Method for high-throughput detection of gene switch ligand induced expression

Abstract

The invention provides a method for high-throughput detection of gene switch ligand induced expression. Specifically, the invention provides a method for detecting gene switch ligand induced expression, a method for detecting target gene expression and application of the detection methods. The detection method provided by the invention has a small quantity demand on plasmids and viruses, and has the remarkable advantages of high distinction degree, low cost, rapidness, high-throughput test and the like, so that the expression of a target gene and the induced expression of a gene switch can be rapidly tested in a high-throughput manner.

Description

Technical Field

[0001] This invention relates to the field of gene and cell therapy, and more specifically to a method for high-throughput detection of gene switch ligand-induced expression. Background Technology

[0002] Gene switches are inducible gene regulatory systems used to control the expression of transgenes. Precise regulation of transgene expression is crucial for various applications, such as gene therapy, large-scale intracellular protein production, cell-based high-throughput screening experiments, functional genomics, and trait regulation in transgenic plants and animals. Several gene switches based on tetracycline inhibitors, immunosuppressive molecules, and steroid hormone receptors (including progesterone and ecdysone receptors) have been developed (Fussenegger, M. (2001) Biotechnol. Prog. 17, 1-51.).

[0003] Gene switch versions are constantly being updated and iterated. The publicly available paper, "A single point mutation in ecdysone receptor leads to increased ligand specificity: Implications for gene switch applications," only describes one method for detecting gene induction: electroporating the gene plasmid into the biceps brachii muscle of C57B / 6 mice, injecting the ligand three days later, and then assessing the activity of secreted alkaline phosphatase (SEAP) in the mouse serum three days after ligand administration. This method is complex, costly, cannot achieve high-throughput screening, and is affected by differences in electroporation efficiency, resulting in unreliable results.

[0004] Given the urgent need for iterative development of existing gene switches, there is a pressing need to achieve highly specific ligand screening and optimization to address a series of issues related to gene switch leakage. Therefore, there is an urgent need in this field to develop a high-throughput method for detecting ligand-induced expression of gene switches. Summary of the Invention

[0005] The purpose of this invention is to provide a high-throughput method for detecting the induced expression of "gene switch" ligands.

[0006] Another object of the present invention is to provide a method for detecting the expression of a target gene.

[0007] Another object of the present invention is to provide the use of the above-described detection method.

[0008] In a first aspect of the present invention, a method for detecting gene switch ligand-induced expression is provided, the method comprising the steps of:

[0009] (i) Construct a first plasmid containing a gene switch functional element;

[0010] (ii) Using the "suspended plate" method, the first plasmid constructed in step (i) was used to co-transfect the host cells with the second and third plasmids, and a normal transfection control group and a ligand-treated experimental group were set up respectively.

[0011] (iii) After transfection, ligand was added to the ligand-treated experimental group. After a period of culture, the expression levels of the normal transfection control group and the ligand-treated experimental group were measured. The expression level index A0 of the normal transfection control group and the expression level index A1 of the ligand-treated experimental group were measured.

[0012] (iv) Calculate the expression level index X = A1 - A0 to characterize the gene switch ligand induced expression, thereby evaluating the gene switch ligand induced expression level or efficiency.

[0013] In another preferred embodiment, the gene switch functional element includes:

[0014] (a) A first expression cassette expressing a first gene switch polypeptide, the first gene switch polypeptide comprising a transactivation domain fused to a first nuclear receptor ligand binding domain;

[0015] (b) A second expression cassette expressing a second gene-switching polypeptide, the second gene-switching polypeptide comprising a DNA-binding domain fused to a second nuclear receptor ligand-binding domain; and

[0016] (c) A third expression cassette expressing a foreign gene product;

[0017] The first gene switch polypeptide and the second gene switch polypeptide are connected by a linker to form a gene switch; and the promoter in the third expression cassette is a promoter regulated by the gene switch.

[0018] In another preferred embodiment, the linker is a transcription linker sequence TL (Internal Ribosome Entry Site (IRES) linker).

[0019] In another preferred embodiment, the gene switch functional element is based on a gene switch functional element selected from the group of receptors: ecdysone receptor, ubiquitous receptor, orphan receptor 1, NER-1, steroid hormone nuclear receptor 1, retinoid X receptor-interacting protein-15, liver X receptor β, steroid hormone receptor-like protein, liver X receptor α, farnesoid X receptor, receptor-interacting protein 14, farnesoid receptor, or a combination thereof.

[0020] In another preferred embodiment, at least one of the first nuclear receptor ligand binding domain and the second nuclear receptor ligand binding domain is selected from the group consisting of: ecdysone receptor, ubiquitous receptor, orphan receptor 1, NER-1, steroid hormone nuclear receptor 1, retinoid X receptor-interacting protein-15, liver X receptor β, steroid hormone receptor-like protein, liver X receptor α, farnesoid X receptor, receptor-interacting protein 14, farnesoid receptor, or combinations thereof.

[0021] In another preferred embodiment, the gene switch functional element is a gene switch functional element based on the ecdysone receptor (EcR).

