KR102815530B1 - Establishment of zebrafish amyotrophic lateral sclerosis model using human-derived mutant FUS gene and drug efficacy analysis using the same - Google Patents

Abstract

The present invention relates to a transgenic zebrafish amyotrophic lateral sclerosis animal model that induces overexpression of the hFUS (R521C) gene, a method for producing the same, and a method for screening amyotrophic lateral sclerosis drugs using the same. The animal model producing method of the present invention can stably and mass-produce a uniform animal model using a binary expression system, and the animal model produced by the method of the present invention can be usefully used in studies on amyotrophic lateral sclerosis, including drug screening, since the disease is rapidly induced.

Description

{Establishment of zebrafish amyotrophic lateral sclerosis model using human-derived mutant FUS gene and drug efficacy analysis using the same}

The present invention relates to a transgenic zebrafish amyotrophic lateral sclerosis animal model that induces overexpression of the hFUS (R521C) gene, a method for producing the same, and a method for screening amyotrophic lateral sclerosis drugs using the same.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, also known as Lou Gehrig's disease. It is a disease in which the motor neurons of the brain and spinal cord die, resulting in loss of motor ability. It is a fatal disease that causes respiratory failure within 3 to 5 years after onset. Since the first causative gene was discovered in 1993, many studies have been conducted, but various causes have been discovered to date. However, the common mechanism of ALS has not been discovered, and as a result, there is no effective treatment. Currently, FDA-approved ALS treatment drugs only extend survival by a few months or slow progression.

Zebrafish is a vertebrate with high genetic and functional similarity to the human genome, and its nervous system and various organ formation processes are very similar to those of humans, making it an important animal model for human disease research, with much research being conducted worldwide. In addition, since the purchase and maintenance costs per individual are very low and a large number of embryos can be secured, it is also an excellent model for screening disease treatment candidates. In addition, most organs, including the nervous system and circulatory system, are formed within 24 hours after fertilization, and it can be used for large-scale screening experiments using 48-well plates or 98-well plates within a few days. Therefore, zebrafish is attracting attention as a disease model for research on nervous system diseases using zebrafish.

Amyotrophic lateral sclerosis has also been studied using zebrafish as a disease model, but the zebrafish disease models developed to date have the disadvantage of not being able to fully utilize the advantages of zebrafish, which are advantageous for large-scale screening, due to the characteristics of degenerative diseases that occur in old age. Therefore, a disease model that can develop the disease within a few days after fertilization is required.

In patients with mutations in the FUS gene, known as the causative gene for familial amyotrophic lateral sclerosis, the disease onset occurs before the age of 45 in more than 60% of cases, and cases in the late teens and early 20s have been reported. In contrast, cases of other causative genes mostly occur after the age of 50. A mutation known to cause amyotrophic lateral sclerosis is FUS (R521C), in which the 521st amino acid is substituted from arginine to cysteine. If this FUS mutation-induced amyotrophic lateral sclerosis is introduced into zebrafish to create a disease model in which the disease onsets at an early age, it will be advantageous for high-throughput drug screening.

CN 2021-11183570

The technical problem to be achieved by the present invention is to provide a transgenic zebrafish as an animal model of amyotrophic lateral sclerosis and a method for producing the same.

In addition, the present invention aims to provide a method for screening for a treatment agent for amyotrophic lateral sclerosis using the animal model.

However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

The present inventors used the IQ system in zebrafish to produce a transgenic zebrafish that overexpresses a mutant FUS protein in which the 521st amino acid of the human FUS protein is substituted from arginine (R) to cysteine (C), and confirmed that the onset of amyotrophic lateral sclerosis was induced by confirming the symptoms of amyotrophic lateral sclerosis and biomarkers of the disease in the transgenic zebrafish.

To solve the above problem, the present invention uses the IQ-system to produce a zebrafish transformed to overexpress a mutant FUS protein in which the 521st amino acid of the human-derived FUS protein is substituted from arginine (R) to cysteine (C).

The IQ system is a binary expression system, and the present invention separately produces an effector line parent (or sub-generation) zebrafish for overexpressing the mutant FUS protein and a driver line parent (or sub-generation) that controls the operation of the effector line, and then crossbreeds the parent generations to obtain offspring, thereby producing a transgenic zebrafish in which the onset of amyotrophic lateral sclerosis is induced.

Specifically, the present invention provides a method for producing an amyotrophic lateral sclerosis zebrafish animal model comprising the following steps (1) to (5):

(1) A first transgenic zebrafish production step comprising the following steps (A) and (B):

(A) A first plasmid vector production step including a cassette for expression of the QFDBD 2*AD gene operating under the HuC promoter as a driver line, and

(B) A step of transforming the first plasmid vector manufactured in step (A) into a zebrafish fertilized egg.

(2) A second transgenic zebrafish production step comprising the following steps (a) and (b):

(a) a second plasmid vector production step comprising a human-derived FUS mutant (hFUS(R521C)) gene operating under the 13QUAS promoter and a cassette for expression of an effector reporter gene as an effector line; and

(b) a step of transforming the second plasmid vector manufactured in step (a) into a zebrafish fertilized egg;

(3) A step of culturing the transformed zebrafish fertilized eggs in steps (1) and (2) and developing them into adults (F0);

(4) a step of mating the first and second transgenic zebrafish adults with each other; and

(5) A step of selecting zebrafish (F1) expressing an effector reporter gene from among the zebrafish obtained after mating in step (4) above.

As one embodiment of the present invention, in the above manufacturing method, the first transgenic zebrafish manufacturing step and the second transgenic zebrafish manufacturing step may be performed simultaneously or sequentially, but if performed sequentially, the order is irrelevant.

