WO2017104967A1 - Zebrafish model having dgat2 mutant gene introduced thereinto and use thereof - Google Patents

Abstract

The present invention provides a recombinant plasmid comprising a human DGAT2 mutant gene, a zebrafish transformed with the plasmid, and a method for screening a therapeutic agent for a peripheral nervous system disease using the same. The use of a zebrafish model of the present invention can effectively screen a therapeutic agent for a peripheral nervous system disease, which has favorable efficacy and few side effects, and can easily and quickly test whether or not a drug candidate causes an in vivo side effect due to the toxicity thereof.

Description

Zebrafish Model with DTVA2 Mutant Gene and Uses thereof

The present invention was made by task number 2013R1A1A1059056 under the auspices of the Ministry of Science, ICT and Future Planning of Korea. Research on the pathogenesis of cancer through “College of Pathology” Sungkyunkwan University (Natural Sciences Campus).

The present invention was made by the task number HI14C3484 under the support of the Ministry of Health and Welfare of the Republic of Korea, the research and management institution of the project is the Korea Health Industry Development Institute, the research project name is "Research Center Hospital Project", the research title is "Stem Cells through Intermediate Research Development of neuro-renewal therapy for patients ”, the host institution is Samsung Seoul Hospital, and the period of research is 2015.7.11-2016.1.10.

The present invention was made by the task number 2014R1A2A2A01004240 under the support of the Ministry of Science, ICT and Future Planning, the research management specialized organization of the project is the Korea Research Foundation, the research project name is "medium-level researcher support project", the research title is "genome in genetic motor sensory neuropathy Exploration of Pathogenesis and Treatment Using Based Cell / Animal Model ”, the host institution is Sungkyunkwan University, and the research period is 2015.5.1-2016.4.30.

This patent application claims priority to Korean Patent Application No. 10-2015-0179383 filed with the Korean Patent Office on December 15, 2015, the disclosure of which is hereby incorporated by reference.

The present invention relates to a zebrafish model into which the DGAT2 mutant gene has been introduced and its use.

Zebrafish is a tropical fish that is widely used as an experimental animal model. Zebrafish can replace animal models such as rats and rats as vertebrates, as well as shorter development time and transparent embryos, making them easier to observe.

In particular, in order to screen for rare disease treatment substances, such as Charco-Marie-Tooth disease, whose pathogenesis is not well known, zebra, which is more economically and timely effective than animal breeding and maintenance costs and costly and time-consuming animals, Fish is much more advantageous. Therefore, there is a need to develop a disease animal model production and screening method capable of simulating in vivo functions and capable of screening on a large scale.

 The extra energy storage in the cell is in the form of triglycerides (TG), and DGAT (diacylglycerol acyltransferase) is an enzyme that acts as the last step in the process of making triglycerides. In mammals, there are two types of DGATs expressed in different genes. There are DGAT1 and DGAT2, both of which are distributed in most organs, but DGAT1 is expressed in the small intestine and DGAT2 is expressed in liver and adipose tissue.

Among them, DGAT2 (Diacylglycrol O-acyltrangerase 2) is a protein produced by the DGAT2 gene. It is composed of 350-400 amino acids and the 200-360th amino acid has catalytic activity. This protein is present in the membrane of the endoplasmic reticlum, and synthesizes diacylglycerols and fatty acyl CoA into triglycerides through ester linkage. Most DGAT2 proteins are present in the membranes of the endoplasmic reticulum, but because they have a highly conserved base that can bind triglycerides, it can be observed that the lipid droplets in adipocytes are located in the fat droplets for expansion. .

Animal models demonstrate the biological significance of DGAT2, which is known as DGAT2 knockout mice after birth by reducing the substrate required for energy metabolism and reducing the amount of triglycerides in the liver and plasma by more than 90% compared to normal mice. You die in a few hours. In addition, due to the reduction of lipid acylceramide in the skin tissue, the skin elasticity is reduced due to the imbalance of skin osmoticity, and the epidermal discharge of the water is increased, thereby showing rapid dehydration. However, DGAT1 knockout mice show a 50% reduction in fat accumulation compared to normal mice but do not significantly affect survival, so the importance of DGAT2 in triglyceride synthesis and metabolism is more important. It is observed to be high.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

We conducted a Charco-Marie-Tus causal gene analysis using patient samples and as a result found mutations in the DGAT2 gene. Based on this, a zebrafish that simulates peripheral nervous system disease symptoms by expressing a human DGAT2 mutant form gene was developed, and the present invention was completed by elucidating that it can quickly and effectively screen related disease target therapeutic substances.

