope of the present invention. Specific steps are as follows:

The second step is to inoculate cells. The well-growing HEK-293 cells were digested with 0.25% trypsin and then planted in different 24-well plates. Seed 6×10 ⁴ cells per well, and added 500 μL containing 10% FBS, 1% (volume/ Volume) of penicillin and streptomycin in DMEM medium.

The third step is transfection. Combine pYH70, pYH71 and pMF111 (BphP1/PpsR2 system), pWS189, pGY32, and pXY34 (BphS system), pYZ301, pYZ303, and pDL6 (PhyB/PIF3 system), pYZ181, pDQ16, and pDL6 (REDMAP system) to 1: Mix it with PEI transfection reagent and serum-free DMEM at a ratio of 1:1 (w/w/w), and place it at room temperature for 15 minutes and then drop it evenly into a 24-well culture plate. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, change into 500 μL DMEM medium containing 10% FBS, 1% (volume/volume) penicillin, streptomycin and 0 or 5 μM PCB for culture.

The fourth step is light. Place the dark group in the dark, and place the light group under an LED with a wavelength of 660 nm/730nm and an illumination intensity of 1mW/cm ² (refer to the experimental materials and methods for specific connection) for 1s, and place it in the dark immediately after the light treatment in.

The results show that under the same illumination conditions, the induction efficiency of the REDMAP system is much higher than the other three red/far-red light systems. For details of the experimental data, please refer to Figure 14 of the specification.

Example 14 The response of REDMAP system to far-red light (730nm) stimulation after different time

The third step is transfection. Divide the 5 24-well plates into 5 groups equally. Within 16 to 24 hours of cell seeding, add pYZ181, pDL6, and pDQ16 to each group at a ratio of 1:1:1 (w/w/w), mix with PEI transfection reagent and serum-free DMEM, and let stand at room temperature for 15 minutes Then evenly drip into the 24-well culture plate. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin, streptomycin, and 5 μM PCB were replaced for culture.

The fourth step is light. After 14-18 hours of liquid exchange, the 5 groups were numbered (respectively numbered 1, 2, 3, 4, and 5), and were ^{irradiated with red light of 660nm wavelength with an intensity of 1mW/cm 2} for 1 minute, and the 24 holes were quickly Place the board in a dark state, and ^{irradiate it with 1mW/cm 2} far-red light with a wavelength of 730nm for 0, 0.01min, 0.1min, 1min, and 10min after 1h (refer to the experimental materials and methods for the specific connection method of the LED light), and immediately after the light treatment Place in the dark.

The results show that the REDMAP system can be turned off with far-red light with a wavelength of 730nm after irradiating it with a red light with a wavelength of 660nm at 1mW/cm2 for 1min. This embodiment illustrates that the REDMAP system has sensitive opening and closing characteristics, and at the same time has precise controllability. For details of the experimental data, please refer to Figure 15 of the specification.

Example 15 The response of the REDMAP system to different intensities of far-red light (730nm) stimulation

This example uses SEAP as the reporter gene to verify the response of the REDMAP system to different intensities of far-red light stimulation, but does not limit the protection scope of the present invention. Specific steps are as follows:

The fourth step is light. After 14-18 hours of liquid exchange, the 5 groups were numbered (respectively numbered 1, 2, 3, 4, and 5), and were ^{irradiated with red light of 660nm wavelength with an intensity of 1mW/cm 2} for 1 minute, and the 24 holes were quickly Place the board in a dark state, and irradiate it with 0, 0.1mW/cm ² , 0.2mW/cm ² , 0.5mW/cm ² , 1mW/cm ² and 730nm wavelength far red light for 1 min after 1 hour (refer to the specific connection method of the LED light Experimental materials and methods), placed in the dark immediately after light treatment.

The results show that the REDMAP system can shut down the system with far red light with a wavelength of ^{730 nm after irradiating it with red light with a wavelength of 660 nm at 1 mW/cm 2 for 1 min.} This embodiment illustrates that the REDMAP system has sensitive opening and closing characteristics, and at the same time has precise controllability. For details of the experimental data, please refer to Figure 16 of the specification.

Example 16 The response of REDMAP system to far-red light (780nm) stimulation of different time and intensity

This example uses SEAP as the reporter gene to verify the response of the REDMAP system to far-red light (780nm) stimulation at different times and intensities, but does not limit the protection scope of the present invention. Specific steps are as follows:

The third step is transfection (the specific steps are the same as in Example 14).