As another embodiment of the present invention, in the first and second transgenic zebrafish production steps, the transformation steps of each of steps (B) and (b) may be performed by microinjecting each of the plasmid vectors produced in steps (A) and (a) together with transposase into a 1-cell stage zebrafish fertilized egg.

As another embodiment of the present invention, the first plasmid vector of step (A) may include a separate first reporter gene expression cassette, wherein step (1) may further include a step (C) of selecting an individual expressing the first reporter gene, wherein the selected individual is the first transgenic zebrafish.

As another embodiment of the present invention, the second plasmid vector of step (a) may include a separate second reporter gene expression cassette, wherein step (2) may further include a step (c) of selecting an organism expressing the second reporter gene, wherein the selected organism is the second transgenic zebrafish.

As another embodiment of the present invention, in the step (b), the hFUS (R521C) gene and the driver reporter gene can be linked to a gene encoding a self-cleaving peptide, and the two genes are organically induced to express under the 13QUAS promoter, and can be expressed as a separate protein by the self-cleaving peptide after expression. The self-cleaving peptide can use a known self-cleaving peptide, and in the specific experiment of the present invention, P2A was used.

In another embodiment of the present invention, the effector reporter gene is for confirming the expression level of hFUS (R521C) or selecting an individual expressing hFUS (R521C), the first reporter gene is for selecting a first transgenic zebrafish, and the second reporter gene is for selecting a second transgenic zebrafish. Any gene that can be monitored without damage to cells or tissues may be used without limitation, but a fluorescent protein gene is preferably used for visual observation. Non-limiting examples of the fluorescent protein include luciferase, green fluorescent protein (GFP), modified green fluorescent protein (mGFP), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP), enhanced red fluorescent protein (ERFP), cyan fluorescent protein (BFP), enhanced cyan fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), or enhanced cyan fluorescent protein (ECFP).

In another embodiment of the present invention, the effector reporter gene, the first reporter gene, and the second reporter gene may each use different fluorescent protein genes, and preferably, they may each encode fluorescent proteins emitting different wavelengths. In specific experiments of the present invention, Zsgreen was used as the effector reporter gene, the EGFP gene was used as the first reporter gene, and the mCherry gene was used as the second reporter gene.

The first transgenic zebrafish and the second transgenic zebrafish of the above manufacturing method each contain DNA constructs of the driver line and the effector line, and the offspring obtained through mating of the two zebrafish contain both DNA constructs of the driver line and the effector line, and QFDBD 2*AD expressed in the driver line drives the effector line to induce overexpression of hFUS (R521C). The hFUS (R521C) induced to be overexpressed accumulates in the zebrafish, and significant symptoms of amyotrophic lateral sclerosis and changes in biomarkers related to the disease can be observed from 5 days after fertilization.

Accordingly, the present invention provides a transgenic zebrafish produced by the above production method as an animal model of amyotrophic lateral sclerosis disease.

As one embodiment of the present invention, the zebrafish animal model includes both a QFDBD 2*AD gene expression cassette and a hFUS (R521C) gene expression cassette driven under a 13QUAS promoter, and the hFUS (R521C) gene expression cassette may further include an effector reporter gene that operates under the 13QUAS promoter, and the reporter gene may be linked to the hFUS (R521C) gene via a self-cleaving peptide upstream or downstream of the hFUS (R521C) gene.

In the present invention, the cassette for expression of the QFDBD 2*AD gene includes a HuC promoter so that the expression of the QFDBD 2*AD gene can be controlled by the HuC promoter so that it can be specifically expressed in the nervous system.

In addition, the present invention provides a method for screening for a therapeutic agent for amyotrophic lateral sclerosis using an animal model of amyotrophic lateral sclerosis disease.

The screening method of the present invention comprises the following steps:

(a) a step of treating a candidate substance to the amyotrophic lateral sclerosis zebrafish animal model;

(I) a step of measuring one or more of the symptoms of amyotrophic lateral sclerosis disease and/or biomarkers of the disease, consisting of the following (1) to (4), in an amyotrophic lateral sclerosis zebrafish animal model treated with the candidate substance and not treated with the candidate substance: and

(d) A step of selecting the candidate substance as a therapeutic substance for amyotrophic lateral sclerosis when the symptoms of amyotrophic lateral sclerosis are improved in an animal treated with the candidate substance compared to an animal not treated with the candidate substance or the biomarker of the disease is changed to a level identical to or similar to that of zebrafish in which the disease is not induced.

As one embodiment of the present invention, in the step (b), the symptoms of amyotrophic lateral sclerosis disease may be decreased mobility and/or denervation at the neuromuscular junction, and the biomarkers of the disease may be increased oxidative stress, increased TNF-α expression, and/or decreased gria1a expression.

The zebrafish animal model of amyotrophic lateral sclerosis of the present invention can contribute to improving the efficiency and accelerating the speed of research on the mechanism of amyotrophic lateral sclerosis, including the symptoms and progression of the disease, since the disease develops from 5 days after fertilization. In addition, the animal model of amyotrophic lateral sclerosis of the present invention rapidly induces the disease in zebrafish, so that large-scale screening of vertebrate-based amyotrophic lateral sclerosis therapeutic agents is possible within a few days, and further, by producing the animal model using a binary expression system, stable maintenance of parent generations in which the disease is not induced makes it easy to manage the individual, and also makes it possible to produce a desired, uniform disease animal model as needed.

