KR102579433B1 - Neu2-deficient mouse as an animal model of lipid metabolism dysfunction and use thereof - Google Patents

Abstract

The present invention relates to Neu2- deficient mice as animal models of lipid metabolism dysfunction and their use. Since the Neu2- deficient mouse according to the present invention exhibits lesions that progress from simple fatty liver to steatohepatitis, it provides not only a model of early fatty liver due to metabolic syndrome, etc., but also an animal model of non-alcoholic steatohepatitis in the background of obesity and diabetes according to modern people's lifestyle. Therefore, it can be useful as a model for developing drugs to prevent or treat such diseases.

Description

NEU2-deficient mouse as an animal model of lipid metabolism dysfunction and use thereof {NEU2-DEFICIENT MOUSE AS AN ANIMAL MODEL OF LIPID METABOLISM DYSFUNCTION AND USE THEREOF}

The present invention relates to Neu2- deficient mice as animal models of lipid metabolism dysfunction and their use.

Neu2 is a gene encoding a protein belonging to the neuraminidase family, which removes sialic acid residues from glycoproteins and glycolipids. Unlike other sugar-related enzymes that are mainly present in the endoplasmic reticulum, Golgi, etc., the encoded protein is located in the cytoplasm. It is known to exist in

Expression of Neu2 shows significant differences between humans and mice. In humans, the expression of Neu2 is observed only in muscle tissues such as myoblasts, and the overall expression is very low, whereas in mice, the expression of Neu2 is observed in various tissues such as mammary cells, kidneys, liver, and lung.

However, to date, research on the substrate and biological functions of Neu2 is insufficient, and the exact function of this gene has not yet been revealed.

Meanwhile, with recent changes in modern people's lifestyles, abnormal lipid metabolism diseases such as hyperlipidemia and non-alcoholic steatohepatitis (NASH) are rapidly increasing, and accordingly, many efforts are being made to develop animal models useful for the development of preventive and therapeutic drugs for these diseases. Research is being done.

However, the previously reported non-alcoholic steatohepatitis animal model is a model of severe liver disease with a phenotype that promotes progression to hepatitis, cirrhosis, and liver cancer, and is limited in its use as an early liver disease research model. Additionally, since the manufacturing method includes the step of causing serious tissue lesions by administering an abnormal high-fat diet or administering chemicals, this model is a chemical-based model. Moreover, in these animal models, it is unclear whether the various sugar chain terminals involved in drug absorption, distribution, metabolism, excretion, etc. have structures similar to those in humans, so it is unclear whether drugs that show efficacy in the model will actually show the same or similar efficacy in humans. It is not guaranteed.

Therefore, in order to develop therapeutic or preventive drugs for hyperlipidemia, diabetes, arteriosclerosis, and non-alcoholic steatohepatitis due to obesity, diabetes, etc. due to changes in modern people's lifestyle, it is necessary to use an abnormal animal model rather than an animal model with serious tissue lesions. Animal models representing the lesions of diseases associated with lipid metabolic dysfunction accompanied by increased triglycerides due to lipid metabolism (e.g., nonalcoholic steatohepatitis) are needed. In addition, if these animal models show similar structures to humans in the various sugar chain terminals involved in drug absorption, distribution, metabolism, excretion, etc., drugs that can more effectively prevent or treat diseases related to lipid metabolism dysfunction can be reliably developed. It can be helpful in doing so.

(Non-patent Document 1)

MC Wallace et al . Standard Operating Procedures in Experimental Liver Research: Thioacetamide model in mice and rats, Laboratory Animals 2015, Vol. 49(S1) 21-29.

(Non-patent Document 2)

Contois, J. H.; Warnick, G.R.; Sniderman, A.D. Reliability of low-density lipoprotein cholesterol, non-high-density lipoprotein cholesterol, and apolipoprotein B measurement. J Clin Lipidol 2011, 5, 264-272.

(Non-patent Document 3)

McCormick, SP et al . Transgenic mice that overexpress mouse apolipoprotein B. Evidence that the DNA sequences controlling intestinal expression of the apolipoprotein B gene are distant from the structural gene. J Biol Chem 1996, 271, 11963-11970.

(Non-patent Document 4)

Santos, A.J.; Nogueira, C.; Ortega-Bellido, M.; Malhotra, V. TANGO1 and Mia2/cTAGE5 (TALI) cooperate to export bulky pre-chylomicrons/VLDLs from the endoplasmic reticulum. J Cell Biol 2016, 213, 343-354.

The object of the present invention is to provide transgenic mice with metabolic syndrome or lipid metabolism dysfunction.

Another object of the present invention is to provide a method for producing transgenic mice with metabolic syndrome or lipid metabolism dysfunction.

Another object of the present invention is a drug candidate for the prevention or treatment of diseases related to lipid metabolism dysfunction, comprising the step of administering a drug candidate for the prevention or treatment of diseases related to lipid metabolism dysfunction to the transgenic mouse. Provides methods for evaluating materials.

According to one aspect of the technology disclosed by the present application, a transgenic mouse lacking the Neu2 gene is provided.

According to another aspect of the technology disclosed by the present application, transgenic mice lacking the Neu2 gene are provided, characterized by an increase in triglycerides in the blood.

According to another aspect of the technology disclosed by the present application, a method for producing a transgenic mouse comprising the step of deleting the Neu2 gene is provided.

According to one aspect of the technology disclosed by the present application, (a) mixing a guide RNA (gRNA) corresponding to the target site of the Neu2 gene with the Cas9 protein; (b) treating a fertilized mouse egg with a mixture of gRNA and Cas9 protein; (c) implanting the fertilized egg of the mouse into a surrogate mouse; (d) selecting a mouse with a deletion of the Neu2 gene among mice given birth by a surrogate mother mouse; and (e) establishing transgenic mice lacking Neu2 through crossbreeding between mice lacking the Neu2 gene; A method for producing a transgenic mouse comprising a is provided.

According to one aspect of the technology disclosed by the present application, a drug for preventing or treating diseases related to lipid metabolism dysfunction (e.g., cardiovascular disease, cerebrovascular disease, ischemic heart disease) in transgenic mice lacking the Neu2 gene. A method is provided for evaluating a drug candidate for the prevention or treatment of diseases associated with lipid metabolism dysfunction, comprising the step of administering the candidate material.

According to another aspect of the technology disclosed by the present application, transgenic mice lacking the Neu2 gene are used for the prevention or treatment of diseases selected from diabetes, arteriosclerosis, hypertension, obesity, hyperlipidemia, fatty liver, hepatitis, stroke, and heart disease. A method for evaluating a drug candidate for preventing or treating the disease is provided, comprising the step of administering the drug candidate.

According to another aspect of the technology disclosed by the present application, cells derived from transgenic mice lacking the Neu2 gene are provided.

According to one aspect of the technology disclosed by the present application, cells derived from fertilized eggs derived from transgenic mice lacking the Neu2 gene or embryos created therefrom are provided.