The fourth step is light. After changing the liquid for 14-18 hours, ^{irradiate the red light with an intensity of 1mW/cm 2} with a wavelength of 660nm for 1 minute, and quickly put 8 24-well plates in the dark state. After 1 hour, use 0-2mW/cm ² with a wavelength of 780nm. Irradiate with far red light for 0-10min (refer to the experimental materials and methods for the specific connection method of the LED lamp), and place it in the dark immediately after the light treatment.

The results show that the REDMAP system can shut down the system with far-red light with a wavelength of 780 nm after irradiating it with red light with a wavelength of 660 nm at 1 mW/cm2 for 1 min. This embodiment illustrates that the REDMAP system has sensitive opening and closing characteristics, and at the same time has precise controllability. For details of the experimental data, please refer to Figure 17 of the specification.

Example 17 Verify whether the REDMAP system can replace PCB by co-transformed PCB synthesis gene cassette (pPKm248)

In this example, SEAP is used as the reporter gene to verify whether the REDMAP system can be replaced by a co-transformed PCB synthesis gene cassette (pPKm248), but the protection scope of the present invention is not limited. Specific steps are as follows:

The third step is transfection. First divide the two 24-well plates into light and dark, and then divide each 24-well plate into two groups. Within 16 to 24 hours of seeding the cells, the first group was added with pYZ181, pDL6, pDQ16, pcDNA3.1(+) in a ratio of 1:1:1:1 (w/w/w/w) with PEI transfection reagent and no Mix the serum with DMEM, add pYZ181, pDL6, pDQ16, pPKm248 in the second group at a ratio of 1:1:1:1 (w/w/w/w) and mix with PEI transfection reagent and serum-free DMEM. , After standing at room temperature for 15 minutes, add dropwise to the 24-well culture plate evenly. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, the first group was cultured with 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin and streptomycin mixture and 5 μM PCB, and the second group was cultured with 500 μL containing 10% FBS , 1% (volume/volume) penicillin, streptomycin mixed solution DMEM medium for culture.

The fourth step is light. After 14-18 hours of liquid exchange, the light group was ^{irradiated with red light of 660nm wavelength with an intensity of 1mW/cm 2} for 1 min (refer to the experimental materials and methods for the specific connection of the LED lamp), and placed in the dark immediately after the light treatment.

The results show that the effect of REDMAP system for synthesizing gene cassettes by co-transformation of PCBs is equivalent to that of adding PCBs to the culture medium, which is replaceable and can be selected according to actual needs. For details of the experimental data, please refer to Figure 18 of the specification.

Example 18 The influence of different red light irradiation intensity and irradiation time on the endogenous gene activation of REDMAP system for CRISPR-dCas9

This example uses ASCL1 as the reporter gene to verify the effect of the REDMAP system on the activation efficiency of endogenous genes under different intensities of red light irradiation. But it does not limit the protection scope of the present invention. Specific steps are as follows:

The third step is transfection. Divide into 6 groups, each with two 24-well plates, divided into dark group and light group. Within 16 to 24 hours of seeding the cells, add pYZ181, pDQ16, pDQ100, pSZ69, pSZ83, pSZ84 to 4:4:1:2:2:2 (w/w/w/w/w/w) in each group. The ratio is mixed with PEI transfection reagent and serum-free DMEM, and after standing at room temperature for 15 minutes, it is evenly dripped into a 24-well culture plate. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin, streptomycin, and 5 μM PCB were replaced for culture.

The fourth step is light. After the liquid was changed, the illumination group was ^{irradiated with red light with an intensity of 0-2mW/cm 2} with a wavelength of 660nm for different times (refer to the experimental materials and methods for the specific connection of the LED lamp), and then cultivated in a dark environment after irradiation.

The fifth step is to detect the reporter gene. Real-time fluorescent quantitative PCR.

The results show that in the REDMAP system based on the CRISPR/dCas9 system, the light intensity is positively correlated with the activation efficiency, which is dependent on the light intensity and time. For details of the experimental data, please refer to attached drawings 19b and 19c of the specification.