It is an object of the present invention to provide a recombinant plasmid comprising a human DGAT2 mutant gene.

Another object of the present invention is to provide a fertilized egg of zebrafish into which the recombinant plasmid is introduced.

Another object of the present invention to provide a zebrafish transformed with the recombinant plasmid.

It is another object of the present invention to provide a method for screening a therapeutic agent for peripheral nervous system diseases.

Other objects and advantages of the present invention will become apparent from the following detailed description and claims.

According to one aspect of the present invention, the present invention provides a recombinant plasmid comprising a human DGAT2 mutant gene represented by SEQ ID NO: 1, and allowing the gene to be expressed in zebrafish.

We conducted a Charco-Marie-Tus causal gene analysis using patient samples and as a result found mutations in the DGAT2 gene. Based on this, the development of zebrafish that simulates peripheral nervous system disease symptoms by expressing human DGAT2 mutant morphology gene, it was found that it can be used to quickly and effectively screen the relevant targets for disease treatment.

The recombinant plasmid of the present invention comprises a human DGAT2 mutant gene represented by SEQ ID NO: 1.

In the present invention, the plasmid may be any plasmid having a promoter capable of expressing the human DGAT2 mutant gene in zebrafish and a multicloning site for cloning, but preferably the pCS2 + vector shown in FIG. 1. to be.

Recombinant plasmids of the invention can be introduced into zebrafish cells using methods known in the art. Examples include, but are not limited to, microinjection, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, and polyethylenglycol (PEG). Direct delivery using precipitation, transfection reagents (e.g., GeneJamer or GenePORTER) (Sussman, R. Direct DNA delivery into zebrafish embryos employing tissue culture techniques.Genesis. 2001 Sep; 31 (1) : 1-5), Paul, et all, Reporter gene expression in fish following cutaneous infection with pantropic retroviral vectors.Mar Biotechnol (NY). 2001 Jun; 3 (Supplement 1): S81-7) may be used.

In one embodiment of the present invention, recombinant plasmids were introduced in the form of mRNA into fertilized eggs of zebrafish in a single cell state by microinjection (see Example 2).

According to another aspect of the present invention, the present invention provides a fertilized egg of zebrafish introduced with the recombinant plasmid.

According to another aspect of the present invention, the present invention provides a transformed zebrafish expressing the human DGAT2 mutant gene represented by SEQ ID NO: 1 sequence is introduced into the recombinant plasmid.

According to one embodiment of the present invention, the zebrafish to which the recombinant plasmid of the present invention is introduced does not exhibit an external defect due to the introduction of a mutant human DGAT2 gene, but abnormally generated terminal motor neurons and terminal motor neuron axon. It is characterized by a decrease in the number of branches produced by the processusum.

The zebrafish of the present invention exhibits neuronal cell development defects due to abnormal development of terminal motor neurons and defects of terminal motor neuron axons, and can be used as a peripheral nervous system model according to these characteristics.

The peripheral nervous system diseases include both neurological diseases caused by terminal nerve damage and disorders, and muscle diseases caused by muscle cell abnormalities.

According to another aspect of the present invention, the present invention provides a method for screening a therapeutic agent for peripheral nervous system diseases, wherein the zebrafish to which the recombinant plasmid is introduced is treated with a test substance and the degree of peripheral nervous system development of the zebrafish is measured.

Specifically, the screening method of the present invention comprises the steps of: (a) administering a peripheral nervous system disease therapeutic agent test substance to zebrafish transformed with a recombinant plasmid comprising a human DGAT2 mutant gene represented by SEQ ID NO: 1; (b) confirming the degree of peripheral nervous system development of zebrafish to which the peripheral nervous system disease treatment agent test agent of step (a) is administered; And (c) selecting a test substance that has significantly recovered the peripheral nervous system development compared with the control zebrafish that did not administer the test substance.