Neu2- deficient mouse according to the present invention exhibits lesions that progress from simple fatty liver to steatohepatitis, and thus has lipid metabolism, such as an early fatty liver model due to metabolic syndrome or non-alcoholic steatohepatitis in the background of obesity or diabetes according to modern lifestyles. Since it provides an animal model for diseases related to functional disorders, it can be useful as a model for developing drugs to prevent or treat such diseases.

Figure 1 shows the results of Neu2 immunostaining and Sambucus nigra lectin (SNL) staining on paraffin blocks prepared by extracting muscle and liver tissues from Neu2 -deficient mice (N2KO) and normal mice (WT).
Figure 2a shows an image obtained by performing H&E staining on a paraffin block prepared by extracting muscle tissue from a Neu2- deficient mouse (N2KO) and a normal mouse (WT), and Figure 2b shows the perimysium from the image shown in Figure 2a. ) shows the results of quantitative analysis of the area of the area and the area of the muscle fiber (Myofiber) area.
Figure 3a shows an image obtained by immunostaining MyoD protein on a paraffin block prepared by extracting muscle tissue from Neu2 -deficient mouse (N2KO) and normal mouse (WT), and Figure 3b shows MyoD protein from the image shown in Figure 3a. Shows the results of counting the number of cells expressing .
Figure 4a shows changes in body weight gain between 6 and 16 weeks of age in normal mice (WT) and Neu2 knockout mice (N2KO), and Figure 4b shows changes in body weight gain in normal mice (WT) and Neu2 knockout mice (N2KO) at 16 and 50 weeks of age. ) shows the results of measuring the body weight.
Figure 5 shows a photograph comparing the size of a 40-week-old normal mouse (WT) and a Neu2- deficient mouse (N2KO).
Figure 6 shows the results of the Rotarod exercise ability test on normal mice (WT) and Neu2- deficient mice (N2KO) at 10, 15, 25, and 45 weeks of age.
Figure 7 shows the results of comparing motor learning ability by repeatedly performing the Rotarod motor ability test in 25-week-old normal mice (WT) and Neu2- deficient mice (N2KO).
Figure 8 shows the results of the Treadmill exercise ability test on normal mice (WT) and Neu2- deficient mice (N2KO) of the same week of age.
Figure 9a shows the results of oxygen consumption rate measurement in the indirect calorimetry test performed on normal mice (WT) and Neu2- deficient mice (N2KO) of the same week of age, and Figure 9b shows the analysis of carbon dioxide generation rate and energy consumption rate in the indirect calorimetry test. Shows the results.
Figure 10 shows the results of monitoring triglycerides in the serum of 10/15/25 week old normal mice (WT) and Neu2- deficient mice (N2KO).
Figure 11 shows the morphological changes in the liver tissues of the two mice by performing H&E tissue staining on paraffin blocks prepared by extracting liver tissues from 10/15/25/45 week old normal mice (WT) and Neu2 deficient mice (N2KO). Indicates the observed results.
Figure 12a shows images obtained by making paraffin blocks from liver tissues of normal mice (WT) and Neu2 -deficient mice (N2KO) at 25 and 45 weeks of age and performing Masson trichrome staining, and Figure 12b shows images obtained from liver tissues of normal mice (WT) and Neu2-deficient mice (N2KO) at 25 and 45 weeks of age. The results of quantitative analysis of the degree of hepatic steatosis and liver fibrosis in 45-week-old normal mice (WT) and Neu2- deficient mice (N2KO) are shown.
Figure 13a shows immunostaining for OXPAT protein, a marker that can identify lipid droplets in the liver tissue extracted from paraffin blocks prepared by extracting liver tissue from normal mice (WT) and Neu2- deficient mice (N2KO). The images obtained through the process are shown, and Figure 13b shows the results of analyzing the content of OXPAT protein in the liver tissue.
Figure 14a shows the results of analyzing the free fatty acid content using 45-week-old mouse liver tissue homogenates (Tissue Lysates), and Figure 14b shows citrate synthase (CS), an enzyme representing the function of mitochondria in 45-week-old mouse liver tissue. Shows the activity measurement results.
Figure 15 shows a schematic diagram for extracting liver tissue from normal mice and Neu2- deficient mice and performing glycopeptide analysis using mass spectrometry.
Figure 16 shows the results of extracting liver tissues from normal mice and Neu2- deficient mice and analyzing glycopeptides using mass spectrometry.
Figure 17 shows the results of analyzing the biological processes related to the 69 glycoproteins analyzed in Figure 16 through Gene Ontology (GO) analysis.

Hereinafter, the present invention will be described in more detail.

As one specific example, the present invention provides transgenic mice lacking the Neu2 gene.

The transgenic mouse lacking the Neu2 gene provides a mouse model with the following characteristics.

(1) When preparing the mouse model, a normal diet rather than an abnormal high-fat diet is fed; In this normal dietary environment, neutral fat in the blood naturally increases and fatty liver lesions progress (that is, among the various forms of fatty liver, it refers to lesions that progress from simple fatty liver to steatohepatitis). Therefore, the mouse model can provide a model for early fatty liver disease caused by metabolic syndrome, etc.

(2) In the mouse model, the progression of non-alcoholic steatohepatitis (steatosis in liver tissue, fibrosis lesions in liver tissue, steady weight gain, etc.) was observed throughout the entire age range from 10 to 50 weeks of age. In this way, it is possible to provide an animal model of non-alcoholic steatohepatitis in the background of obesity or diabetes according to modern people's lifestyle, especially an animal model that develops cirrhosis accompanied by hyperlipidemia, fatty liver lesions, and fibrosis due to aging.

(3) The mouse model lacking Neu2 , a mouse-specific gene with low or no expression in humans among proteins belonging to the glycohydrolyzing enzyme family, can better represent human responses than existing mouse models in various drug efficacy tests. You can.

In one embodiment of the present invention, the Neu2- deficient transgenic mouse of the present invention is characterized by an increase in triglycerides in the blood.

The mouse used to produce the transgenic mouse in the present invention can be of any known type that can be appropriately selected and used by a person skilled in the art.

In one embodiment, C57BL/6J mice can be used as mice used to produce transgenic mice.

The Neu2 gene is located on the first chromosome of mice and encodes the sialidase (also referred to as neuraminidase) protein. To date, four sialidase proteins (Neu1, Neu2, Neu3, and Neu4) are known as mammalian sialidase. However, it is known that Neu1, Neu3 and Neu4 proteins are expressed in the brain, whereas Neu2 protein is not expressed in the brain. Mice lacking the Neu1 , Neu3 and/or Neu4 genes are known to be used as models for neurological diseases (PLoS One. 2015 Nov 16;10(11):e0143218. FASEB J. 2017 Aug;31(8):3467 -3483). It is not known whether Neu1 to Neu4 deletions are associated with dyslipidemia. In addition, it is known that three isoforms related to Neu2 exist: silaidase-2 isoform 1, sialidase-2 isoform 2, and sialidase-2 isoform 3.