[0022] In another preferred embodiment, with the participation of a ligand, the second gene switch polypeptide (such as LTF) forms a stable high-affinity interaction with the first gene switch polypeptide (such as CAP) at the inducible promoter, inducing the receptor protein to recruit transcription factors and drive the expression of the foreign gene.

[0023] In another preferred embodiment, the DNA binding domain comprises at least one of GAL4 (GAL4 DBD), LexA DBD, transcription factor DBD, steroid / thyroid hormone nuclear receptor superfamily member DBD, bacterial LacZ DBD, and yeast DBD.

[0024] In another preferred embodiment, the trans-activation domain comprises at least one of the VP16 trans-activation domain and the B42 acid activator trans-activation domain.

[0025] In another preferred embodiment, the first gene-switching polypeptide comprises a VP16 transactivation domain fused to a retinoid receptor X (RXR) nuclear receptor ligand-binding domain, and the second gene-switching polypeptide comprises a GAL4 DBD fused to an EcR nuclear receptor ligand-binding domain.

[0026] In another preferred embodiment, the ligand binds to the EcR ligand-binding domain.

[0027] In another preferred embodiment, the ligand is a diacylhydrazine.

[0028] In another preferred embodiment, the ligand is selected from RG-115819, RG-115932, RG-115830, or a combination thereof.

[0029] In another preferred embodiment, the ligand is an amide ketone, an oxadiazolline, or a combination thereof.

[0030] In another preferred embodiment, the first plasmid further contains a transcriptional enhancement element WPRE, an inverted terminal repeat sequence ITR, or a combination thereof.

[0031] In another preferred embodiment, the first plasmid comprises one or more promoters operatively linked to the polynucleotide or fragment thereof, enhancers, introns, transcription termination signals, polyadenylation signals, origin of replication, resistance genes, selectivity markers, exogenous genes, nucleic acid restriction sites, and / or homologous recombination sites.

[0032] In another preferred embodiment, the promoter includes a tissue-specific promoter, an inducible promoter, a constitutive promoter, or a combination thereof.

[0033] In another preferred embodiment, the promoter is selected from the group consisting of: Ubc promoter, EFS promoter, MSCV promoter, SV40 promoter, mPGK promoter, hPGK promoter, CMV promoter, EF1A promoter, CAG promoter, CBh promoter, SFFV promoter, FRP promoter, or combinations thereof.

[0034] In another preferred embodiment, the promoter is a Ubc promoter and / or an FRP promoter.

[0035] In another preferred embodiment, the promoter regulated by the gene switch (the promoter that is operatively linked to the exogenous gene in the third expression cassette) is FRP [6×Ga1RE: minimal promoter].

[0036] In another preferred embodiment, the first expression box, the second expression box, and the third expression box are each independently driven by a constitutive or inducible promoter.

[0037] In another preferred embodiment, the resistance gene is an ampicillin (AmpR) resistance gene.

[0038] In another preferred embodiment, the exogenous gene is a reporter gene (i.e., the exogenous gene product is a reporter protein).

[0039] In another preferred embodiment, the reporter gene is selected from the group consisting of: chloramphenicol acetyltransferase encoding gene, β-galactosidase encoding gene, β-glucuronidase encoding gene, Renilla luciferase encoding gene, alkaline phosphatase encoding gene, firefly luciferase encoding gene, green fluorescent protein encoding gene, red fluorescent protein encoding gene, or combinations thereof.

[0040] In another preferred embodiment, the reporter gene is a green fluorescent protein (EGFP) encoding gene.

[0041] In another preferred embodiment, the polyadenylation signal is selected from the group consisting of bovine growth hormone (bGH) polyadenylation signal, simian virus 40 polyadenylation signal (SV40pA), synthetic polyadenylation signal, or a combination thereof.

[0042] In another preferred embodiment, the polyadenylation signal is a bovine growth hormone (bGH) polyadenylation signal.

[0043] In another preferred embodiment, the “suspension transfer dish” method refers to transferring the host cells cultured in suspension to an adherent dish for static culture, followed by co-transfection with three plasmids.

[0044] In another preferred embodiment, the wall-mounted vessel includes a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate.

[0045] In another preferred embodiment, the host cell is a HEK293 cell.

[0046] In another preferred embodiment, the time for adhering static culture is 0.5-4 hours, more preferably 1-3 hours, and even more preferably 1.5-2 hours.

[0047] In another preferred embodiment, the second plasmid is a plasmid containing the AAV rep and AAV cap genes (a plasmid that packages AAV serotypes).

[0048] In another preferred embodiment, the third plasmid is an auxiliary plasmid containing an AAV genome open reading frame (an auxiliary AAV packaging plasmid).

[0049] In another preferred embodiment, the incubation time of the transfection system is 5-60 minutes, more preferably 10-30 minutes, and even more preferably 15-20 minutes.

[0050] In another preferred embodiment, the culture time after transfection is 12-96 hours, more preferably 48-72 hours, and even more preferably 72 hours.

[0051] In another preferred embodiment, the expression level determination includes a fluorescence assay.