The test substance to be analyzed in the present invention includes various substances. For example, the test substance includes, but is not limited to, chemicals, proteins, peptides, antibodies, nucleic acids, and natural extracts. The test substance analyzed by the screening method of the present invention may be a single compound or a mixture of compounds (eg, a natural extract or a cell or tissue culture). The test substance can be obtained from a library of synthetic or natural compounds. Methods of obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA), and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories (USA). ) And MycoSearch (United States). Samples can be obtained by a variety of combinatorial library methods known in the art, for example biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution required By a synthetic library method, a “1-bead 1-compound” library method, and a synthetic library method using affinity chromatography screening. Methods of synthesizing molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994 and the like.

The step of confirming the degree of peripheral nervous system development of zebrafish in step (b) includes, but is not limited to, various assays such as morphological observation through a microscope and various marker analysis capable of confirming peripheral nervous system neuronal activity.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a recombinant plasmid containing a human DGAT2 mutant gene, a zebrafish transformed with the plasmid, and a method for screening a therapeutic agent for peripheral nervous system diseases using the same.

(b) Using the zebrafish model of the present invention, it is possible to effectively screen effective peripheral nervous system disease treatment drugs with less side effects, and to test the presence or absence of side effects in vivo according to the toxicity of drug candidates easily and quickly. have.

1 is a cleavage map of a plasmid recombined so that the mutant form gene of DGAT2 found in a patient can be expressed in zebrafish.

 2 is a graph showing the results of observing the development of zebrafish 3 days after the recombinant DGAT2 plasmid was introduced.

A: head of zebrafish, P: tail of zebrafish

3 is a diagram showing the results of observing changes in the motor neuron cells of zebrafish 3 days after the introduction of the recombinant DGAT2 plasmid.

D: back of zebrafish, V: belly of zebrafish

Figure 4 is a graph quantifying the degree of change in the axon of the motor neuron cells of zebrafish 3 days after the introduction of the recombinant DGAT2 plasmid.

5 is a graph showing the results of observing the development of zebrafish 15 days after the recombinant DGAT2 plasmid was introduced.

A: head of zebrafish, P: tail of zebrafish

6 is a diagram illustrating a site for observing peripheral nervous system cell changes in zebrafish.

Figure 7 shows the results of observing changes in the motor neuron cells of zebrafish 15 days after the recombinant DGAT2 plasmid was introduced.

FIG. 8 is a graph quantifying the degree of change in axon of motor neuron cells of zebrafish 15 days after the recombinant DGAT2 plasmid was introduced.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Example 1 Breeding of Zebrafish and Preparation of Embryos

Wild type zebrafish were bred under conditions (temperature: 28-28.5 ° C., contrast: lighted from 9 am to 8 pm, other times off, food: brine shrimp). The embryos were divided into two partitions each day by zebrafish before mating, and then brightened the next morning to remove mating between females and males. The zebrafish eggs obtained through the mating were transferred to a mold made of agar gel.

Example 2: Preparation of Human DGAT2 Gene Expression Zebrafish Model

Normal and mutant forms of the human DGAT2 gene were cut with EcoRI restriction enzymes in the pCMV-Myc vector and then transferred to the pCS2 + vector to construct a new recombinant plasmid that can be expressed in zebrafish (FIG. 1).

The recombinant plasmid prepared in the above manner was microinjected in the form of mRNA into zebrafish embryos in a single cell state. mRNA was synthesized in vitro using the mMESSAGE mMACHINE SP6 kit (Ambion Inc., Austin, TX, USA). Zebrafish fertilized eggs were transferred to agarose gel molds and the synthesized mRNA was microinjected with a micropipette mounted on a micromanipulator (World Precision Instruments Inc., Sarasota, FL, USA).

Example 3 Investigation of Developmental Defects in Zebrafish Three Days After Incorporation of the Human DGAT2 Mutant Gene

The present inventors observed the phenotype of the zebrafish model to which the human DGAT2 mutation and the normal gene prepared in Example 2 were introduced 3 days after birth.