In one embodiment of the present invention, the Neu2 gene may be NCBI Gene: 23956.

In another embodiment of the present invention, the Neu2 gene deletion may occur in the exon region of the Neu2 gene.

Deletion of the Neu2 gene can be artificially created by nuclease. For example, deletion of the Neu2 gene may use one or more of the following nucleases.

(a) zinc finger nuclease (ZFN);

(b) transcription activator-like effector nuclease (TALEN);

(c) meganuclease; or

(d) RNA-Guided Endonuclease (RGEN)

As an example, deletion of the Neu2 gene can be artificially engineered by RNA-Guided Endonuclease (RGEN).

A representative embodiment of the present invention is to introduce sequence-induced double-strand breaks by introducing components of the CRISPR system, including the CRISPR-related nuclease Cas9 and sequence-specific guide RNA (gRNA), into mouse fertilized egg cells. and inducing said cleavage using the ability of the CRISPR system to induce.

Components of the CRISPR system, including the CRISPR-related nuclease Cas9 and sequence-specific guide RNA (gRNA), can be introduced into cells as encoded on one or more vectors, such as plasmids.

The CRISPR/Cas9 system includes transcripts and other elements involved in inducing the expression or activity of Cas genes. The CRISPR/Cas9 system may be a Type I, Type II, or Type III system. The methods and compositions disclosed herein utilize the CRISPR/Cas9 system by utilizing the CRISPR complex (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-specific cleavage of nucleic acids.

In one embodiment of the present invention, deletion of the Neu2 gene can be achieved by guide RNA and Cas9 protein corresponding to the target site in the exon region of the Neu2 gene. The sequence of the target site within the exon region of the Neu2 gene may be one or more regions of the nucleic acid sequence of the Neu2 gene.

According to another aspect of the technology disclosed by the present application, the base sequence of the target site of the Neu2 gene may be SEQ ID NO: 1.

The guide RNA may be, but is not limited to, nucleotides of 18 to 25 bp, 18 to 24 bp, 18 to 23 bp, 19 to 23 bp or 20 to 23 bp.

For example, the following guide RNA may be provided:

Additionally, Cas proteins typically contain at least one RNA recognition or binding domain. This domain can interact with guide RNA (gRNA, described in more detail below).

Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Casl0d, CasF , CasG, CasH, Csy1, Csy2, Csy3, Cse1(CasA), Cse2(CasB), Cse3(CasE), Cse4(CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1 and Cu1966, and their homologs or variants. A modified version may be included.

Cas proteins can be derived from type II CRISPR/Cas systems. For example, a Cas protein may be a Cas9 protein or may be derived from a Cas9 protein.

Cas proteins can also be fusion proteins. For example, a Cas protein can be fused to a cleavage domain, epigenetic modification domain, transactivation domain, or transcriptional repressor domain. The fused domain or heterologous polypeptide can be located at the N terminus, C terminus, or internal to the Cas protein.

Additionally, Cas proteins can be fused to heterologous polypeptides. Heterologous peptides include, for example, nuclear localization signals (NLS) such as SV40 NLS for targeting the nucleus, mitochondrial localization signals for targeting mitochondria, ER retention signals, etc. These signals can be located at the N terminus, C terminus, or anywhere within the Cas protein.

Cas proteins can also be linked to a cell permeable domain. For example, the cell-penetrating domain is derived from the HIV-1 TAT protein, the TLM cell-penetrating motif from human hepatitis B virus, MPG, Pep-1, VP22, a cell-penetrating peptide from herpes simplex virus, or a polyarginine peptide sequence. It can be.

Cas proteins may also contain heterologous polypeptides, such as fluorescent proteins, purification tags, or epitope tags to facilitate tracking or purification.

Cas proteins can be provided in any form. For example, Cas protein may be provided in the form of a protein such as Cas protein complexed with gRNA. Alternatively, the Cas protein may be provided in the form of a nucleic acid encoding the Cas protein, such as RNA (e.g., messenger RNA (mRNA)) or DNA. Optionally, the nucleic acid encoding the Cas protein can be codon optimized for efficient translation into the protein in a particular cell or organism.

A nucleic acid encoding a Cas protein can be stably integrated into the genome of a cell and can be operably linked to a promoter that is active in the cell. Alternatively, the nucleic acid encoding the Cas protein can be operably linked to the promoter of the expression construct.

In another embodiment of the invention, the present invention can provide artificially engineered cells or embryos comprising one or more of the artificially engineered Neu2 gene and the products expressed therefrom. The cell or embryo may develop into an entity characterized by a deletion of the Neu2 gene in the nucleic acid sequence.

“Guide RNA” or “gRNA” includes an RNA molecule that binds to a Cas protein and targets the Cas protein to a specific location within the target DNA. The guide RNA may include two segments, a “DNA targeting segment” and a “protein binding segment”. “Segment” includes a segment, section, or region of a molecule, such as a consecutive stretch of nucleotides in RNA. Some gRNAs contain two separate RNA molecules, crRNA, the “activator RNA,” and tracrRNA, the “targeter RNA.” Other gRNAs are single RNA molecules (single RNA polynucleotides), also referred to as “single molecule gRNA”, “single guide RNA” or “sgRNA”.

crRNA and the corresponding tracrRNA hybridize to form gRNA. crRNA provides a single-stranded DNA targeting segment that hybridizes to the CRISPR RNA recognition sequence. If used for modification within cells, the complete sequence of a given crRNA or tracrRNA molecule can be designed to be specific for the type of RNA molecule for which it will be used.

The DNA targeting segment (crRNA) of a given gRNA contains a nucleotide sequence complementary to the sequence of the target DNA.

The DNA targeting segment of the gRNA interacts sequence-specifically with the target DNA through hybridization (i.e., base pairing). As such, the nucleotide sequence of the DNA targeting segment can vary and determines the location within the target DNA at which the gRNA and target DNA will interact. The DNA targeting segment of the gRNA of interest can be modified to hybridize to the desired sequence in the target DNA.

The DNA targeting segment can be from about 12 nucleotides to about 100 nucleotides in length. For example, the DNA targeting segment can be about 12 nucleotides (nt) to about 80 nt, about 12 nt to about 50 nt, about 12 nt to about 40 nt, about 12 nt to about 30 nt. , may have a length of from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, or from about 12 nt to about 19 nt.

The tracrRNA can be in any form (e.g., full-length tracrRNA or active partial tracrRNA) and of various lengths.

The complementarity ratio between the DNA targeting sequence in the target DNA and the CRISPR RNA recognition sequence is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) %, at least 97%, at least 98%, at least 99%, or 100%).

The guide RNA may have additional desirable characteristics, such as modified or regulated stability; intracellular targeting; tracking using fluorescent labels; It may include modifications or sequences that provide binding sites for proteins or protein complexes.

Examples of such modifications include, for example, 5' caps (e.g., 7-methylguanylate cap (m7G)); 3' polyadenylated tail (i.e., 3' poly(A) tail); riboswitch sequences (e.g., allowing for regulated stability and/or regulated accessibility by proteins and/or protein complexes); stability control sequence; dsRNA may include a sequence that forms a double helix structure (i.e., hairpin), etc.