Figure 1 shows the DNA constructs of the Driver line and Effector line designed for the experiment.
Figure 2 shows the expression of hFUS mutant protein in the cytoplasm of neurons of zebrafish induced with amyotrophic lateral sclerosis, as confirmed by fluorescence.
Figure 3 shows the results of evaluating the motility of zebrafish induced with amyotrophic lateral sclerosis and normal zebrafish 5 days after modification.
Figure 4a shows the confirmation of denervation of the neuromuscular junction and muscles in zebrafish induced with amyotrophic lateral sclerosis, and Figure 4b shows the results of confirming the correlation between presynaptic markers and postsynaptic markers using Pearson correlation.
Figure 5 shows a comparison of the levels of oxidative stress in zebrafish induced with amyotrophic lateral sclerosis and normal zebrafish 5 days after modification.
Figure 6 shows the results of confirming the expression levels of synapse-related proteins and TNF involved in neuroinflammation in zebrafish induced with amyotrophic lateral sclerosis 5 days after modification.
Figure 7 is a schematic diagram of a drug screening experiment using zebrafish induced with amyotrophic lateral sclerosis.
Figure 8 shows the results of measuring motility in zebrafish induced with amyotrophic lateral sclerosis treated with edaravone on days 3 and 5 after fertilization.
Figure 9 shows the results of confirming a decrease in the expression of TNF-alpha and an increase in the expression of gria1a in zebrafish induced with amyotrophic lateral sclerosis treated with riluzole.

The present inventors have produced a zebrafish that overexpresses a human-derived FUS mutant (R521C) gene using the IQ system to produce an animal model exhibiting symptoms of amyotrophic lateral sclerosis at the larvae stage in order to develop a treatment for amyotrophic lateral sclerosis more quickly and efficiently, and have completed the present invention by confirming that the animal model effectively functions to verify the effectiveness of drug candidates for the development of a treatment for amyotrophic lateral sclerosis.

The Q-system is a gene regulation system derived from red bread mold (Neurospra crassa), and consists of the QF transcription activator and the QUAS effector sequence. The Q-system is a binary expression system, similar to the conventional Gal4-UAS system used in zebrafish transformation, and is used to regulate the expression of specific genes. Unlike the Gal4-UAS system, the Q-system has the advantage of being less affected by promoter silencing and strongly promoting gene expression, but has the problem of toxicity of the QF activator.

The present inventors have developed the IQ-system, which can complement the shortcomings of the Q-system and induce gene expression more strongly. The IQ-system removes the toxicity of the QF activator and, at the same time, adds a transcriptional activation domain to exhibit high activity, and can induce gene expression more strongly by overlapping 13 QUAS corresponding to the promoter.

The IQ system is a binary expression system. The inventors designed it by distinguishing an effector line expressing a disease-inducing gene (FUS mutant gene) and a driver line regulating the operation of the effector line. Specifically, a human-derived FUS mutant gene (hFUS (R521C)) operably linked under a promoter with 13 repeated QUAS sequences was constructed as an effector line, and a QFDBD 2*AD gene operably linked under a HuC promoter was constructed as a driver line for activating the effector line. The structure of each line is as follows.

Effector Line 13QUAS:hFUS(R521C)

Diver Line HuC:QFDBD 2*AD

To confirm the activity of the effector line, the effector line may be designed to include a reporter gene operably linked under the 13QUAS promoter identical to the hFUS (R521C) gene, and the reporter gene and the hFUS (R521C) gene may be linked to a self-cleaving peptide gene.

Each effector line and diver line were constructed as DNA plasmids using gene cloning technology, and each plasmid was transformed into a separate zebrafish using transposase.

In order to identify the transformed individual with each plasmid, each plasmid may contain a reporter gene operably linked under a promoter separate from the above-mentioned promoter. At this time, it is preferable that the reporter genes of the diver line and the effector line be designed so that they can be distinguished from each other. Zebrafish of the transformed parent generation confirmed through the expression of the reporter gene were selected and crossed with each other to select an individual expressing the reporter gene, thereby creating an animal model of amyotrophic lateral sclerosis.

In the present invention, the zebrafish in which the driver line operates is named as the first transgenic zebrafish, the zebrafish in which the effector line operates is named as the second transgenic zebrafish, and the offspring generation of the first and second transgenic zebrafish in which the driver line and the effector line operate simultaneously is named as amyotrophic lateral sclerosis-induced zebrafish. However, since one purpose of the present invention is to provide an amyotrophic lateral sclerosis animal model, an animal model unless specifically mentioned herein means an amyotrophic lateral sclerosis-induced zebrafish, and the animal model may be used interchangeably with terms such as amyotrophic lateral sclerosis animal and amyotrophic lateral sclerosis zebrafish, and when it is simply described as a 'transgenic zebrafish' without specifically referring to the parental zebrafish or the first and second transgenic zebrafish, it can be understood to mean amyotrophic lateral sclerosis zebrafish.

Meanwhile, in the present invention, the DNA cassette for expression of a gene insert may additionally include an additional configuration for transcriptional activation. For example, the inventors of the present invention added a region encoding a VP16 domain to the driver line to promote gene expression specifically in neuron cells.

In a specific experiment of the present invention, the Effector Line and Driver Line having the following structure were used to visually confirm the activation of the Effector Line and the first and second transgenic zebrafish (Fig. 1).

Effector Line 13QUAS:Zsgreen-p2a-hFUS(R521C)-terminator-Crystallin alpha:mcherry)

Driver Line HuC:QFDBD 2*AD-VP16-Crystallin alpha:EGFP

In order to be used for research on disease mechanisms or screening for therapeutic agents, it is necessary to be able to stably and uniformly produce disease animal models, and the disease must be induced quickly in the produced animals in terms of time and cost. While it takes about 6 months for the disease to develop and its symptoms to be confirmed in the conventional amyotrophic lateral sclerosis animal model produced using zebrafish, it was confirmed that the amyotrophic lateral sclerosis animal model produced by the method of the present invention exhibited disease symptoms, histological and molecular changes on the 5th day after fertilization, and thus it can be effectively used for new drug screening.