[0052] In another preferred embodiment, the fixed exposure parameters for the fluorescence measurement under a 10x microscope are 1-5 seconds, more preferably 2-4 seconds, and even more preferably 2 seconds.

[0053] In another preferred embodiment, the fluorescence index is selected from the group consisting of: fluorescence region value, fluorescence percentage, fluorescence intensity, or a combination thereof.

[0054] In another preferred embodiment, the method further includes: constructing a control plasmid that does not contain gene switch functional elements and whose other elements are the same as those of the first plasmid.

[0055] In a second aspect of the present invention, a method for detecting the expression of a target gene is provided, the method comprising the steps of:

[0056] (i) Construct a first plasmid containing the target gene and a control plasmid without the target gene;

[0057] (ii) Using the "suspended plate" method, the first plasmid and control plasmid constructed in step (i) were co-transfected with the second plasmid and the third plasmid to host cells, respectively, corresponding to the experimental group and the control group;

[0058] (iii) After a period of time following transfection, the expression levels of the experimental group and the control group were measured, and the expression level index B0 of the control group and the expression level index B1 of the experimental group were measured; and

[0059] (iv) Calculate the expression level index Y = B1 - B0, which characterizes the expression of the target gene, and thus evaluate the expression level or efficiency of the target gene.

[0060] In another preferred embodiment, the expression level determination includes fluorescence assay, Western blotting, immunohistochemistry, ELISA, and Lowry assay.

[0061] In another preferred embodiment, the first plasmid and the control plasmid also contain a reporter gene.

[0062] In another preferred embodiment, the first plasmid contains a gene-switching functional element, said gene-switching functional element comprising:

[0063] (a) A first expression cassette expressing a first gene switch polypeptide, the first gene switch polypeptide comprising a transactivation domain fused to a first nuclear receptor ligand binding domain;

[0064] (b) A second expression cassette expressing a second gene switch polypeptide, the second gene switch polypeptide comprising a DNA binding domain fused to a second nuclear receptor ligand binding domain;

[0065] (c) A third expression cassette expressing the target gene; and / or

[0066] (d) Expression of the fourth expression cassette of the reporter gene.

[0067] The first gene switch polypeptide and the second gene switch polypeptide are connected by a linker to form a gene switch; and the promoters in the third expression cassette and the fourth expression cassette are promoters regulated by the gene switch.

[0068] In another preferred embodiment, the third expression box and the fourth expression box may be the same expression box or different expression boxes.

[0069] In another preferred embodiment, the reporter gene may be fused with the target gene for expression.

[0070] In another preferred embodiment, the linker is a transcription linker sequence TL.

[0071] In another preferred embodiment, at least one of the first nuclear receptor ligand binding domain and the second nuclear receptor ligand binding domain is selected from the group consisting of: ecdysone receptor, ubiquitous receptor, orphan receptor 1, NER-1, steroid hormone nuclear receptor 1, retinoid X receptor-interacting protein-15, liver X receptor β, steroid hormone receptor-like protein, liver X receptor α, farnesoid X receptor, receptor-interacting protein 14, farnesoid receptor, or combinations thereof.

[0072] In another preferred embodiment, the gene switch functional element is a gene switch functional element based on the ecdysone receptor (EcR).

[0073] In another preferred embodiment, with the participation of a ligand, the second gene switch polypeptide (such as LTF) forms a stable high-affinity interaction with the first gene switch polypeptide (such as CAP) at the inducible promoter, inducing the receptor protein to recruit transcription factors and drive the expression of the foreign gene.

[0074] In another preferred embodiment, the DNA binding domain comprises at least one of GAL4 (GAL4 DBD), LexA DBD, transcription factor DBD, steroid / thyroid hormone nuclear receptor superfamily member DBD, bacterial LacZ DBD, and yeast DBD.

[0075] In another preferred embodiment, the trans-activation domain comprises at least one of the VP16 trans-activation domain and the B42 acid activator trans-activation domain.

[0076] In another preferred embodiment, the first gene-switching polypeptide comprises a VP16 transactivation domain fused to a retinoid receptor X (RXR) nuclear receptor ligand-binding domain, and the second gene-switching polypeptide comprises a GAL4 DBD fused to an EcR nuclear receptor ligand-binding domain.

[0077] In another preferred embodiment, the ligand binds to the EcR ligand-binding domain.

[0078] In another preferred embodiment, the ligand is a diacylhydrazine.

[0079] In another preferred embodiment, the ligand is selected from RG-115819, RG-115932, RG-115830, or a combination thereof.

[0080] In another preferred embodiment, the ligand is an amide ketone, an oxadiazolline, or a combination thereof.

[0081] In another preferred embodiment, the control plasmid does not contain the target gene, and all other elements are identical to the first plasmid.

[0082] In another preferred embodiment, the reporter gene is selected from the group consisting of: chloramphenicol acetyltransferase encoding gene, β-galactosidase encoding gene, β-glucuronidase encoding gene, Renilla luciferase encoding gene, alkaline phosphatase encoding gene, firefly luciferase encoding gene, green fluorescent protein encoding gene, red fluorescent protein encoding gene, or combinations thereof.