Example 19 REDMAP system is used for reversibility analysis of endogenous gene activation of CRISPR-dCas9

This example uses ASCL1 as the reporter gene to verify the reversibility of the endogenous gene activation of CRISPR-dCas9 using the REDMAP system. But it does not limit the protection scope of the present invention. Specific steps are as follows:

The third step is transfection. Divide five 24-well plates into five groups. Within 16 to 24 hours of seeding the cells, add pYZ181, pDQ16, pDQ100, pSZ69, pSZ83, pSZ84 to 4:4:1:2:2:2 (w/w/w/w/w/w) in each group. The ratio is mixed with PEI transfection reagent and serum-free DMEM, and after standing at room temperature for 15 minutes, it is evenly dripped into a 24-well culture plate. The total volume of each well is 50μL, and the mass ratio of particles to PEI is 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin, streptomycin, and 5 μM PCB were replaced for culture.

The fourth step is light. After changing the solution for 14-18h, place all groups under LED with a wavelength of 660nm and an illumination intensity of 200μW/cm ² for 1s. After the light is over, place them in a dark place for incubation. After 1h, place groups 2-5 at a wavelength of 200μW/cm 2. 730nm intensity of 1mW/cm ² LED light for 1 min, 36h later, 4 and 5 groups are placed in the wavelength of 660nm, light intensity of 200μW/cm ² LED light for 1s.

The fifth step is to use real-time fluorescent quantitative PCR to detect the reporter gene every 12h, and change the medium every 24h.

The results show that the REDMAP system based on the CRISPR/dCas9 system has good reversible regulation. For details of the experimental data, please refer to Figure 19d of the specification.

Example 20 The efficiency of REDMAP system used for CRISPR-dCas9 to activate multiple endogenous genes

This example uses IL1RN, RHOXF2, TTN, and MIAT as reporter genes to verify the efficiency of the REDMAP system for CRISPR-dCas9 to activate multiple endogenous genes, but does not limit the protection scope of the present invention. Specific steps are as follows:

The third step is transfection. Divide the two 24-well plates into a dark group and a light group, and each group is divided into five groups. Within 16 to 24 hours of seeding the cells, add pYZ181, pDQ16, pDQ100, pSZ69, pSZ92, pSZ93 in the first group, pYZ181, pDQ16, pDQ100, pSZ69, pSZ105, pSZ106 in the second group, and add in the third group pYZ181, pDQ16, pDQ100, pSZ69, pSZ107, pSZ108, pYZ181, pDQ16, pDQ100, pSZ69, pYZ417, pYZ418 were added to the fourth group, and the fifth group was blank without transfected plasmids, all with 4:4:1 The ratio of :2:2:2 (w/w/w/w/w/w) is mixed with PEI transfection reagent and serum-free DMEM. After standing at room temperature for 15 minutes, it is evenly dripped into a 24-well culture plate. The total volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume/volume) penicillin, streptomycin, and 5 μM PCB were replaced for culture.

The fourth step is light. After changing the solution for 14-18h, all groups were placed under ^{an LED with a wavelength of 660nm and a light intensity of 1mW/cm 2} for 1 min. After the light was over, they were placed in a dark place for cultivation.

The results show that the REDMAP system based on the CRISPR/dCas9 system can efficiently activate different types of endogenous genes. For details of the experimental data, please refer to attached drawings 19e-h of the description.

Example 21 The investigation of different red light illumination time and light intensity on the working condition of REDMAP system in mice

In this example, Luciferase is used as the reporter gene to verify the effect of different red light illumination time and light intensity on the working condition of the REDMAP system in mice, but does not limit the protection scope of the present invention. Specific steps are as follows:

The second step is hydrodynamic transfection. The REDMAP system plasmid was injected into mice using hydrodynamic tail vein injection, and each mouse was injected with 100μg CMV-ΔPhyA-Gal4-P2A-FHY1-VP64-pA, 100μg CMV-pcyA-2A-HO-2A-FD-2A -FNR-pA and 50μg 5×UAS-P _TATA- Luciferase-pA.

The fourth step is light. The tail vein was injected for about 8 hours, and the mice were exposed ^{to red light with different light intensity (0-10mW/cm 2} ) for a certain light time (0-360s).