The inventors of the present invention treated the manufactured amyotrophic lateral sclerosis zebrafish with Riluzole and edaravone, which are approved by the FDA as amyotrophic lateral sclerosis treatments, to confirm changes in motor ability, and confirmed that the progression of amyotrophic lateral sclerosis was delayed in the same pattern as the efficacy of the above drugs confirmed in clinical trials, thereby verifying that the animal model of the present invention can be used as a platform for developing a therapeutic substance for amyotrophic lateral sclerosis. Specifically, when screening is performed through motility analysis, drug efficacy can be evaluated within a week, and analysis can be performed within about 30 minutes per 48-well plate.

The amyotrophic lateral sclerosis animal model of the present invention comprises an AFDBD 2*AD gene operating under a HuC promoter as a Driver Line and an hFUS (R521C) gene operating under a 13QUAS promoter as an Effector Line. The Effector Line may include a reporter gene and the hFUS (R521C) gene operating simultaneously under the 13QUAS promoter, and the reporter gene and the hFUS (R521C) gene may be linked by a self-cleaving peptide gene.

In the present invention, the “reporter gene” is a gene encoding a protein used to monitor the introduction of a plasmid vector according to an example of the present invention or the operation of the IQ-system, and can be used without limitation as long as it is a gene that can be monitored without damage to transformed cells or tissues. Preferably, it may be luciferase, green fluorescent protein (GFP), modified green fluorescent protein (mGFP), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP), enhanced red fluorescent protein (ERFP), cyan fluorescent protein (BFP), enhanced cyan fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), or enhanced cyan fluorescent protein (ECFP). In specific experiments, the present inventors used mCherry, which is an mRFP, and Zsgreen, which is an mGFP, as reporter genes.

In the present invention, the “vector” refers to a genetic construct including a regulatory element, i.e., a promoter, etc., which is an expression vector capable of expressing a target gene in a suitable host cell, so that a gene insert contained in the vector is operably linked to be expressed. The vector according to the present invention may preferably be a plasmid vector, a cosmid vector, a viral vector, etc., but the present inventors used a DNA plasmid vector as a vector in specific experiments.

In the present invention, “operably linked” means that a nucleic acid expression control sequence performing a general function and a nucleic acid sequence encoding a gene of interest are functionally linked. In the present invention, each component is considered to be operably linked to each other unless it is explicitly stated that it is a direct linkage, and a gene insert whose expression is regulated by a specific promoter in the present specification is described as being “operably linked to a specific promoter” or “operating under a specific promoter.”

In the present invention, the operable linkage of the genetic insert, i.e., the transgene and the promoter, can be produced using a genetic recombination technique well known in the art, and the site-specific DNA cleavage and linkage can be performed using enzymes, etc. generally known in the art.

The term "promoter" used in the present invention is a part of DNA that is involved in binding of RNA polymerase so that transcription can be initiated. It is generally located adjacent to and upstream of a target gene, and is a site where RNA polymerase or a transcription factor, which is a protein that induces RNA polymerase, binds, and can induce the enzyme or protein to be located at the correct transcription start site. That is, it is located at the 5' site of a gene to be transcribed in the sense strand, and induces RNA polymerase to bind to that site directly or through a transcription factor and initiate mRNA synthesis for the transgene, and has a specific gene sequence.

Hereinafter, embodiments will be described in detail with reference to the attached drawings. However, since various modifications may be made to the embodiments, the scope of the patent application rights is not limited or restricted by these embodiments. It should be understood that all modifications, equivalents, or substitutes to the embodiments are included in the scope of the rights.

The terms used in the examples are for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, the terms "comprises" or "has" and the like are intended to specify the presence of a feature, number, step, operation, component, or combination thereof described in the specification, but should be understood to not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments belong. Terms defined in commonly used dictionaries, such as those defined in common dictionaries, should be interpreted as having a meaning consistent with the meaning they have in the context of the relevant art, and shall not be interpreted in an idealized or overly formal sense, unless expressly defined in this application.

Hereinafter, preferred examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following examples.

[Example]

Example 1: Zebrafish breeding and care

All experimental procedures in this study were approved by the Korea University Institutional Animal Care & Use Committee (IACUC) and were performed in accordance with the animal experiment regulations of the National Veterinary Research and Quarantine Service. Wild-type zebrafish, Tg(13QUAS:Zsgreen-p2a-hFUS(R521C)-terminator-Crystallinalpha:mcherry), Tg(HuC:QFDBD 2*AD-VP16), and Tg(13QUAS:Zsgreen-pA) transgenic zebrafish were used in this study. Adult and embryonic zebrafish were raised in a system with a 14:10 h light/dark cycle and a water temperature of 28.5°C. To define morphological characteristics, the stages of zebrafish embryos and larvae were defined by morphological characteristics in hours post-fertilization (hpf) or days post-fertilization (dpf). Drug candidates and control drugs were treated with Egg water from 9 hours post-fertilization. Egg water treated with the same concentration of DMSO as the experimental group was used as a negative control.