Guide RNA may be provided in any form. For example, the gRNA can be provided in the form of RNA as two molecules (isolated crRNA and tracrRNA) or as one molecule (sgRNA) and optionally in complex with a Cas protein. gRNA can also be provided in the form of DNA encoding RNA. DNA encoding a gRNA may encode a single RNA molecule (sgRNA) or separate RNA molecules (e.g., isolated crRNA and tracrRNA).

DNA encoding a gRNA can be stably integrated into the genome of a cell and can be operably linked to a promoter that is active in the cell. Alternatively, the DNA encoding the gRNA can be operably linked to the promoter of the expression construct.

In one embodiment of the present invention, mice deficient in Neu2 are produced by a method for producing transgenic mice including the step of deleting the Neu gene using a nuclease.

In another embodiment of the invention, mice deficient in Neu2 are prepared by performing the following sequential steps:

(a) mixing guide RNA (gRNA) corresponding to the target site of the Neu2 gene with Cas protein;

(b) treating a mouse fertilized egg with a mixture of gRNA and Cas protein;

(c) implanting the fertilized egg of the mouse into a surrogate mouse;

(d) selecting a mouse with a deletion of the Neu2 gene among mice given birth by a surrogate mother mouse; and

(e) Establishing transgenic mice lacking Neu2 through crossbreeding between mice lacking the Neu2 gene.

The base sequence of the target region of the Neu2 gene can be used without limitation as long as it is a base sequence that is common to isoforms of the Neu2 gene. In particular, the base sequence of the target region of the Neu2 gene may be the base sequence of SEQ ID NO: 1.

Any known genetic testing method can be used without limitation to determine whether the Neu2 gene is defective in the mouse.

The transgenic mouse lacking the Neu2 gene according to the present invention is capable of producing offspring through mating and has the advantage of easy reproducibility as it is possible to transmit the genetic characteristics to future generations.

In another embodiment of the present invention, prevention of diseases related to lipid metabolism dysfunction, comprising administering a drug candidate for the prevention or treatment of diseases related to lipid metabolism dysfunction to the transgenic mouse lacking the Neu2 gene of the present invention. Alternatively, a method for evaluating therapeutic drug candidates is provided. At this time, the disease related to lipid metabolism dysfunction may be selected from diabetes, arteriosclerosis, hypertension, obesity, hyperlipidemia, fatty liver, hepatitis, stroke, and heart disease, but is not limited thereto.

For example, the animal model according to the present invention can be used in research necessary to treat or prevent diseases related to lipid metabolism dysfunction, for example, as a method of screening test drugs.

As one specific example, the screening method of the present invention

1) administering candidate substances and/or therapeutic candidates for preventing diseases related to lipid metabolism dysfunction to transgenic mice lacking the Neu2 gene;

2) After administration of the candidate substance, it may include analyzing the tissue of the mouse by comparing it with a control group that did not administer the candidate substance.

The candidate material is preferably, but not limited to, any one selected from the group consisting of peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma. The compound may be a new compound or a well-known compound. These candidate substances may form salts.

Methods for administering the above candidate substances include, for example, oral administration, intravenous injection, subcutaneous administration, intradermal administration, or intraperitoneal administration, and can be appropriately selected according to the symptoms of the target animal, the properties of the candidate substance, etc. Additionally, the dosage of the candidate substance can be appropriately selected depending on the administration method or the properties of the candidate substance.

In one embodiment of the present invention, the present invention provides cells derived from the transgenic mouse.

In another embodiment of the present invention, the cell may be a cell derived from a fertilized egg of the mouse or an embryo created therefrom.

Example

Hereinafter, the present invention will be explained in more detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited to these only.

Experimental method 1. Rotarod motor ability test

The Rotarod motor ability test is a test that measures the mouse's instantaneous muscle strength and motor balance abilities through exercises that mainly use fast twitch muscles.

This test was repeated three times a day for five consecutive days, and mice were acclimatized by running a similar experimental protocol on the same machine.

This test uses a Rotarod device (BS Technolab, Korea) with five 3 cm diameter rods connected to each other. In the Rotarod device, the rod is rotated by accelerating from 4 rpm to 50 rpm for 5 minutes, then the rod is steadily rotated at 50 rpm and the time until the mouse loses balance on the rod and falls to the floor is measured.

Experimental method 2. Treadmill exercise ability test

The Treadmill exercise ability test is a test that measures the endurance of mice through exercises that mainly use slow twitch muscles.

This test is repeated weekly, the incline angle of the treadmill is maintained at 10 degrees, and the frequency and intensity of the electric shock are set to 2Hz and 1.22mA, respectively. The “fatigue area” is set to the same length as the electrical stimulation lead, and before the motor ability test, the test mouse is trained to become accustomed to the treadmill by gradually increasing the speed from 3 m/min to 5 m/min for 10 minutes. do.

During this test, the mouse subject to the test is asked to exercise at a speed of 5 m/min for 10 minutes, and the speed is increased by 1 m/min every 2 minutes. Afterwards, the maximum speed is maintained at 12 m/min and the test is performed.

A “fatigue state” is defined as when a mouse exercising on a treadmill falls behind and stays in the fatigue zone for more than 5 seconds, and the time until the mouse exercising on a treadmill reaches a fatigue state is measured.

Experiment Method 3. Indirect Calorimetry Test

The indirect calorimetry test is a basal metabolic rate measurement test that analyzes the respiratory exchange rate (RER) by measuring the concentration of gases related to breathing in mice. It is a test that can predict obesity/diabetes and metabolic diseases in mouse models.

In this test, each mouse is placed in a metabolic cage (OxyletPro System-Physiocage (Panlab, Harvard Apparatus, USA)), and oxygen consumption (VO ₂ ) and carbon dioxide production (VCo ₂ ) are measured and recorded at 20-minute intervals.

The RQ ratio is calculated by dividing oxygen consumption by carbon dioxide production, and the energy expenditure (EE) is corrected by applying the value of 0.75 square body weight.

Example 1. Neu2 Preparation of knockout mice (N2KO)

C57BL/6J mice were selected as mice for the production of Neu2- deficient mice.

Exons commonly held by the three Neu2 genes so that all three Neu2 gene isoforms (silaidase-2 isoform 1, sialidase-2 isoform 2, and sialidase-2 isoform3) known to date can be removed with a single gene edit. was selected as the target site for gene editing.

Guide RNA (Toolgen, Korea) corresponding to the selected target site of gene editing and Cas9 protein (Toolgen, Korea) were mixed and injected into fertilized eggs of C57BL/6J mice. The fertilized egg was implanted into a female ICR mouse, which was a surrogate mother mouse. A genotype test was performed on the offspring mice born to the surrogate ICR mouse, and only the offspring mice in which gene editing occurred were selected. Through crossbreeding between the selected offspring mice, homozygotes mice with a deletion of the Neu2 gene were finally produced. .