[0083] In another preferred embodiment, the reporter gene is a green fluorescent protein (EGFP) encoding gene.

[0084] In another preferred embodiment, the first plasmid and the control plasmid further contain a transcriptional enhancement element WPRE, an inverted terminal repeat sequence ITR, or a combination thereof.

[0085] In another preferred embodiment, the first plasmid and the control plasmid contain one or more promoters operatively linked to the polynucleotide or fragment thereof, enhancers, introns, transcription termination signals, polyadenylation signals, origins of replication, selectivity markers, nucleic acid restriction sites, and / or homologous recombination sites.

[0086] In a third aspect of the invention, the method as described in the first aspect of the invention is provided for:

[0087] (a) Detecting the level or efficiency of gene switch ligand-induced expression; and / or

[0088] (b) Screening for gene switch ligands suitable for expression.

[0089] In a fourth aspect of the invention, the method as described in the first aspect or the second aspect of the invention is provided for use in:

[0090] (a) Detecting the expression efficiency of the target gene;

[0091] (b) Screening for target gene fragments suitable for expression;

[0092] (b) Detect the conversion or driving expression efficiency of the promoter element;

[0093] (c) Screening for suitable promoter elements for expression;

[0094] (d) Detect the transfection or expression efficiency of the vector; and / or

[0095] (e) Screening for suitable vectors for expression.

[0096] In another preferred embodiment, the vector includes a viral vector, preferably an AAV vector.

[0097] In another preferred embodiment, the AAV carrier includes, but is not limited to: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV-DJ.

[0098] In another preferred embodiment, the use further includes detecting the transfection or expression efficiency of viral capsids, and / or screening for viral capsids suitable for expression.

[0099] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0100] Figure 1 , 2 The plasmid maps of plasmid VG-RST-GFP and control plasmid VG-142 are shown respectively.

[0101] Figure 3 The images shown are fluorescence images obtained with exposure parameters of 1, 2, 3, 4, and 5 s using a fluorescence microscope.

[0102] Figure 4 The fluorescence analysis results of the single-plasmid transfection group and the three-plasmid transfection group based on the control plasmid VG-142 are shown.

[0103] Figure 5 The fluorescence analysis results of single-plasmid transfection and triple-plasmid transfection groups based on plasmid VG-RST-GFP are shown.

[0104] Figure 6 The fluorescence analysis results of well 1 (control group without ligand induction) and well 2 (experimental group with ligand induction) in the RC2-RST-GFP triple plasmid transfection group are shown. Among them, A is the fluorescence image of well 1 in the RC2-RST-GFP group, B is the fluorescence image of well 2 in the RC2-RST-GFP group; C is a single-channel image of well 1 in the RC2-RST-GFP group, D is a single-channel image of well 2 in the RC2-RST-GFP group; E is the image of well 1 in the RC2-RST-GFP group after threshold adjustment, and F is the image of well 2 in the RC2-RST-GFP group after threshold adjustment.

[0105] Figure 7 The values ​​and percentages of the fluorescent regions in well 1 (the control group without ligand induction) and well 2 (the experimental group with ligand induction) of the RC2-RST-GFP triple plasmid transfection group are shown. Detailed Implementation

[0106] Through extensive and in-depth research, the inventors have developed a high-throughput method for detecting "gene switch" ligand-induced expression at the cellular level. By constructing the functional element of the switch on a core plasmid capable of packaging AAV with three plasmids, and performing three-plasmid transfection, the entire transfected cell system exhibits both fluorescent (or other marker gene) expression of plasmid-transfected cells and fluorescent (or other marker gene) expression of AAV-infected HEK293 cells after viral packaging. By setting up a control group without ligand induction and an experimental group with ligand induction, the expression intensity of the experimental group is subtracted from the background expression present in plasmid transfection (i.e., the expression intensity of the control group), allowing for the calculation of the fluorescent (or other marker gene) expression intensity of AAV-infected HEK293 cells after viral packaging. This allows for the evaluation of the "gene switch" ligand-induced expression effect.

[0107] This method is a plate-based cell-level experiment that requires minimal amounts of plasmids and viruses. It innovatively utilizes a "suspension plate" cell culture and transfection method, making it simple to operate and enabling rapid, high-throughput testing of the induced expression of "gene switches." This invention was developed based on these principles.

[0108] the term

[0109] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0110] As used herein, the terms "first plasmid" and "core plasmid" are used interchangeably and refer to plasmids containing the target gene (e.g., gene switch functional elements).

[0111] As used in this article, the "second plasmid" and "third plasmid" can be co-transfected with the "first plasmid" and simultaneously introduced into suitable host cells, where they are expressed and packaged into AAV viral particles.