Example 2: Preparation of Plasmid DNA

To construct a human FUS mutant gene (hFUS (R521C), cytosine at position 1561 of the open reading frame (ORF) of the wild-type human FUS gene (pENTR4_FUS Plasmid #26366, addgene) was substituted with thiamine using polymerase chain reaction (PCR, KOD FX, TOYOBO). The FUS ORF primers used for polymerase chain reaction are as follows.

hFUS ORF forward primer 5’-atggcctcaaacgattatac-3’ hFUS(R521C) ORF reverse primer 5’-ttaatacggcctctccctgcaa-3’

To construct the IQ-system Effector diver plasmid containing the hFUS (R521C) gene, the 13QUAS: Zsgreen-p2a-terminator-Crystallin alpha: mcherry plasmid vector provided in a previous study ( Communications biology vol. 4,1 1405. 16 Dec. 2021) was used. The hFUS (R521C) gene was inserted after p2a and before the terminator of the vector using the One-Step sequence-ligation-independent cloning (SLIC) technique. In the plasmid vector, the 13QUAS promoter consisted of the base sequence of SEQ ID NO: 2, and the terminator consisted of the base sequence of SEQ ID NO: 4. The enzyme used to cut the plasmid was BglII (#R0144S, NEB). The primers used to construct the gene to be inserted are as follows.

SLIC_hFUS(R521C)_F 5’ - GAGAACCCTGGACCTAGATCtATGGCCTCAAACGATTATAC - 3’ SLIC_hFUS(R521C)_R 5’ - ATACCGTCGACCTCGAGATCTTAATACGGCCTCTCCCTGCA -3’

T4 DNA polymerase (#M0203S, NEB) was used to insert the hFUS (R521C) gene into the plasmid. The constructed effector plasmid DNA has the structure below.

plasmid: 13QUAS:Zsgreen-p2a-hFUS(R521C)-terminator-Crystallin alpha:mcherry

Example 3: Production of transgenic zebrafish

To produce transgenic zebrafish that specifically express the ALS-causing gene hFUS (R521C) in neurons, an effector line of the IQ-system was produced. The effector plasmid produced in Example 2 was microinjected together with transposase into 1-cell zebrafish fertilized eggs, and the eggs were raised to adults (Founders, F0). Transgenic zebrafish were produced by selecting individuals expressing red fluorescent protein (mcherry) in the eyes among the zebrafish (F1) produced by mating with wild-type zebrafish.

Transgenic zebrafish that overexpress hFUS (R521C) specifically in neurons were produced from the offspring by crossing with zebrafish transformed with the driver line, HuC:QFDBD 2*AD-VP16-Crystallin:EGFP (Fig. 1). In the driver line, the QFDBD 2*AD gene consisted of the base sequence of SEQ ID NO: 1, and VP-16 consisted of the base sequence of SEQ ID NO: 3.

Example 4: Section immunohistochemical staining

Zebrafish embryos and fry were anesthetized with an anesthetic (ethyl 3-aminobenzoate methanesulfonate salt, 200 mg/L (MS222, Sigma)) and fixed in 4% paraformaldehyde/PBS (1X phosphate buffered saline) at 4°C for 12–18 h. The fixed embryos and fry were immersed in a 1.5% agarose/5% sucrose solution to make blocks, and then stored in a 30% sucrose solution at 4°C for 12–18 h until the blocks equilibrated and sank. After placing the sunken blocks in a stainless steel cup containing methyl butanol (2-methyl butane) in liquid nitrogen, they were placed onto a plastic mold to remove moisture, and then frozen in methyl butanol. The frozen block was cut into 14-16 ㎛ sections using a cryostat microtome and attached to glass slides. The glass slides with the sections attached were washed with PBS for about 30 minutes and then reacted with 5% sheep serum, 5% bovine serum albumin/PBS (Blocking solution) for more than 1 hour. The primary antibody was diluted in blocking solution and reacted at 4℃ for 12-18 hours. After that, the slides were washed with PBS for 1-2 hours and reacted with blocking solution for 1 hour. After that, the secondary antibody was diluted in blocking solution and reacted at 4℃ for 12-18 hours. After the reaction, DAPI diluted in blocking solution was reacted at room temperature for 15 minutes to stain the nuclei of the tissue cells. After washing with PBS for 1-2 hours, the slide glass was covered with a coverslip with 150-200 ㎕ of 75% glycerol/25% PBS solution, and then fluorescence photography was performed. The primary and secondary antibodies used in the experiment are as follows.

Primary antibody: rabbit anti-FUS antibody (1:1000, Invitrogen, Cat. No. PA552610). Secondary antibody: Alexa 568-conjugated secondary antibodies (1:1000, Invitrogen, Cat. No. A-11011). Nuclear staining reagent: DAPI (D1306, Invitrogen).

Example 5: Whole-tissue-fixed immunohistochemical staining

Zebrafish were treated with PTU from day 1 to day 5 after fertilization. Zebrafish fry were anesthetized with an anesthetic (ethyl 3-aminobenzoate methanesulfonate salt, 200 mg/L (MS222, Sigma)) and fixed in 4% paraformaldehyde/PBS (1X phosphate buffered saline) at 4℃ for 12–18 hours. The fixed fry were washed with PBS for about 30 minutes and then

It was placed in acetone and reacted at -20℃ for 7 minutes. After that, it was washed twice with PBS and reacted with 10mg/ml collagenase for 40 minutes. After that, it was washed twice with PBS and fixed for 20 minutes in 4% paraformaldehyde/PBS (1X phosphate buffered saline). After that, it was washed twice with PBS and reacted with 5% sheep serum, 5% bovine serum albumin/PBS (Blocking solution) for more than 1 hour, then the primary antibody was diluted in blocking solution and reacted at 4℃ for 12~18 hours. After that, the fry were washed with PBS for 1~2 hours and reacted with blocking solution for 1 hour. After that, the secondary antibody was diluted in blocking solution and reacted at 4℃ for 12~18 hours. After washing with PBS for 1-2 hours, the sections were fixed on 1.5% low melting agar and subjected to fluorescence photography. The primary and secondary antibodies used in the experiment were as follows. Primary antibody: mouse anti-acetyl tubulin antibody (mouse anti-acetyl tubulin, 1:1000, Sigma-Aldrich T6793-2ML) Secondary antibody: 568-conjugated secondary antibodies (568-conjugated secondary antibodies, 1:1000, Invitrogen, Cat. No. A-11004).