The normal mouse (WT) used in the following experimental examples is a normal C57BL/6J mouse.

Experimental Example 1. Neu2 Verification of inhibition of Neu2 expression in muscle and liver tissues of knockout mice

To verify whether Neu2 expression is suppressed in the muscle and liver tissues of mice by Neu2 gene deletion, muscle and liver tissues were extracted from the Neu2- deficient mouse (N2KO) and normal mouse (WT) in Example 1, and for each tissue Neu2 immunostaining and Sambucus nigra lectin (SNL) staining were performed respectively to confirm the presence or absence of Neu2 protein (Neu2 immunostaining) and sialylated Glycoconjugates (glycoproteins and glycolipids) (SNL staining) in each tissue.

For the Neu2 immunostaining, FFPE (formalin-fixed, paraffin-embedded) tissue blocks were cut to a thickness of 4 μm and paraffin was removed by soaking in 100% xylene, 100% EtOH, 95% EtOH, 85% EtOH, 75% EtOH. Rehydration was carried out step by step. For immunostaining, the tissue was immersed in citrate buffer (pH 6.0) and heated at 121°C for 3 minutes to induce antigen retrieval. Endogenous peroxidase activity was blocked by leaving in 0.3% hydrogen peroxide at room temperature for 10 minutes, and then washed with PBS buffer. Afterwards, the tissue was treated with a serum-free blocking solution at room temperature for 20 minutes to block non-specific antibody binding, and reacted with an antibody against Neu2 overnight at 4°C. After antibody reaction, the cells were washed with PBS buffer, reacted with HRP-labeled Rabbit Ig antibody for 30 minutes, and then washed with PBS buffer. To develop Neu2 protein, the reaction was performed in VECTASTAIN ABC reagent for 30 minutes, then developed with DAB solution and washed with PBS buffer. To stain cell nuclei, they were stained with Mayer's hematoxylin for 30 seconds and washed with PBS buffer. The tissue was dehydrated step by step in 75% EtOH, 85% EtOH, 95% EtOH, and 100% EtOH and then immersed in 100% xylene to make it transparent. Permount was added onto the stained tissue and a cover glass was placed to complete the tissue sample.

For SNL staining, FFPE (formalin-fixed, paraffin-embedded) tissue blocks were cut to a thickness of 4 μm, soaked in 100% xylene to remove paraffin, and then incubated step by step in 100% EtOH, 95% EtOH, 85% EtOH, and 75% EtOH. It was rehydrated. For immunostaining, the tissue was immersed in citrate buffer (pH 6.0) and heated at 121°C for 3 minutes to induce antigen retrieval. Endogenous peroxidase activity was blocked by standing in 0.3% hydrogen peroxide for 10 minutes at room temperature, and then washed with PBS buffer. Afterwards, the tissue was reacted with Sambucus nigra lectin (SNL) reagent at 4°C overnight and washed with PBS buffer. For SNL color development, after reacting with VECTASTAIN ABC reagent for 30 minutes, color was developed with DAB solution and washed with PBS buffer. To stain cell nuclei, they were stained with Mayer's hematoxylin for 30 seconds and washed with PBS buffer. The tissue was dehydrated step by step in 75% EtOH, 85% EtOH, 95% EtOH, and 100% EtOH and then immersed in 100% xylene to make it transparent. Permount was added onto the stained tissue and a cover glass was placed to complete the tissue sample.

The results of mouse tissue staining analysis performed as above are shown in Figure 1. As can be seen from Figure 1, Neu2 protein expression was decreased in muscle and liver tissue of Neu2 -deficient mice (IHC: Anti-Neu2), and expression of sialylated Glycoconjugates was increased (SNL Staining).

Experimental Example 2. Neu2 Analysis of changes in mouse muscle tissue due to genetic deletion

Experimental Example 2-1. Analysis of morphological changes in muscle tissue

In order to analyze changes in the shape of mouse muscle tissue due to Neu2 gene deletion, paraffin blocks of muscle (EDL) tissue extracted from a 25-week-old normal mouse (WT) and the Neu2- deficient mouse (N2KO) of Example 1 were prepared and then paraffinized. Microsections were made and hematoxylin-eosin (H&E) tissue staining was performed.

For H&E tissue staining, FFPE (formalin-fixed, paraffin-embedded) tissue blocks were cut into 4μm-thick pieces, soaked in 100% xylene to remove paraffin, and then washed in 100% EtOH, 95% EtOH, 85% EtOH, and 75% EtOH. Rehydration was carried out step by step. To stain cell nuclei, they were stained with hematoxylin for 1 minute, soaked in 1% hydrogen chloride solution, drained, and washed with PBS buffer. Color was developed by soaking in 1% ammonia solution and washed with PBS buffer. To stain the cytoplasm, the cells were stained with Eosin for 2 minutes and washed with PBS buffer. The tissue was dehydrated step by step in 75% EtOH, 85% EtOH, 95% EtOH, and 100% EtOH, and then immersed in 100% xylene to make it transparent. Permount was added onto the stained tissue and a cover glass was placed to complete the tissue sample.

The results of analyzing the morphological changes in muscle tissue on which H&E staining was performed as described above are shown in Figure 2a. As can be seen from Figure 2a, it was observed that the shape of muscle tissue of 25-week-old Neu2- deficient mouse (N2K) was different from that of normal mouse (WT).

In addition, in order to analyze the difference in changes in tissue shape, the entire tissue was imaged using Whole Slide Scanner equipment and quantitative analysis was performed. Specifically, the area of the perimysium was quantitatively analyzed using the positive pixel counting algorithm, and to measure the average myocyte size, the area of the myofiber area was divided by the number of myocytes. The results are shown in Figure 2b. As can be seen from Figure 2b, compared to normal mice (WT), the area of the myocardium in Neu2- deficient mice (N2KO) increased 5.7-fold, and the average size of muscle cells decreased 0.7-fold.

Considering these results, it was confirmed that Neu2 gene deletion inhibits muscle cell growth and development.

Experimental Example 2-2. Myofiber differentiation and development inhibition assay

To investigate whether Neu2 gene deletion leads to inhibition of muscle fiber differentiation and development, immunostaining of myoblast crystal protein (MyoD), a key factor in muscle fiber differentiation, was performed. MyoD protein is a transcription protein that regulates muscle differentiation. It is expressed in myoblasts and its expression is reported to decrease as muscle fibers (Myofibers) grow.