The fifth step is to detect the reporter gene. After 10 hours of illumination, each mouse was injected with 100 μL of firefly luciferin at a concentration of 100 mM before detection, and imaging was performed on a small animal in vivo imaging system (IVIS Lumina III; model no. CLS136334; PerkinElmer) for 10 to 30 minutes.

The sixth step is statistics. The luminous intensity statistics of the imaging photos obtained in the fifth step are performed. For the experimental data, please refer to attached drawings 20 and 21 of the specification.

The results show that the REDMAP system can precisely and controllably control the expression of reporter genes (luciferase) in mice. By counting the luciferase signals, the present invention finds that the REDMAP system has a good efficiency in inducing gene expression in mice. For details of the experimental data, please refer to attached drawings 20 and 21 of the specification.

Example 22 Research on the working condition of AAV packaged REDMAP system in mice

This example uses Luciferase as the reporter gene to verify the working condition of the REDMAP system packaged by AAV in mice, but does not limit the protection scope of the present invention. Specific steps are as follows:

The second step is to prepare and purify the AAV virus.

In the third step, the mice were divided into dark group and light group. The packaged AAV was injected into mice using the tail vein, and each mouse was injected with 1.5×10^11vg AAV-CMV-Δ PhyA-Gal4-P2A-FHY1-VP64-pA and 1.5×10^11vg AAV-5× UAS-P _hCMVmin -Luciferase-pA.

The fourth step is light. After 2, 5, 8, and 11 weeks, the PCB was injected with 3.5 mg/kg and ^{irradiated with 5mW/cm 2} red light with a wavelength of 660nm for 1 min.

The fifth step is to detect the reporter gene. After 8 hours of illumination, each mouse was injected with 100 μL of firefly luciferin at a concentration of 100 mM before detection, and imaging was performed on a small animal in vivo imaging system (IVIS Lumina III; model no. CLS136334; PerkinElmer) for 10 to 30 minutes.

The sixth step is statistics. The luminous intensity statistics are performed on the imaging photos obtained in the fifth step, and the experimental data are detailed in Figure 22 of the specification.

The results show that the REDMAP system delivered by adeno-associated virus can also activate the expression of reporter genes (luciferase) in mice. By counting the luciferase signal, the present invention found that the adeno-associated virus delivery REDMAP system has good long-term induction in mice. The efficiency of gene expression. For details of the experimental data, please refer to Figure 22 of the specification.

Example 23 Therapeutic effect of REDMAP system in diabetic mouse model

In this example, insulin and blood glucose are used as reports to verify the blood glucose control of type I diabetic mice by the REDMAP system, but does not limit the protection scope of the present invention. Specific steps are as follows:

The second step is transfection. The HEK-293 cells were cultured overnight in a 10 cm dish, and pYZ181, pDQ16 and pYZ393 were transfected at a ratio of 1:1:1 (w/w/w, 12μg/dish), and the medium was changed after 6h.

The third step is to prepare microcapsules containing engineered cells. After the transfected cells are digested, the cells are encapsulated in microcapsules according to the operating steps, with an average of 200 cells per capsule. The prepared microcapsules were incubated with 10μM PCB for one hour and then injected into the mouse subcutaneously. Subsequently, the diabetic mice injected with the microcapsules were randomly divided into a light group and a dark group, with 5 mice in each group.

The fourth step is light. After transplantation, an intensity of light per day group 5s 660nm wavelength red light 20mW / cm ^2, the in situ injection of 20μL 2.5mM before the irradiation of the PCB; darkness has been set in the dark.

The fifth step is to detect the blood glucose level of the mice. The blood glucose and insulin content of each mouse were detected 48h after transplantation. For details of the experimental data, please refer to Figure 23c of the specification.

The sixth step is to detect the insulin content of mice. The insulin content of each mouse was detected 48h after transplantation. For details of the experimental data, please refer to Figure 23d of the specification.

The results showed that the blood sugar of diabetic mice in the light treatment group was significantly lower than that of the control group (dark), and the insulin level in the light treatment group increased significantly. It can be seen that the REDMAP system can well control the blood glucose in mice by controlling the release of insulin. For details of the experimental data, please refer to Figure 23 of the specification.

Example 24 Verification of the effect of the REDMAP system in regulating the Ras-MAPK signaling pathway

This example uses Western Blot to detect the phosphorylation of Erk protein to verify the effect of the REDMAP system in regulating the Ras-MAPK signaling pathway, but does not limit the protection scope of the present invention. Specific steps are as follows:

The second step is to inoculate cells. ^{Inoculate 3×10 5} HEK-293 cells/well in a 6-well plate and divide them into 7 groups.