The control group without microinjection of mRNA and zebrafish in which normal human DGAT2 gene mRNA was introduced had a straight tail and a straight movement. It also showed no significant change in movement (FIG. 2).

Example 4 Investigation of Peripheral Nervous System Defects in Zebrafish with Human DGAT2 Mutant Gene

The present inventors used a zebrafish embryo into which a human DGAT2 mutant gene was introduced to determine SV2 and BTX as markers, and to perform fluorescent staining. After that, a specific region of the body was selected to observe the degree of activation of motor neurons, and the axons of neurons were observed through a confocal microscope.

As a result, in the normal zebrafish without injection of the mRNA and the zebrafish in which the normal human DGAT2 gene was introduced, both markers were normally expressed and showed activation of the terminal motor neuron, whereas the mutant human DGAT2 mRNA was injected in a small amount. In the case of zebrafish, it was observed that terminal motor neuron cells were abnormally generated while the expression patterns of these marker proteins were abnormal (FIG. 3 and FIG. 4).

Example 5: Investigation of developmental defects in zebrafish 15 days after introduction of human DGAT2 mutant gene

The inventors observed the phenotype of the zebrafish model into which the human DGAT2 mutation and the normal gene prepared in Example 2 were introduced 15 days after birth.

The control group without microinjection of mRNA and zebrafish in which normal human DGAT2 gene mRNA was introduced had a straight tail and a straight movement. It also showed no significant change in movement (FIG. 5).

Therefore, the present inventors determined the markers of SV2 and BTX which can confirm the activation of peripheral motor motor neurons using a zebrafish embryo into which a human DGAT2 mutant gene was introduced, and performed fluorescent staining. Thereafter, a specific region of the trunk was selected to observe the degree of activation of motor neurons, and the axon fascicle and branch of motor neurons were divided and observed through a confocal microscope (FIG. 6).

Example 6 Investigation of Peripheral Nervous System Defects in Zebrafish with Human DGAT2 Mutant Gene

The present inventors defined the motor neurons defined in Example 5 in the case of normal zebrafish without mRNA injection and zebrafish in which the normal human DGAT2 gene was introduced, and in the case of zebrafish in which the mutant human DGAT2 mRNA was injected in a small amount. The fascicle and branch portions of axons were analyzed.

As a result, in the case of normal zebrafish without mRNA injection and zebrafish into which the normal human DGAT2 gene was introduced, more than about 25 branches were generated per fascicle of the terminal motor neuron axon, whereas mutant humans In the case of zebrafish in which DGAT2 mRNA was injected microscopically, it was observed that the number of these branches decreased to less than 15 (FIGS. 7 and 8).

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (7)

1. A recombinant plasmid comprising a human DGAT2 mutant gene represented by SEQ ID NO: 1 and allowing the gene to be expressed in zebrafish.
2. A fertilized egg of zebrafish into which the recombinant plasmid of claim 1 is introduced.
3. A transformed zebrafish, wherein the recombinant plasmid of claim 1 is introduced to express a human DGAT2 mutant gene represented by SEQ ID NO: 1.
4. 4. The zebrafish of claim 3 wherein the zebrafish has neuronal cell defects.
5. The zebrafish according to claim 4, wherein the neuronal cell development defect is abnormal development of terminal motor neurons or terminal motor neuron axon defects.
6. Screening methods for treating peripheral nervous system diseases comprising the following steps:

   (a) administering a peripheral nervous system disease therapeutic agent test substance to zebrafish transformed with a recombinant plasmid comprising a human DGAT2 mutant gene represented by SEQ ID NO: 1;

   (b) confirming the degree of peripheral nervous system development of zebrafish to which the peripheral nervous system disease treatment agent test agent of step (a) is administered; And

   (c) selecting a test substance that significantly restores the peripheral nervous system development compared to the control zebrafish that did not administer the test substance.
7. The method of claim 6, wherein the test substance of step (a) is any one selected from the group consisting of chemicals, proteins, peptides, antibodies, nucleic acids and natural extracts.

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