For MyoD protein immunostaining, FFPE (formalin-fixed/paraffin-embedded) tissue blocks were cut to a thickness of 4 μm and then immersed in 100% xylene to remove paraffin, followed by 100% EtOH, 95% EtOH, 85% EtOH, and 75% EtOH. Rehydration was carried out step by step. For immunostaining, the tissue was immersed in citrate buffer (pH 6.0) and heated at 121°C for 3 minutes to induce antigen retrieval. Endogenous peroxidase activity was blocked by leaving in 0.3% hydrogen peroxide at room temperature for 10 minutes, and then washed with PBS buffer. Afterwards, in order to block non-specific reactions between tissues and antibodies, treatment was performed with serum-free blocking solution and endogenous mouse Ig blocking reagent at room temperature for 20 minutes, followed by incubation with antibodies against MyoD at 4°C overnight. reacted. After antibody reaction, it was washed with PBS buffer and reacted with HRP-labeled Mouse IgG antibody for 30 minutes, then washed with PBS buffer. To develop Neu2 protein, the reaction was carried out in VECTASTAIN ABC reagent for 30 minutes, followed by color development with DAB solution and washing with PBS buffer. To stain cell nuclei, they were stained with Mayer's hematoxylin for 30 seconds and washed with PBS buffer. The tissue was dehydrated step by step in 75% EtOH, 85% EtOH, 95% EtOH, and 100% EtOH, and then immersed in 100% xylene to make it transparent. Permount was added onto the stained tissue and a cover glass was placed to complete the tissue sample.

The results of immunostaining of MyoD protein as described above are shown in Figures 3a and 3b. As can be seen from Figures 3a and 3b, the number of cells expressing MyoD in the muscle tissue of Neu2- deficient mice (N2KO) was 5.6 times greater than that of normal mice (WT). These results are interpreted to mean that the differentiation of myoblasts into muscle fibers was suppressed due to Neu2 gene deletion, and as a result, the number of cells positive for MyoD expression was maintained.

Experimental Example 3. Neu2 Phenotypic analysis through behavioral analysis of defective mice

Experimental Example 3-1. Body Weight Measurement

To investigate the effect of Neu2 gene deletion on body weight, the body weight of normal mice (WT) and Neu2 deletion mice (N2KO) was measured from 6 to 16 weeks of age and at 50 weeks of age, and the results are shown in Figures 4a and 4. Shown in 4b. Additionally, a photo comparing the sizes of two 40-week-old mice is shown in Figure 5.

In the case of Neu2 knockout mice (N2KO), a significant increase in body weight was observed after 10 weeks of age compared to normal mice (WT), and a maximum weight gain of 10% was observed in mice at 16 weeks of age (see Figure 4a). In addition, this weight gain phenomenon was consistently observed even after 40 weeks of age (see Figure 5), and 50-week-old Neu2- deficient mice (N2KO) were confirmed to have increased body weight by more than 30% compared to normal mice (WT) born from the same mother ( see Figure 4b).

Experimental Example 3-2. Rotarod motor skills test

According to the procedure described in Experimental Method 1, the Rotarod exercise ability test was performed on 10-, 15-, 25-, and 45-week-old normal mice (WT) and Neu2- deficient mice (N2KO) three times a day for 3 days. did. The results are shown in Figure 6.

As can be seen from Figure 6, the motor ability of Neu2 knockout mice (N2KO) is reduced by 15% at 10 weeks of age, 19% at 15 weeks of age, 26% at 25 weeks of age, and 38% at 45 weeks of age compared to normal mice (WT). This indicates that the exercise capacity of Neu2- deficient mice (N2KO) tends to decrease as the age increases compared to normal mice (WT). It can be assumed that these results are caused by the inhibition of muscle growth and obesity due to the Neu2 gene defect mentioned above.

Interestingly, in normal mice (WT), exercise capacity tended to increase with age up to 25 weeks of age, whereas exercise capacity did not increase in Neu2- deficient mice (N2KO).

Additionally, in order to confirm the effect of motor learning ability, additional analysis was performed by repeating the procedure described in Experimental Method 1 on 25-week-old mice. That is, the Rotarod exercise ability test was repeated 6 times a day for 3 days in 25-week-old normal mice (WT) and Neu2- deficient mice (N2KO). The results are shown in Figure 7. As can be seen from Figure 7, the exercise capacity of normal mice (WT) increased by 20% on the 3rd day compared to the exercise capacity on the 1st day, while the Neu2 knockout mouse (N2KO) showed no significant difference in exercise capacity. These results suggest that Neu2 knockout mice (N2KO) not only show reduced motor skills but also show reduced motor learning skills.

Experimental Example 3-3. Treadmill exercise test

According to the procedure described in Experimental Method 2, the Treadmill exercise ability test was performed on normal mice (WT) and Neu2- deficient mice (N2KO) of the same age (15 and 45 weeks of age) once a week for 5 weeks. The results are shown in Figure 8.

As can be seen from Figure 8, compared to normal mice (WT), Neu2- deficient mice (N2KO) at 15 and 45 weeks of age showed a 15% decrease in exercise capacity, and there was no difference in exercise ability by week of age.

Experimental Example 3-4. Indirect Calorimetry Test

According to the procedure described in Experimental Method 3, an indirect calorimetry test was performed on normal mice (WT) and Neu2- deficient mice (N2KO) of the same age (25 and 45 weeks of age). The metabolic rate of each mouse was analyzed by calculating measurements for 4 consecutive days. The results are shown in Figures 9a and 9b.

As can be seen from Figures 9A and 9B, compared to normal mice (WT), Neu2- deficient mice (N2KO) showed a tendency to decrease the oxygen consumption rate, carbon dioxide generation rate, and energy consumption rate at both 25 and 45 weeks of age. In addition, all metabolic capacity measurements tended to decrease more at 45 weeks of age than at 25 weeks of age. Energy consumption rate was analyzed to decrease by up to 9% at 25 weeks of age and up to 21% at 45 weeks of age.

Experimental Example 4. Neu2 Analysis of the effects of genetic deletion on mouse liver tissue

Experimental Example 4-1. Increase in neutral fat in the blood

Triglycerides (TG) were monitored in the serum of 10/15/25 week old normal mice (WT) and Neu2 knockout mice (N2KO). Mouse serum was coagulated for 15 minutes at room temperature, then centrifuged at 1,000 MA, USA), and all reagents and procedures were performed according to the manufacturer's protocol.

Figure 10 shows the TG monitoring results.

As can be seen from Figure 10, the level of neutral fat was significantly increased in Neu2 knockout mice (N2KO) compared to normal mice (WT) from 10 to 25 weeks of age. These results suggest that Neu2 knockout mice (N2KO) may have a higher risk of hyperlipidemia, diabetes, and arteriosclerosis due to increased neutral fat compared to normal mice (WT). Therefore, related lesions were additionally observed in liver tissue.

Experimental Example 4-2. Onset of fatty liver lesions

Liver tissues were extracted from 10/15/25/45 week old normal mice (WT) and Neu2 -deficient mice (N2KO), and H&E tissue staining was performed on the extracted liver tissues in the same manner as in Experimental Example 2-1, and the two mice were Morphological changes in liver tissue were observed. The results are shown in Figure 11.