The third step is transfection. The pYZ339 and pYZ340 were mixed with PEI transfection reagent and serum-free DMEM at a ratio of 1:1 (w/w). After standing at room temperature for 15 minutes, they were evenly dropped into the culture plate. The total volume of each well is 200μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 1000 μL of DMEM medium containing 10% FBS, 1% (volume/volume) of penicillin, streptomycin and 5 μM PCB were replaced for culture.

The fourth step is light. On the next day after transfection, place the cells under red light (1mW/cm ² ) with a wavelength of 660nm for 1 min. After 90 min, place them under 730nm far-red light (1mW/cm ² ) for 1 min. Cultivate immediately in a dark place. Cells were collected at 0 min, 10 min, 45 min, and 90 min after light.

The fifth step, protein extraction, Western Blot to detect the level of egg phosphorylation.

The results show that the REDMAP system can quickly and sensitively regulate the Ras-MAPK signaling pathway. For details of the experimental data, please refer to Figure 24b of the specification.

Example 25 Verification of the effect of the REDMAP system in regulating the artificial Ras-MAPK signal pathway

This example uses SEAP as the reporter gene to verify the effect of the REDMAP system in regulating the artificial Ras-MAPK signal pathway, but does not limit the protection scope of the present invention. Specific steps are as follows:

The second step is to inoculate cells.

The third step is transfection. Mix pYZ339, pYZ340, pTetR-ElK1 and pMF111 with PEI transfection reagent and serum-free DMEM at a ratio of 5:5:1:5 (w/w), and then evenly drop them into the culture plate after standing at room temperature for 15 minutes . The total volume of each well is 200μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, 1000 μL of DMEM medium containing 10% FBS, 1% (volume/volume) of penicillin, streptomycin and 5 μM PCB were replaced for culture.

The fourth step is light. The next day after transfection, the cells were ^{irradiated with red light (1 mW/cm 2} , 1 min on, 5 min off) with a wavelength of 660 nm for 48 hours.

The results show that the REDMAP system can be used to efficiently regulate the artificial Ras-MAPK signaling pathway. For details of the experimental data, please refer to Figure 24d of the manual.

Table 1. Plasmid construction table

The * in the following sequence represents the stop codon

SEQ ID NO. 1: Amino acid sequence of PhyA

SEQ ID NO. 2: Amino acid sequence of FHY1

SEQ ID NO. 3: FHL amino acid sequence

SEQ ID NO. 4: Amino acid sequence of insulin

SEQ ID NO.5: Amino acid sequence of PhyA (1-598)

SEQ ID NO. 6: Amino acid sequence of ΔPhyA(1-617)

SEQ ID NO.7: Amino acid sequence of PhyA-GAL4

SEQ ID NO. 8: Amino acid sequence of PhyA-TetR

SEQ ID NO.9: Amino acid sequence of FHY1-VP64

SEQ ID NO.10: Amino acid sequence of FHL-VP64

SEQ ID NO.11: Amino acid sequence of FHY1-VPR

SEQ ID NO.12: Amino acid sequence of FHL-VPR

SEQ ID NO.13: Amino acid sequence of FHY1-VP16

SEQ ID NO. 15: Amino acid sequence of NLS

SEQ ID NO. 16: Amino acid sequence of NES

SEQ ID NO.17: Amino acid sequence of connecting peptide

SEQ ID NO.18: Amino acid sequence of connecting peptide

SEQ ID NO.19: Nucleotide sequence of _{(UAS) 5}

SEQ ID NO. 20: Nucleotide sequence of _{(TetO) 7}

SEQ ID NO.21: The nucleotide sequence of hCMVmin

SEQ ID NO.22: The nucleotide sequence of TATA

SEQ ID NO.23: Amino acid sequence of PhyA-CAAX

SEQ ID NO.24: Amino acid sequence of FHY1-SOScat

The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that those skilled in the art can think of are all included in the present invention, and the appended claims are the protection scope.