Staining of the neuromuscular junction was performed using the same staining method as above. However, the primary antibody used was mouse anti-SV2 antibody (SV2, 1:50, DSHB). The secondary antibody used was 568-conjugated secondary antibodies (568-conjugated secondary antibodies, 1:1000, Invitrogen, Cat. No. A-11004), which was treated together with α-bungarotoxin (α-bungarotoxin, Alexa Fluor 647 conjugate, 1:1000, Invitrogen, B35450).

Example 6: Zebrafish behavior analysis

Zebrafish fry behavioral analysis was performed using Danio Vision (Noldus) with an automatic behavioral tracking analysis program, Ethovision XT12 (Noldus). In addition, all behavioral experiments were performed from 10:00 AM to 3:00 PM, and experiments were conducted using siblings obtained from the same parents. Behavioral analysis experiments were performed at the 5-day post-fertilization stage, when locomotor activity becomes active in wild-type individuals and locomotor activity declines in disease models. The fry were raised in a rearing room with a 14-h light/10-h dark cycle at 28.5°C, and transferred to a 48-well plate 3 h before the experiment. To measure the locomotion of the fry, the 5-day post-fertilization fry were acclimated to the light for 10 min, and then recorded for 5 min under the same conditions.

Example 7: Quantitative reverse transcription polymerase chain reaction analysis

Quantitative reverse transcription-polymerase chain reaction was performed using a LightCycler 96 instrument (Roche). The cDNA used in the experiment was synthesized from RNA extracted from wild-type offspring and disease model fry 5 days after fertilization at the same stage as the behavioral experiment. Each reaction mixture containing 2.5 μl of template cDNA, 0.2 μM primer, and 2X Fast Start Essential DNA Green Master Mix (Roche) was amplified through a program step consisting of 95 °C for 10 min, 40 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s. The expression of β-actin, tumor necrosis factor a (tnfa), synaptosome associated protein 25a (snap25a), and glutamate receptor (gria1a) genes was measured.

Example 8: Analysis of active oxygen stress concentration

Analysis of reactive oxygen concentration was performed using a SpectraMax M2 instrument (molecular devices) with the analysis program SoftMax Pro. Zebrafish were treated with PTU from day 1 to day 5 after fertilization. On day 5 after fertilization, CellROX Deep Red Reagent (C10422, Invitrogen) was treated at a concentration of 5 μM for 30 minutes. After anesthesia using an anesthetic (ethyl 3-aminobenzoate methanesulfonate salt, 200 mg/L (MS222, Sigma)), 15 fish were placed per well in a 96-well plate, and fluorescence at 640/665 nm was measured.

Example 9: Statistical Analysis

All statistical analyses were performed using the GraphPad Prism 7 software program (GraphPad Software). In the comparison between two groups, the Student unpaired t-test was performed when the population was normally distributed, and the Mann-Whitney U test was performed when the population was non-normally distributed. In the comparison of three or more groups, one-way ANOVA was performed, and the Tukey test was performed as a post-hoc test. In all statistical data, statistical significance was considered to be valid when the two-tailed probability of P was less than 0.05.

[Experimental example]

Experimental Example 1. Validation of Amyotrophic Lateral Sclerosis Disease Model

Previous studies have shown that the FUS protein, which should be located in the nucleus in normal cells, is incorrectly located in the cytoplasm in neurons of ALS patients with mutant FUS. In the zebrafish amyotrophic lateral sclerosis disease model produced in the present invention, it was confirmed that the mutant human FUS protein was expressed in the cytoplasm of neurons (Fig. 2).

The most representative symptom of amyotrophic lateral sclerosis, decreased motility, is the most important characteristic commonly observed in ALS patients and other disease models. Through behavioral analysis of the zebrafish amyotrophic lateral sclerosis disease model produced in the present invention, it was confirmed that the motility was significantly reduced compared to the control group on the 5th day after fertilization (Fig. 3). In addition, while this decrease in motility occurs in adults in other disease models, in the disease model produced in the present invention, the disease developed early on the 5th day after fertilization.

Many studies have reported neuromuscular junction dysfunction and muscle denervation in patients with amyotrophic lateral sclerosis and mouse disease models. The same phenomenon was confirmed in the zebrafish disease model produced in the present invention. The correlation between presynaptic markers and postsynaptic markers was measured in zebrafish 5 days after fertilization, and it was confirmed that the correlation was reduced in the disease model compared to the control group (Pearson correlation) (Fig. 4). This is data showing that denervation occurred at the neuromuscular junction.

High oxidative stress is detected in postmortem tissues of patients with amyotrophic lateral sclerosis. In addition, increased oxidative stress due to mitochondrial dysfunction is a symptom that is receiving attention in the study of the mechanism of amyotrophic lateral sclerosis. In the zebrafish amyotrophic lateral sclerosis disease model produced in the present invention, increased oxidative stress due to active oxygen was confirmed on the 5th day after fertilization (Fig. 5).