As can be seen from Figure 11, steatotic lesions were observed in the liver tissue of Neu2- deficient mice over 45 weeks of age. Interestingly, triglyceride levels were increased in Neu2 knockout mice (N2KO) at all ages from 10 to 25 weeks of age, but steatosis in liver tissue was observed in mice older than 45 weeks of age. In the case of Neu2 knockout mice (N2KO), it is assumed that steatosis lesions occurred in liver tissue due to an imbalance between excessive inflow and outflow of increased triglycerides in serum.

Experimental Example 4-3. Fibrotic lesions in liver tissue

Since non-alcoholic fatty liver disease (steatosis) is generally divided into simple fatty liver and cirrhosis accompanied by fibrosis, to determine whether fatty liver of Neu2 knockout mice (N2KO) is accompanied by fibrosis, 25- and 45-week-old mice were tested. Paraffin blocks were prepared from liver tissues of normal mice (WT) and Neu2- deficient mice (N2KO), and Masson trichrome staining was performed.

For Masson's trichrome staining, FFPE (formalin-fixed, paraffin-embedded) tissue blocks were cut into 4μm-thick pieces, soaked in 100% xylene to remove paraffin, and then incubated step by step in 100% EtOH, 95% EtOH, 85% EtOH, and 75% EtOH. It was rehydrated. For adhesion, the slide was soaked in Bouin's solution preheated to 56°C for 15 minutes, then sufficiently cooled and washed with tap water. It was stained with Weigert's Iron Hematoxylin solution for 5 minutes and washed with deionized water (DW). They were then stained with Biebrich Scarlet-Acid Fucshin solution for 5 minutes and washed with deionized water (DW). The stained tissue was immersed in Phosphotungstic/Phosphomolybdic Acid solution and Aniline Blue solution for 5 minutes each, and then in acetic acid (1%) solution for 2 minutes. Subsequently, it was dehydrated step by step in 75% EtOH, 85% EtOH, 95% EtOH, and 100% EtOH, and then immersed in 100% xylene to make it transparent. Permount was added onto the stained tissue and a cover glass was placed to complete the tissue sample. Masson's trichrome staining was performed using StatLab's Trichrome Stain Kit, and all reagents and procedures were performed according to the manufacturer's protocol.

The results of Masson's trichrome staining for mouse liver tissue are shown in Figures 12A and 12B. As a result of analyzing the image in Figure 12a, it was confirmed that steatosis in the liver tissue of 45-week-old Neu2- deficient mice (N2KO) increased more than 9-fold and fibrosis increased more than 2.8-fold compared to normal mice (WT) (Figure 12b). At this time, some fibrosis was observed in the liver tissue of a 45-week-old normal mouse (WT), but this could be seen as fibrosis due to mouse aging. In addition, steatosis and fibrotic lesions were not observed in 25-week-old mouse liver tissue stained under the same conditions, and the image analysis results also showed no significant differences (FIGS. 12a and 12b).

Experimental Example 4-4. Increased lipid droplet marker (OXPAT) in liver tissue

Fatty liver can be caused by an imbalance between the inflow and outflow of neutral fat in the blood. The causes of this imbalance are lipid metabolism disorders, continuous influx of excessive free fatty acids, dysfunction of mitochondria responsible for fatty acid oxidation, and excessive fatty acid production in liver cells. etc. may be the cause. Based on this, in order to determine the cause of fatty liver observed in Neu2 -deficient mice (N2KO), immunostaining for OXPAT protein, a marker that can identify lipid droplets in liver tissue, was performed. OXPAT is reported to be a protein that binds to fat droplets and plays a role in delivering fatty acids to mitochondria.

For OXPAT immunostaining, FFPE (formalin-fixed, paraffin-embedded) tissue blocks were cut into 4-μm-thick pieces, soaked in 100% xylene to remove paraffin, and incubated step by step in 100% EtOH, 95% EtOH, 85% EtOH, and 75% EtOH. It was rehydrated. For immunostaining, the tissue was immersed in citrate buffer (pH 6.0) and heated at 121°C for 3 minutes to induce antigen retrieval. Endogenous peroxidase activity was blocked by leaving in 0.3% hydrogen peroxide at room temperature for 10 minutes, and then washed with PBS buffer. Afterwards, the tissue was treated with a serum-free blocking solution at room temperature for 20 minutes to block non-specific antibody binding, and reacted with an antibody against OXPAT overnight at 4°C. After antibody reaction, it was washed with PBS buffer, and after 30 minutes of reaction with HRP-labeled Rabbit IgG antibody, it was washed with PBS buffer. To develop OXPAT protein, the reaction was carried out in VECTASTAIN ABC reagent for 30 minutes, followed by color development with DAB solution and washing with PBS buffer. To stain cell nuclei, they were stained with Mayer's hematoxylin for 30 seconds and washed with PBS buffer. The tissue was dehydrated step by step in 75% EtOH, 85% EtOH, 95% EtOH, and 100% EtOH, and then immersed in 100% xylene to make it transparent. Permount was added onto the stained tissue and a cover glass was placed to complete the tissue sample.

OXPAT immunostaining results are shown in Figures 13a and 13b.

As can be seen from Figure 13a, a quantitative increase in OXPAT protein was observed in the liver tissue of 25-week-old and 45-week-old Neu2- deficient mice (N2KO). As a result of analyzing the image in Figure 13a, compared to normal mice (WT), 25-week-old Neu2- deficient mice (N2KO) showed a quantitative increase of 1.8-fold, and 45-week-old Neu2- deficient mice (N2KO) showed a quantitative increase of 1.4-fold (Figure 13b) ).

Experimental Example 4-5. Increased free fatty acid in liver tissue and decreased mitochondrial CS enzyme activity

In order to investigate the quantitative changes in free fatty acid (Free Fatty Acid) and mitochondrial function in liver tissue due to Neu2 deletion, the free fatty acid content and the function of mitochondria as a major enzyme were examined using 45-week-old mouse liver tissue homogenates (Tissue Lysates). The activity of a representative citrate synthase (CS) was measured.

To measure free fatty acid content, quick-frozen tissue was washed with PBS buffer solution, 200 ㎕ of chloroform-Triton X-100 was added, and homogenized on ice. The homogenized sample was centrifuged at maximum speed in a microcentrifuge for 10 minutes to recover only the organic phase (lower phase), dried at 50°C to remove chloroform, and then completely removed in a vacuum dryer for 30 minutes. Samples were prepared by adding 200 ㎕ of free fatty acid assay buffer to the dried fatty acid sample and shaking for 5 minutes. 50㎕ of the prepared sample was placed in a 96-well plate, 2㎕ of ACS reagent was added, light was blocked, and reaction was performed at 37°C for 30 minutes. Add 50㎕ of the prepared reaction reagent, mix evenly, block light, react at 37°C for 30 minutes, and measure at 570nm using Mithras (Berthold) equipment. The measured absorbance value was quantitatively calculated using the Standard Curve. Free fatty acid was measured using DoGenBio's EZ-Free Fatty Acid Assay Kit, and all reagents and procedures were performed according to the manufacturer's protocol.