[0112] Gene switch system

[0113] The gene switch system, referring to patents CN101868241A and CN110462040A, comprises a gene switch polypeptide including: a first gene switch polypeptide containing a transactivation domain fused to a first nuclear receptor ligand-binding domain (this domain is expressed as a coactivator protein CAP, expressed as a fusion protein by the transactivation domain AD and the heterodimeric chaperone-binding domain HP); and a second gene switch polypeptide containing a DNA-binding domain fused to a second nuclear receptor ligand-binding domain (this domain is expressed as a ligand-dependent transcription factor LTF, expressed as a fusion protein by the DNA-binding domain DBD and the ligand-binding domain LBD), wherein the first and second gene switch polypeptides are linked by a linker. With the participation of the ligand, LTF forms a stable high-affinity interaction with CAP at the inducible promoter, leading to dimerization of the DNA-binding domain and the transactivation domain. CAP and LTF combine to form a ligand-dependent transcription factor complex (LDTFC), which, upon ligand combination, activates the promoter (FRP). This new conformation can induce receptor proteins to recruit transcription factors, driving the expression of target genes.

[0114] It should be understood that the method of the present invention is not limited to the types of gene switch systems. When different gene switch systems are used, the efficiency, expression level, and expression intensity of the corresponding gene switch ligand-induced expression can be detected using the method of the present invention, requiring only a change in the gene switch functional element in the constructed first plasmid.

[0115] When the expression of the exogenous gene product in the third expression cassette of the first plasmid is absent, compared to the expression of the exogenous gene product in the presence of the ligand, the expression of the exogenous gene product is reduced or eliminated. In some cases, the expression of the exogenous gene product can be restored by providing an additional amount of ligand.

[0116] Ecdysone and its receptor

[0117] Ecdysone (20-hydroxyecdysone, 20E) is a typical steroid hormone that dominates the regulation of important physiological processes in insects, such as molting, metamorphosis, and reproduction. The 20E receptor EcR-USP has been identified for nearly 20 years, and there have been many important breakthroughs in the transcriptional regulation mechanism of the 20E and its receptor complex. Existing studies have shown that: (1) the 20E receptor is formed by the nuclear receptor EcR and USP; (2) the EcR-USP heterodimer acquires DNA-binding activity with the assistance of a molecular chaperone protein complex; (3) 20E activates the EcR-USP heterodimer and initiates the transcription of downstream genes by releasing co-repressor factors and recruiting co-activators; (4) the 20E-EcR-USP ligand-receptor complex induces the expression of 20E primary response genes, and the 20E secondary response genes induced by the transcription factors encoded by the 20E primary response genes amplify the 20E signal, thereby regulating the physiological processes of insects such as molting, metamorphosis, and reproduction.

[0118] The ecdysone receptor (EcR) is a member of the nuclear receptor superfamily and is classified as subfamily 1,H. Other members of this nuclear receptor subfamily 1,H include: ubiquitous receptor (UR), orphan receptor 1 (OR-1), steroid hormone nuclear receptor 1 (NER-1), RXR interacting protein-15 (RIP-15), liver X receptor β (LXRβ), steroid hormone receptor-like protein (RLD-1), liver X receptor (LXR), liver X receptor α (LXRα), farnesoid X receptor (FXR), receptor interacting protein 14 (RIP-14), and farnesoid receptor (HRR-1).

[0119] Cell culture and transfection method using "suspension dishes"

[0120] This invention innovatively employs a "suspension transfer dish" cell culture and transfection method. Specifically, HEK293 cells cultured in a shaker using a component-defined serum-free suspension medium are sampled and counted to calculate the required cell suspension volume. The cells are diluted with DMEM high-glucose medium containing 2% bovine serum and transferred to adherent dishes (such as 12-well plates), which are then placed in a cell culture incubator for static culture. After 1.5-2 hours of static adherent culture, AAV-packaged tri-plasmid transfection is performed.

[0121] Compared to single-plasmid transfection methods, which involve transfecting a single plasmid of the target plasmid into adherent or suspension 293 cells, single-plasmid transfection results in low expression efficiency, making it impossible to evaluate expression based on marker genes (fluorescence or other enzymes). Furthermore, for traditional single-plasmid transfection methods, the culture and passage of adherent cells are less efficient than the "suspension transfection dish" method used in this invention. Traditional suspension transfection methods require large quantities of cells and plasmids, and their efficiency needs optimization, making high-throughput testing impossible.

[0122] The "suspended plate" transfection method of this invention: ① uses three plasmids for transfection, which has a higher expression efficiency than single plasmid transfection, thus enabling expression evaluation based on marker genes (fluorescence or other enzymes); ② uses a "suspended plate" cell culture and transfection method, overcoming the problem of low passage efficiency of adherent cells in adherent transfection methods; ③ uses a "suspended plate" cell culture and transfection method, overcoming the problems of low efficiency of suspension transfection and high demand for cells and plasmids in suspension transfection methods. The method of this invention can achieve high-throughput testing.

[0123] Compared to traditional viral infection methods, which involve large-scale plasmid extraction, transfection and packaging of AAV with a relatively large number of three plasmids, virus purification, and detection of viral titer via qPCR, as well as SDS-PAGE electrophoresis and silver staining to detect viral purity and VP1 / 2 / 3 expression, the present method involves infecting 293 cells with the virus at an appropriate MOI, adding ligands, and then photographing the fluorescence image under fixed exposure parameters after 72 hours. The light intensity of the fluorescence image at the same magnification is analyzed using grayscale software to evaluate whether the switch induces gene expression and the difference in light intensity that induces expression, thereby evaluating the efficiency of gene switch induction. The preparation cycle from plasmid to adeno-associated virus is too long, the demand for virus detection is high, and the cost is also higher.