Neurodegenerative diseases including amyotrophic lateral sclerosis are known to cause neuroinflammation. Among the cytokines involved in neuroinflammation, TNFα is known to be greatly increased in actual amyotrophic lateral sclerosis patients and mouse models. In the zebrafish disease model produced in the present invention, the expression level of TNFα was confirmed through quantitative reverse transcription polymerase chain reaction analysis, and it was confirmed that it significantly increased compared to the control group (Fig. 6). This is data that can indicate an increase in the inflammatory response. In addition, the expression level of genes known to be related to amyotrophic lateral sclerosis was confirmed to be imbalanced in the expression of RNA of proteins related to synapses. Synaptic RNA imbalance has been reported in previous studies on amyotrophic lateral sclerosis.

Experimental Example 2. Drug Efficacy Analysis Using Zebrafish Amyotrophic Lateral Sclerosis Disease Model

The experiment performed in Experimental Example 1 can be applied to drug efficacy analysis for new drug screening. The efficacy of a drug that inhibits disease progression in amyotrophic lateral sclerosis was evaluated in a zebrafish amyotrophic lateral sclerosis disease model produced in the present invention. Drugs were treated to zebrafish from day 3 to day 5 after fertilization. The drugs were dissolved in embryo medium containing 0.1% dimethyl sulfoxide (DMSO) and treated. Thereafter, analysis was performed on zebrafish on days 3 and 5 after fertilization (Fig. 7). The drugs used were Riluzole 5uM and edaravone 10uM, FDA-approved amyotrophic lateral sclerosis treatment drugs.

The drug efficacy in recovery of motor ability was evaluated using the zebrafish disease model produced in the present invention. In the disease model on the third day after fertilization, a significant decrease in motor ability occurred, whereas the motor ability of the group treated with the edaravone drug recovered to a statistically significant level. However, in the zebrafish on the fifth day after fertilization, a decrease in motor ability occurred at the same level as the disease model that was not treated with the drug (Fig. 8). These results indicate that edaravone significantly delays the progression of the disease in the disease model. This is the same as the efficacy of edaravone in actual clinical practice. If a drug efficacy evaluation is conducted using this experimental method, a new drug that exhibits a better therapeutic effect than currently commercially available drugs can be screened.

The efficacy of Riluzole, an amyotrophic lateral sclerosis treatment, was evaluated by quantitative reverse transcription polymerase chain reaction analysis to determine the expression levels of TNFa, a cytokine involved in neuroinflammation, and synapse-related genes. As a result, it was confirmed that the expression abnormalities of TNFa and gria1a were significantly recovered compared to the group that was not treated with the drug (Fig. 9).

The efficacy of new drug candidates can be evaluated in the above manner, and in addition, the drug efficacy can be evaluated by applying the disease model disability quantification method shown in the experiments performed in Experimental Example 1.

Although the embodiments have been described with limited drawings as described above, those skilled in the art can apply various technical modifications and variations based on the above. For example, even if the described techniques are performed in a different order than the described method, and/or the components of the described system, structure, device, circuit, etc. are combined or combined in a different form than the described method, or are replaced or substituted by other components or equivalents, appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are also included in the scope of the claims described below.

<110> Korea University Research and Business Foundation <120> Demyelinating disease animal model using transgenic zebrafish and manufacturing method <130> APC-2022-0327 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 681 <212> DNA <213> Artificial Sequence <220> <223> QFDBD 2*AD <400> 1 atgccgccta aacgcaagac actcaatgcc gctgccgaag ccaatgccca cgctgatggc 60 catgctgatg gcaatgctga tggtcatgtc gctaacactg cagcaagcag caacaacgcc 120 cgttttgcgg acttgaccaa cattgacaca cccggcctcg gccctaccac gacgacgtta 180 cttgtcgagc ccgctcgttc gaaacgccag agagtctcga gggcctgtga tcagtgtcga 240 gctgcacgtg aaaagtgtga tggaatccag ccggcttgct tcccctgtgt gtcgcagggc 300 cggtcgtgta cctaccaggc cagtcccaag aagcgaggag tccagacggg ctacatccgc 360 actctcgaac tggctctggc ttggatgttc gagaacgttg cccgcagcga ggacgccctc 420 cacaatcttt tggtccgtga tgctggccag ggcagcgctc tcctggtcgg caaagactcg 480 cctgctgcag aacgcctgca tgcaagatgg gcgacgagtc gagtcaacaa aagcatcacc 540 cgtcttctct caggtcaggc cgcacaagat ccatctgaag acggccaatc cccgtccgaa 600 gacataaatg tccaagatca ggccgcacaa gatccatctg aagacggcca atccccgtcc 660 gaagacataa atgtccaaga t 681 <210> 2 <211> 244 <212> DNA <213> Artificial Sequence <220> <223> 13QUAS promoter <400> 2 gggtaatcgc ttatcctcgg ataaacaatt atcctcacgg gtaatcgctt atccgctcgg 60 gtaatcgctt atcctcgggt aatcgcttat cctcgggtaa tcgcttatcc tcggataaac 120 aattatcctc acgggtaatc gcttatccgc tcgggtaatc gcttatcctc ggataaaacaa 180 ttatcctcac gggtaatcgc ttatccgctc gggtaatcgc ttatcctcgg gtaatcgctt 240 atcc 244 <210> 3 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> VP16 <400> 3 caggccgcac aagatccatc tgaagacggc caatccccgt ccgaagacat aaatgtccaa 60 gatcaggccg cacaagatcc atctgaagac ggccaatccc cgtccgaaga cataaatgtc 120 caagat 126 <210> 4 <211> 1205 <212> DNA <213> Artificial Sequence <220> <223> terminator <400> 4 ctagtaatta agtctcagcc accgttaact gaacatgtca aaacctgtgg agactgttga 60 gatttgatgt tctgaaaaga taaagcctat aaataaaatg ttgcccaaat ttcctgcctg 120 atgtttttct ttgtctttgc tacatggctt tgctgctcgg atcggctcac tctgtgtatg 180 ccacgttcac tttgtactct ccttctcacg gtaggtttat tatttttaga tgtgcagtta 240 gtttctgtga aataacacac cacacactga tattgtctgt gcattgactt ggtgagtgca 300 cattgttttt gatcttgaca tatttatatt tgattgatca ggtgaactgt gtgaatctaa 360 agtgctccat acagatgttc tgcattgaaa atattctcat tttattagtg gaagtgagtg 420 tatgccacat ccaatcaatt tcagcaaaca ccccagtatg atttaatgca aaaaaatgaa 480 ggtatcaaac acgcattact actttgcagt taaatattta acatttattc caacacgaaa 540 aaaagcagta aataacactt tgacaaacac gtcaggacat cttatttttg tcaccctcac 600 aggcaattta gtataatata ttatatatat atatatatca tataataata ttcagtataa 660 tatatatata tatatcatat tataatattc agtataatat aaaacacaaa cacatatatg 720 tataatataa tataacattt ttatttattg agatgcctct atggaccgtg ttataagaag 780 taaagatcag gagaagtaaa catgaagtgt aattatgaat actgatgtta aattaagcta 840 tgatgagttt tcactgttaa tttaccatct caattaaatg ttgatgcctc catgaccaag 900 ttaagcagat gagactgaga caactgtaga agacaagatg ttcactttgc tgaatatagc 960 tggcttgaca gttatctatg actctataaa tatatatata tttttttttt tataaaatga 1020 tttatttata actatatatc catttctcag acaggtgctt catatccctc actcccgtag 1080 ctgtccatgc tggatctgtc cccgttgttt ttaaaaagct aaataagtta ttaacatgac 1140 tgcatccagc gagccaaacc tgtctggtgt acagctacca gagaagcttg agatcctagt 1200 caccg 1205