Claims

A red light and far-red light regulated gene expression REDMAP system is characterized in that the switch includes a photoreceptor and an effector, and the photoreceptor includes the Arabidopsis thaliana photosensitive protein PhyA and its interacting protein FHY1/FHL.
The REDMAP system of claim 1, wherein the photoreceptor is constructed by constructing the fusion protein of PhyA and DNA binding protein, the fusion protein of FHY1/FHL and transcription activation domain, and the nuclear localization of the aforementioned two fusion proteins. /Nuclear export signal and the connecting peptide between the aforementioned two fusion proteins are obtained.
The REDMAP system of claim 1, wherein the effector includes an operon, an inducible weak promoter and a target gene.
The REDMAP system of claim 1, wherein the amino acid sequence of the Arabidopsis thaliana light-sensitive protein PhyA is shown in SEQ ID NO. 1, and the amino acid sequence of the protein FHY1 that interacts with it is shown in SEQ ID NO. 2 As shown, the amino acid sequence of the FHL is shown in SEQ ID NO.3.
The REDMAP system of claim 1, wherein the PhyA includes PhyA (1-598aa) and ΔPhyA (1-617aa), the amino acid sequences of which are shown in SEQ ID NO. 5-6, respectively.
The REDMAP system of claim 2, wherein the DNA binding protein is a protein that can bind to a specific DNA sequence, including GAL4 and TetR; the fusion protein of PhyA and DNA binding protein includes PhyA-GAL4, PhyA -TetR, the amino acid sequence of which is shown in SEQ ID NO. 7-8, respectively.
The REDMAP system of claim 2, wherein the transcription activation domain is a transcription factor with the function of recruiting RNA polymerase, including VP64, VPR, VP16; the fusion of the FHY1/FHL and the transcription activation domain Proteins, including FHY1-VP64, FHL-VP64, FHY1-VPR, FHL-VPR, FHY1-VP16, FHL-VP16, and their amino acid sequences are shown in SEQ ID NO. 9-14, respectively.
The REDMAP system of claim 2, wherein the nuclear localization/nuclear export signals of the two fusion proteins are NLS and NES, and their amino acid sequences are shown in SEQ ID NO. 15-16; and/or, The amino acid sequence of the connecting peptide between the two fusion proteins is shown in SEQ ID NO.17.
The REDMAP system according to claim 1, wherein the red light has a wavelength of 660±10 nm, and the far-red light has a wavelength of 720-900 nm.
The REDMAP system of claim 3, wherein the operon can interact with the DNA binding protein, and the operon includes (UAS) 5 , (TetO) 7 and any copy number thereof, and the The nucleotide sequences of (UAS) 5 and (TetO) 7 are shown in SEQ ID NO. 19-20, respectively; the inducible weak promoters include hCMVmin and TATA, and their nucleotide sequences are shown in SEQ ID NO. 21, respectively. -22 shown.
A method for constructing a REDMAP system is characterized in that it comprises the following steps:

(1) Building a photoreceptor

Construct the fusion protein of PhyA and DNA binding protein, the fusion protein of FHY1/FHL and transcription activation domain, the nuclear localization/nuclear export signal of the aforementioned two fusion proteins, and the connecting peptide between the aforementioned two fusion proteins as photoreceptors;

(2) Building effectors

Construct an inducible promoter effector that responds to DNA binding proteins, including an operon, an inducible weak promoter and a target gene, namely PREDMAP- Reporter; wherein the inducible promoter PREDMAP includes an operon and an inducible weak promoter Son; Among them, Reporter is the target gene, which is any meaningful protein.
The method of claim 11, wherein the PhyA is as described in claim 4 or 5; and/or, the DNA binding protein, the fusion protein of PhyA and DNA binding protein is according to claim 6. And/or, the FHY1/FHL is as described in claim 4; and/or, the transcription activation domain, the fusion protein of the FHY1/FHL and the transcription activation domain is as claimed 7; and/or, the nuclear localization/nuclear export signal is as described in claim 8; and/or, the connecting peptide is as described in claim 8; and/or, the operon, The inducible weak promoter is as described in claim 10.
A REDMAP system constructed by the method of claim 11.
A eukaryotic expression vector and/or host cell and/or engineered cell and/or engineered cell transplantation vector, characterized in that the eukaryotic expression vector and/or host cell and/or engineered cell and/or The engineered cell transplantation vector comprises the REDMAP system according to any one of claims 1-10, 13.
The REDMAP system according to any one of claims 1 to 10 and 13, or the eukaryotic expression vector and/or host cell and/or engineered cell and/or engineered cell transplantation vector according to claim 14 Preparation of a kit for regulating or inducing cell gene expression and/or regulating protein-protein interaction kits.
The application according to claim 15, wherein the product that regulates or induces cell gene expression includes insulin, and the amino acid sequence of the insulin is shown in SEQ ID NO.4.
A kit, characterized in that the kit contains the REDMAP system according to any one of claims 1-10,13.
A method for regulating gene expression in a host cell using the REDMAP system according to any one of claims 1-10 or 13, characterized in that the method comprises the following steps:

a) Constructing the REDMAP system according to any one of claims 1-10 or 13 in a host cell eukaryotic plasmid expression vector;

b) Introducing into the host cell via an expression vector;

c) Inducing or regulating the effector-encoded reporter gene in the host cell through red light and far-red light irradiation, so as to realize the expression of the encoding gene of the REDMAP system in the host cell.
The method of using the REDMAP system according to any one of claims 1-10 or 13 to perform transgene regulation and expression in a transplanted vector, characterized in that the method comprises the following steps:

a) preparing a eukaryotic plasmid expression vector containing the REDMAP system according to any one of claims 1-10 or 13;

b) using the eukaryotic plasmid expression vector of step a) to prepare engineered cells containing the REDMAP system of any one of claims 1-10 or 13;

c) using the engineered cells of step b) to prepare an engineered cell transplantation vector containing the REDMAP system of any one of claims 1-10 or 13;

d) Inducing expression of the engineered cell transplantation vector by red light and far-red light, so as to realize the expression of the gene encoded by the REDMAP system in the transplantation vector.
A method for the REDMAP system to regulate the interaction between proteins and proteins. For regulating the cell signaling pathway Ras-MAPK, the method is characterized in that the method includes:

a) Fusion of PhyA protein and membrane anchor signal peptide CAAX sequence into PhyA-CAAX, and then anchored on the cell membrane after expression, the nucleotide sequence of PhyA-CAAX is shown in SEQ ID NO.23;

b) FHY1 protein and Ras protein guanine nucleotide exchange domain SOScat are fused and expressed as FHY1-SOScat, and the nucleotide sequence of FHY1-SOScat is shown in SEQ ID NO.24;

c) Under the irradiation of red light/far-red light, FHY1 protein will bind/dissociate with PhyA protein, and SOScat will be recruited/detached from the cell membrane, thereby activating/inhibiting the Ras-MAPK signaling pathway.
A method of artificially constructed Ras-MAPK signal pathway regulated by the REDMAP system, characterized in that the method comprises:

a) PhyA-CAAX, FHY1-SOScat, and recombinant transcription factor pTetR-ElK1, TetR-response report pMF111 are transfected into the host cell;

b) Under the irradiation of red light/far-red light, activate/inhibit the Ras-MAPK signaling pathway, thereby starting the expression of the reporter gene.
A REDMAP system-mediated method for regulating endogenous gene expression in host cells based on CRISPR-dCas9, characterized in that the method comprises:

a) Construction of the REDMAP system effector of the CRISPR-dCas9 transcriptional regulatory element MS2-p65-HSF1, namely P REDMAP- MS2-p65-HSF1;

b) Co-transform the REDMAP system with the CRISPR-dCas9 protein element and introduce it into the host cell;

c) Inducing and regulating the host cell by irradiation with red light and far-red light, realizing the expression of the transcriptional regulatory element MS2-p65-HSF1 encoded by the effector, and finally realizing the regulation of the endogenous target gene.
A method for treating diabetes with REDMAP system, characterized in that the method comprises:

a) Construct effector of insulin REDMAP system;

b) Preparation of a transplantation vector for regulating insulin expression by the REDMAP system, wherein the preparation method of the transplantation vector includes:

Preparing a eukaryotic plasmid expression vector containing the REDMAP to regulate insulin expression;

Using the eukaryotic plasmid expression vector to prepare engineered cells containing the REDMAP system to regulate insulin expression;

Using the engineered cells to prepare a microcapsule-encapsulated cell transplantation carrier;

c) Control the expression level of insulin through the irradiation of red light/far-red light, thereby lowering blood sugar.