[0124] The "suspension plate" transfection method of the present invention has the following advantages: ① It does not require large-scale plasmid extraction, but only micro- or small-scale extraction, thus saving the preparation cycle in the detection cycle; ② It does not require virus purification, thus saving the preparation cycle in the detection cycle; ③ It does not require detection of virus titer by qPCR, nor does it require detection of virus purity and VP1 / 2 / 3 expression by silver staining of proteins after SDS-PAGE electrophoresis, thus reducing detection requirements and detection costs.

[0125] The experiments of this invention tested the expression and infection expression effects of virus prepared by transfection with a single plasmid and packaging. From the perspectives of expression discrimination, ease of operation, cost, and throughput, this invention has significant advantages such as high discrimination, low cost, rapid and high-throughput testing.

[0126] Detection methods

[0127] The present invention provides a method for detecting gene switch ligand-induced expression as described in the first aspect of the present invention.

[0128] The present invention also provides a method for detecting the expression of a target gene as described in the second aspect of the present invention.

[0129] The cell culture and transfection method using the "suspension plate" of this invention improves the efficiency of culture passage and transfection, and relatively reduces the detection requirements and costs. Therefore, the above detection method can rapidly and with high throughput test the induced expression of "gene switches" or the expression of target genes.

[0130] Main advantages of the invention

[0131] 1. The method of the present invention can rapidly and with high throughput test the induced expression of "gene switches", realize more efficient and rapid cell-level gene switch testing and evaluation, accelerate the iteration speed of gene switches, enable gene switches to be applied as early as possible, and benefit the public.

[0132] 2. The method of the present invention can detect the expression of "gene switch" ligands at the cellular level based on cell culture plates with high throughput, requiring less plasmid or virus and with high efficiency.

[0133] 3. The method of the present invention can identify the induced expression effect of the switch by the intensity of fluorescence expression in the well plate under fixed parameters, and the operation is simple.

[0134] 4. The method of the present invention can utilize commercially available free and open-source image analysis software (such as ImageJ), requires no device dependence, and is easy to operate.

[0135] 5. Compared with single plasmid transfection methods or traditional viral infection methods, the method of this invention innovatively uses a "suspension plate" cell culture and transfection method, which improves the efficiency of culture passage and transfection, and relatively reduces the detection requirements and costs, thereby enabling high-throughput and simpler testing.

[0136] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0137] Example 1: Construction of plasmids containing gene switch functional elements

[0138] like Figure 1 , 2 As shown, the inventors constructed the plasmid VG-RST-GFP(ITR.Ubc). CAP.TL.LTF.FRP The plasmids VG-RST-GFP and VG-X142 (ITR.Ubc.EGFP.WPRE.BGH.ITR) were constructed on the pUC57 plasmid backbone vector via recombinant cloning, and the plasmids VG-RST-GFP and VG-X142 were extracted.

[0139] The functional elements contained in plasmid VG-RST-GFP are: Ubc promoter, gene switch element-co-activating protein CAP sequence, transcription linker sequence TL, gene switch element-ligand-dependent transcription factor LTF sequence, inducible promoter FRP, green fluorescent protein EGFP sequence, post-transcriptional regulatory sequence WPRE, and bovine growth hormone polyadenylation signal (BGHpA) between two terminal inverted repeat sequences ITR.

[0140] Example 2: Determination of Exposure Parameters for Fluorescence Microscopy Equipment

[0141] Using the control plasmid VG-X142 (ITR.Ubc.EGFP.WPRE.BGH.ITR), HEK293 cells were suspended in a serum-free container using a "suspension transfer" method. The cells were then sampled and counted, diluted with 2% FBS high-glucose DMEM, and transferred to 12-well plates. After static adhesion culture for 1.5-2 hours, three plasmids were transfected and packaged. 72 hours later, fluorescence microscopy exposure parameters were determined based on fluorescence photography at exposure times of 1, 2, 3, 4, and 5 seconds.

[0142] like Figure 3 As shown, the fluorescence imaging effect is clearest when the exposure time is 2 seconds; therefore, the exposure time under 10x magnification is fixed at 2 seconds. Referring to the method for confirming exposure parameters in this embodiment, those skilled in the art can adaptively adjust the optimal exposure parameters for different fluorescence microscopes and objectives of different magnifications.

[0143] Example 3: Comparison of three-plasmid transfection and single-plasmid transfection using the "suspended plate" method (based on control plasmid) VG-142)

[0144] Using the control plasmid VG-142 (ITR.Ubc.EGFP.WPRE.BGH.ITR), HEK293 cells were suspended in a serum-free container using a "suspension transfer" method. The cells were then sampled and counted, diluted with 2% FBS high-glucose DMEM, and transferred to 12-well plates. After static adhesion and incubation for 1.5-2 hours, plasmid transfection was performed. Single and triple plasmid transfections with the same plasmid dosage were tested. Fluorescence photography with a 2-second exposure was conducted 24, 48, and 72 hours after transfection.