To measure CS activity, quick-frozen tissues were washed with PBS buffer and lysed with 10 times the tissue volume of lysis reagent (25mM Tris-HCl pH 7.4, 150mM NaCl, 1% NP-40, 1% Na-deoxycholate, 0.1% SDS, 1mM Na ₃ VO ₄ , 10mM NaF, 0.4mM PMSF, Protease inhibitor, phosphatase inhibitors) were homogenized using liquid nitrogen and a mortar. The homogenized sample was centrifuged at 13,000 xg for 10 minutes at 4°C, and only the supernatant was collected and used to measure CS activity. Samples and CS analysis reagents were stored in an ice bucket before use. 50㎕ of tissue sample was placed in a 96-well plate, and 50㎕ of reaction reagent (Assay Buffer, Developer, Substrate Mix mixed reagent) was added and immediately measured using Mithras (Berthold) equipment in kinetic mode at 412nm for 30 minutes. The measured absorbance value was used to calculate activity using the Standard Curve and CS activity quantification formula. CS activity was measured using Biovision's Citrate Synthase Activity Colorimetric Assay Kit, and all reagents and procedures were performed according to the manufacturer's protocol.

The results of measuring free fatty acid content and CS enzyme activity are shown in Figures 14a and 14b, respectively.

As can be seen from Figures 14a and 14b, compared to normal mice (WT), the amount of free fatty acid in the liver tissue of Neu2- deficient mice (N2KO) increased by 1.3-fold, and CS activity decreased by 0.7-fold.

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Combining the results of Experimental Examples 4-1 to 4-5 above, the following conclusions can be drawn: Neu2- deficient mice (N2KO) were observed to exhibit abnormal lipid metabolism, and this abnormal lipid metabolism was associated with serum triglyceride levels. You can see that it brings about an increase. In addition, increased serum neutral fat flows excessively into the liver tissue, deteriorating mitochondrial function, and the decreased mitochondrial function causes an imbalance in which the inflow of neutral fat does not flow out smoothly. As a result, it is believed that fatty liver and cirrhotic lesions occur in Neu2 knockout mice (N2KO).

Example 2. As a dyslipid disease model Neu2 Exploring the possibility of utilizing defective mice

As a result of analyzing the serum of normal mice and Neu2- deficient mice born from the same mother at 10, 15, and 25 weeks of age, it was observed that the level of neutral fat in the serum continued to increase in Neu2- deficient mice, and in Neu2- deficient mice over 45 weeks of age. Fatty liver and liver fibrosis lesions were observed in liver tissue, suggesting that Neu2- deficient mice have impaired lipid metabolism. In addition, the reduced metabolic capacity of Neu2- deficient mice compared to normal mice and the induction of obesity due to weight gain suggest that these Neu2- deficient mice have the potential as a model to study lipid metabolic syndrome, which causes hyperlipidemia, diabetes, and arteriosclerosis. do.

Specifically, to verify the utility of Neu2- deficient mice as a metabolic syndrome research model, liver tissues from normal mice and Neu2- deficient mice were extracted and glycopeptide analysis was performed using mass spectrometry. A schematic diagram for such glycopeptide analysis is shown in Figure 15, and the results of analyzing liver tissue glycopeptides in this way are shown in Figure 16.

As can be seen from Figure 16, when liver tissue glycopeptides of Neu2- deficient mice were analyzed using mass spectrometry, sialylation of 10 (14%) of the total 69 glycoproteins increased, and 31 ( 45%), the sialylation of glycoproteins decreased, and there was no change in the sialylation of 28 glycoproteins.

For the 69 glycoproteins analyzed above, the related biological processes were analyzed through Gene Ontology (GO) analysis. The results are shown in Figure 17.

As can be seen from Figure 17, lipid metabolism-related GO terms related to lipid transport, regulation of lipid metabolism, steroid metabolism, and triglyceride metabolism were analyzed. , out of a total of 69 analyzed sialylated glycoproteins, 15 (22%) sialylated glycoproteins were identified as being involved in lipid metabolic processes.

Therefore, Neu2 gene deletion resulted in sialylation changes in a wide range of lipid metabolism-related proteins. Among the 15 sialylated proteins related to lipid metabolism analyzed in Neu2- deficient mice, the eight major glycoproteins in which quantitative changes were observed are summarized in Table 1.

[Table 1] Siylated glycoproteins related to lipid metabolism identified in the liver of Neu2- deficient mice

Among the proteins summarized in Table 1, proteins such as Apolipoprotein B (ApoB), 3-Keto-steroid reductase, and Transport and Golgi organization protein 1 (TANGO1) are reported to be proteins directly involved in the transport and synthesis of lipid components in the body. In particular, according to Non-Patent Documents 2 and 3, ApoB is a lipoprotein that transports fat molecules including triglycerides and cholesterol in the body, and high concentrations of triglycerides and cholesterol and high levels of ApoB contribute to the formation of plaques that cause vascular disease (atherosclerosis). reported as the main cause. In addition, TANGO1 is known as a protein that exports large lipid particles, and according to Non-Patent Document 4, it was reported that TANGO1 protein interacts with ApoB to export large lipid particles from the endoplasmic reticulum. On the other hand, paraoxonase 1 is a protein known to be a major anti-atherosclerosis component of high-density lipoprotein (HDL). This enzyme is regulated by PPAR-γ and is secreted from the liver and is known to reduce atherosclerosis. The results of the analysis of sialylation of glycoproteins related to lipid metabolism due to Neu2 gene deletion through this example may provide important clues to understanding the mechanism of lipid metabolism disorders seen in Neu2 deficient mice.

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Claims (9)

A transgenic mouse with a defect in the Neu2 gene, which has a malfunction in lipid metabolism. The transgenic mouse according to claim 1, characterized by an increase in triglycerides in the blood. A method for producing the transgenic mouse of claim 1, comprising the step of deleting the Neu2 gene using a nuclease. (a) mixing guide RNA (gRNA) corresponding to the target site of the Neu2 gene with Cas protein;
(b) treating a mouse fertilized egg with a mixture of gRNA and Cas protein;
(c) implanting the fertilized egg of the mouse into a surrogate mouse;
(d) selecting a mouse with a deletion of the Neu2 gene among mice given birth by a surrogate mother mouse; and
(e) establishing transgenic mice lacking Neu2 through crossbreeding between mice lacking the Neu2 gene;
A method for producing a transgenic mouse comprising. The method of claim 4, wherein the base sequence of the target region of the Neu2 gene is SEQ ID NO: 1. A method of evaluating a drug candidate for the prevention or treatment of a disease related to lipid metabolism dysfunction, comprising administering a drug candidate for the prevention or treatment of a disease related to lipid metabolism dysfunction to the transgenic mouse of claim 1. The method of claim 6, wherein the disease related to lipid metabolism dysfunction is selected from diabetes, arteriosclerosis, hypertension, obesity, hyperlipidemia, fatty liver, hepatitis, stroke, and heart disease. Cells derived from the transgenic mouse of claim 1. The cell of claim 8, wherein the cell is derived from a fertilized egg or an embryo created therefrom.