Claims (14)

delete delete delete delete delete (1) A first transgenic zebrafish production step comprising the following steps (A) and (B):
(A) A first plasmid vector production step containing a cassette for expression of the QFDBD 2*AD gene operating under the HuC promoter, and
(B) A step of transforming the first plasmid vector manufactured in step (A) into a zebrafish fertilized egg.
(2) A second transgenic zebrafish production step comprising the following steps (a) and (b):
(a) a second plasmid vector production step including a human-derived FUS mutant (hFUS (R521C)) gene operating under the 13QUAS promoter and a cassette for expression of an effector reporter gene; and
(b) a step of transforming the second plasmid vector manufactured in step (a) into a zebrafish fertilized egg;
(3) A step of culturing the transformed zebrafish fertilized eggs in steps (1) and (2) and developing them into adults (F0);
(4) a step of mating the first and second transgenic zebrafish adults with each other; and
(5) A method for producing an amyotrophic lateral sclerosis zebrafish animal model, comprising: a step of selecting zebrafish (F1) expressing a reporter gene from among the zebrafish obtained after mating in step (4);
A manufacturing method wherein steps (1) and (2) above can be performed simultaneously or sequentially, and if performed sequentially, the order of steps (1) and (2) is irrelevant.
In Article 6,
A method for producing an animal model, wherein steps (B) and (b) above are performed by microinjecting each plasmid vector produced in steps (A) and (a) together with transposase into a 1-cell zebrafish fertilized egg. In Article 6,
In the above step (A), the first plasmid vector comprises a first reporter gene operably linked under a promoter,
A method for producing an animal model, wherein step (1) further comprises a step (C) of selecting an individual expressing a first reporter gene after step (B). In Article 6,
In step (a) above, the second plasmid vector comprises a second reporter gene operably linked under a promoter,
A method for producing an animal model, wherein the step (2) further comprises a step (c) of selecting an individual expressing a second reporter gene after the step (b).
In Article 6,
A method for producing an animal model, wherein in the step (b) above, the hFUS (R521C) gene and the effector reporter gene are linked to a gene encoding a self-cleaving peptide. In clause 6, clause 8, or clause 9,
A method for producing an animal model, wherein the above effector reporter gene is a gene encoding a fluorescent protein. In Article 11,
A method for producing an animal model, wherein the reporter gene is at least one selected from the group consisting of luciferase, green fluorescent protein (GFP), modified green fluorescent protein (mGFP), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP), enhanced red fluorescent protein (ERFP), cyan fluorescent protein (BFP), enhanced cyan fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), and enhanced cyan fluorescent protein (ECFP). Zebrafish animal model of amyotrophic lateral sclerosis prepared by the method of claim 6. (a) A step of treating a candidate substance to an amyotrophic lateral sclerosis zebrafish animal model of Article 13;
(I) a step of measuring one or more of the symptoms of amyotrophic lateral sclerosis disease and/or biomarkers of the disease, consisting of the following (1) to (5), in an amyotrophic lateral sclerosis zebrafish animal model treated with the candidate substance and not treated with the candidate substance: and
(1) Motility
(2) Denervation at the neuromuscular junction
(3) Oxidative stress
(4) TNF-α expression level
(5) gria1a expression level
(d) A method for screening drugs for amyotrophic lateral sclerosis, comprising: a step of selecting the candidate substance as a therapeutic substance for amyotrophic lateral sclerosis when an animal treated with the candidate substance shows increased motility, a higher incidence of denervation at the neuromuscular junction, a decrease in oxidative stress, a decrease in the expression level of TNF-α, and/or an increase in the expression level of gria1a compared to an animal not treated with the candidate substance.