[0145] like Figure 4 As shown, group VG-142 was a single-plasmid transfection group, and group RC2-142 was a three-plasmid transfection group. Numbers 1 and 2 represent replicates for each group. The results showed a significant difference in fluorescence expression between single-plasmid and three-plasmid transfections with the same core plasmid amount. The expression level of single-plasmid transfection was lower with the same amount of plasmid, indicating that the efficiency of three-plasmid transfection using the "suspended plate" method is much higher than that of single-plasmid transfection.

[0146] Example 4: Comparison of three-plasmid transfection and single-plasmid transfection using the "suspended transfection dish" method (based on plasmid VG-) RST-GFP)

[0147] Based on the ecdysone receptor switching system, the inventors constructed the plasmid VG-RST-GFP(ITR.Ubc) described in the patent principle. CAP.TL.LTF.FRP HEK293 cells (EGFP-WPRE-BGH-ITR) were suspended in a serum-free container using a "suspension transfer" method. Cells were then sampled and counted, diluted with 2% FBS high-glucose DMEM, and transferred to 12-well plates. After static adhesion and incubation for 1.5-2 hours, plasmid transfection was performed, testing both single and triple plasmid transfections with the same plasmid dosage. For both single and triple plasmid transfections, ligand was added to well 2 after 24 hours. Fluorescence imaging was performed at 24, 48, and 72 hours post-transfection, using a 2-second exposure.

[0148] like Figure 5 As shown, the VG-RST-GFP group was a single-plasmid transfection group, and the RC2-RST-GFP group was a triple-plasmid transfection group. Well 1 was the normal transfection control for each group, and well 2 underwent ligand addition after 24 hours. Additionally, due to initial ligand overdosing, significant crystal precipitation of the compound occurred, but the amount of crystal precipitation did not increase or decrease over time. The results indicate that there was a significant difference in fluorescence expression between single-plasmid and triple-plasmid transfections with the same core plasmid amount. Single-plasmid transfection with the same amount of plasmid resulted in lower expression levels, indicating that triple-plasmid transfection using the "suspended plate" method is far more efficient than single-plasmid transfection. Furthermore, compared to well 1 of the RC2-RST-GFP group without drug addition, well 2 with drug addition showed significantly enhanced fluorescence expression, achieving a strong differentiation between plasmid expression and viral leakage expression in the background. Therefore, it is possible to accurately measure and evaluate the ligand-induced expression level of the "gene switch" gene.

[0149] Example 5: Detection of the expression efficiency induced by the "gene switch" ligand

[0150] As mentioned above, the detection method is as follows: HEK293 cells cultured in a shaker using a serum-free suspension medium with defined components are removed for cell sampling and counting. The required amount of cell suspension is calculated according to Table 1.

[0151] Table 1

[0152]

[0153] Cells were diluted in DMEM high-glucose medium containing 2% bovine serum and transferred to adherent culture dishes. The dishes were then placed in a cell culture incubator for static culture. After 1.5-2 hours of static adhesion, AAV-packaged triple plasmid transfection was performed. This transfection method was a "suspended plate" method. Ligands were added after transfection, and fluorescence images were taken 72 hours later under fixed exposure parameters. ImageJ software was used to analyze the grayscale of the control group (without ligand induction) and the experimental group (with ligand induction) at the same magnification. This evaluated whether gene switching induced expression and the difference in light intensity that induced expression, thus assessing the efficiency of gene switching induction.

[0154] In this embodiment and Figure 6 In the study, the RC2-RST-GFP group was a three-plasmid transfection group. Well 1 was the normal transfection control for each group (the control group without ligand induction), and well 2 was treated with ligand after 24 hours (the experimental group with ligand induction). Fluorescence images were selected after 96 hours of culture (i.e. 72 hours after drug induction), and the results were analyzed using the image analysis software ImageJ.

[0155] The ImageJ analysis process is as follows:

[0156] (1) Open the fluorescence image in ImageJ: for example Figure 6 As shown in A and B, A is the fluorescence image of well 1 of the RC2-RST-GFP group after 72 hours without drug addition, and B is the fluorescence image of well 2 of the RC2-RST-GFP group after 72 hours with drug addition.

[0157] (2) Extracting individual channels (Image-Color-Split Channels): such as Figure 6 As shown in C and D, C is a single-channel image of well 1 of the RC2-RST-GFP group 72 hours after no drug was added, and D is a single-channel image of well 2 of the RC2-RST-GFP group 72 hours after drug was added.

[0158] (3) Adjust the threshold and select an appropriate area (Image-Adjust-Threshold): The software will automatically select a default value. Using the default value consistently can eliminate errors caused by manually selecting the threshold in different photos. For example... Figure 6 As shown in E and F, E is the image of well 1 in the RC2-RST-GFP group after adjusting the threshold 72 hours without drug administration, and F is the image of well 2 in the RC2-RST-GFP group after adjusting the threshold 72 hours with drug administration.

[0159] (4) Set the parameters to be measured (Analyze-Set Measurements).

[0160] (5) Analyze-Measure: Results are as follows Figure 7 As shown, where Figure 7 A shows that the fluorescence region value of well 1 in the RC2-RST-GFP group (the control group without ligand induction) is 19313, with a fluorescence percentage of 1.946%; among which Figure 7 B shows that the fluorescence region value of well 2 in the RC2-RST-GFP group (the experimental group induced by ligand addition) is 110370, and the fluorescence percentage is 11.066%.

[0161] The background fluorescence of plasmid expression and viral infection leakage in well 1 of the RC2-RST-GFP group (control group without ligand induction) and well 2 of the RC2-RST-GFP group (experimental group with ligand induction) is shown. Therefore, the fluorescence region value of ligand-induced expression due to viral infection is: Fluorescence region value of well 2 of the RC2-RST-GFP group (experimental group with ligand induction) - Fluorescence region value of well 1 of the RC2-RST-GFP group (control group without ligand induction) = 110370 - 19313 = 91057; the fluorescence percentage is: Fluorescence percentage of well 2 of the RC2-RST-GFP group (experimental group with ligand induction) - Fluorescence percentage of well 1 of the RC2-RST-GFP group (control group without ligand induction) = 11.066% - 1.946% = 9.12%.

[0162] This enables rapid, simple, and high-throughput detection of the expression efficiency induced by "gene switch" ligands.

[0163] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method of detecting gene switch ligand induced expression, characterized by, The method comprises steps of: (i) constructing a first plasmid containing a gene switch functional element; (ii) performing three-plasmid co-transfection on host cells using the first plasmid constructed in step (i) and a second plasmid and a third plasmid by a "suspension dish" method, and setting a normal transfection control group and a ligand addition experiment group, respectively; (iii) adding a ligand to the ligand addition experiment group after the transfection is completed, and performing expression determination on the normal transfection control group and the ligand addition experiment group after a period of culture, to determine an expression index A0 of the normal transfection control group and an expression index A1 of the ligand addition experiment group; and (iv) calculating an expression index X = A1-A0 representing ligand-induced expression of the gene switch, to evaluate the expression level or efficiency of the ligand-induced expression of the gene switch.

2. The method of claim 1, wherein, The gene switch functional element comprises: (a) a first expression cassette expressing a first gene switch polypeptide comprising a transactivation domain fused to a first nuclear receptor ligand binding domain; (b) a second expression cassette expressing a second gene switch polypeptide comprising a DNA binding domain fused to a second nuclear receptor ligand binding domain; and (c) a third expression cassette expressing an exogenous gene product; wherein the first gene switch polypeptide and the second gene switch polypeptide are connected by a linker to form a gene switch, and the promoter in the third expression cassette is a promoter regulated by the gene switch.

3. The method of claim 1, wherein, The first plasmid further contains a transcription enhancer element WPRE, a long terminal repeat LTR, or a combination thereof.

4. The method of claim 1, wherein, The "suspension dish" method refers to transferring host cells in suspension culture to a substrate dish for substrate culture, and then performing three-plasmid co-transfection.

5. The method of claim 1, wherein, The second plasmid is a plasmid containing AAV rep and AAV cap genes.

6. The method of claim 1, wherein, The third plasmid is an auxiliary plasmid containing an AAV genome open reading frame.

7. The method of claim 2, wherein, At least one of the first nuclear receptor ligand binding domain and the second nuclear receptor ligand binding domain is selected from the group consisting of an ecdysone receptor, a ubiquitous receptor, an orphan receptor 1, NER-1, a steroid hormone nuclear receptor 1, a retinoid X receptor interacting protein-15, a liver X receptor beta, a steroid hormone receptor-like protein, a liver X receptor alpha, a farnesoid X receptor, a receptor interacting protein 14, a farnesol receptor, or a combination thereof.

8. A method for detecting the expression of a target gene, characterized in that, The method comprises steps of: (i) constructing a first plasmid containing a target gene and a control plasmid not containing the target gene; (ii) performing three-plasmid co-transfection on host cells using the first plasmid and the control plasmid constructed in step (i) and a second plasmid and a third plasmid by a "suspension dish" method, to correspond to an experiment group and a control group, respectively; (iii) performing expression determination on the experiment group and the control group after a period of time after the transfection is completed, to determine an expression index B0 of the control group and an expression index B1 of the experiment group; and (iv) calculating an expression index Y = B1-B0 representing expression of the target gene, to evaluate the expression level or efficiency of the target gene.

9. Use of the method according to claims 1-7, characterized in that, for: (a) detecting the expression level or efficiency of a gene switch ligand; and / or (b) screening a gene switch ligand suitable for expression.

10. The method according to claims 1 to 7 or the use of the method according to claim 8, characterized in that for: (a) detecting the efficiency of expression of a gene of interest; (b) screening for a fragment of a gene of interest suitable for expression; (b) detecting the transformation or driving expression efficiency of a promoter element; (c) screening for a promoter element suitable for expression; (d) detecting the transfection or expression efficiency of a vector; and / or (e) screening for a vector suitable for expression.

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