KR101489844B1 - 14-3-3 sigma null mouse, development of in vivo septic shock research model using the same and use of the same - Google Patents

Abstract

The present invention relates to a 14-3-3 sigma gene-deficient mouse, a method for developing an in vivo septicemia study model and its application.

Description

14-3-3 sigma gene-deficient mouse, method for developing a research model of in vivo septicemia using the same, and application thereof {14-3-3 sigma null mouse, development of in vivo septic shock research model using the same and use of the same}

The present invention relates to a 14-3-3 sigma gene-deficient mouse, a method for developing an in vivo septicemia study model and its application.

Congenital immunity and inflammation are the primary defense mechanisms against pathogens. Abnormal regulation of innate immune and inflammatory responses responsible for these defenses is known to be responsible for a variety of immune-related diseases such as sepsis, asthma, and autoimmune diseases. In addition, if the innate immune and inflammatory responses are not precisely resolved, persistent cell damage will occur and ultimately provide an important cause of chronic diseases such as cancer and diabetes.

Thus, innate immunity and inflammation control technology is a key technology for the treatment and prevention of chronic inflammatory diseases as well as infectious diseases and immune diseases of pathogens.

Recent research trends distinguish two types of inflammatory reactions. In other words, it is classified into therapeutic inflammation involving innate immunity and pathological inflammation caused by chronic inflammatory reaction. (Aggawal et al., 1986). In this study, we investigated the pathogen-induced inflammation of the cell, which is induced by inflammatory cells. , Biochem Pharmacol., 2006).

In fact, this research direction has been accepted by researchers as having considerable relevance in terms of the pathogenesis of cell damage and disease. This concept suggests that strict regulation of the actual inflammatory response will play an important role in the prevention of disease, and therefore fundamental key proteins that are altered in response to the anti-inflammatory signal that resolves the inflammatory response or that are shifted from inflammatory to anti- Something is becoming an important issue.

14-3-3 sigma is an adapter function protein belonging to the 14-3-3 family. It is known that 14-3-3 sigma has cancer-inhibiting activity unlike other isoforms. It is also known that 14-3-3 sigma mitigates the anticancer drug resistance activity of cancer cells by inhibiting the activity of Akt.

[Related Prior Art Patent Literature]

European Patent Publication No. 1 127 942A1

The present invention has been made in view of the above needs, and an object of the present invention is to develop a research model of in vivo sepsis.

In order to accomplish the above object, the present invention provides a method for producing a transgenic mouse for the treatment of sepsis by defeating the 14-3-3 sigma gene.

In one embodiment of the present invention, the 14-3-3 sigma gene preferably has the nucleotide sequence of SEQ ID NO: 1, but it is preferable that the 14-3-3 sigma gene has at least one substitution, deletion, inversion and / or translocation mutation in the gene, All mutant genes that achieve the desired effect are also included within the scope of the present invention.

The present invention also provides a transgenic mouse for treating sepsis in which a 14-3-3 sigma gene is defective.

The present invention also relates to a method for screening a 14-3-3 sigma gene for a transgenic mouse, And measuring the survival rate of the transgenic mouse in the presence of the test substance, wherein when the survival rate of the transgenic mouse is increased in the presence of the test substance as compared with the case where the test substance is not present Wherein the test substance is a candidate substance for treating septicemia.

In one embodiment of the present invention, the candidate substance is preferably an extract or compound derived from a natural product, but is not limited thereto.

In another embodiment of the present invention, the method further comprises, but is not limited to, treating the transgenic mice with lipopolysaccharide (LPS) prior to candidate treatment.

The present invention also provides a method for screening candidate substances for the treatment of inflammation, comprising the steps of: treating a transgenic mouse lacking the 14-3-3 sigma gene as a test substance; And measuring the expression level of the inflammatory cytokine of the transgenic mouse in the presence of the test substance, wherein the inflammatory cytokine expression level of the transgenic mouse in the presence of the test substance as compared to the absence of the test substance Wherein the test substance is a candidate substance for treating inflammation when the expression level of the cancer is decreased.

In one embodiment of the invention, the inflammatory cytokine is preferably a cytokine selected from the group consisting of IL-1beta, IL-6 and Cycloxygenase-2, More preferably Cycloxygenase-2, but is not limited thereto.

In another embodiment of the present invention, the expression level of the inflammatory cytokine is preferably measured by mRNA or a tissue immunostaining method, but is not limited thereto.

The present invention also provides a kit for treating sepsis comprising a transgenic mouse in which a 14-3-3 sigma gene is deficient.

The present invention also provides a kit for the analysis of inflammatory cytokines including a transgenic mouse in which a 14-3-3 sigma gene is deleted.

The mouse of the present invention will be further described.

The present invention provides a 14-3-3 sigma knockout mammal comprising one or more cells in which the 14-3-3 sigma polypeptide is functionally inactivated. The 14-3-3 sigma knockout of the present invention is a 14-3-3 sigma knockout mammal, The functional division of the -3 sigma gene or the loss of one or more functional mutations of the 14-3-3 sigma gene and is caused as a result of a portion of the gene including deletion or replacement of the 14-3-3 sigma gene. Mutations include single point mutations and target coding or non-coding regions of 14-3-3 sigma. Preferably, such knockout animals are non-human mammals such as pigs, sheep or rodents. More preferably, the knockout animal is a mouse or rat. These knockout animals are used not only for screening procedures to identify agonists and / or antagonists of 14-3-3 sigma, but also for testing their efficacy as disease treatments in vivo.

Detailed methods for generating non-human knockout animals are described in further detail below. Transgenic constructs can be introduced into animal germ cells to produce knockout mammals. For example, one or more of the constructs are introduced into the genome of a mammalian embryo by standard transduction techniques. In an exemplary embodiment, the knockout non-human animal of the invention is transfected into the germ cells of a non- Lt; / RTI >

Embryonic target cells at various stages of initiation can be used to introduce transgenes. Other methods are used differently depending on the stage of development of embryonic target cells.

The particular line (s) of the animal used to practice the present invention is selected for general good health, good embryo production, good prothrombin time in the embryo, and good reproductive fitness. Moreover, haplotype is a significant factor.

Introduction of a transgene into an embryo can be accomplished by methods known in the art, such as, for example, injection, electroporation or lipofection. For example, a 14-3-3 sigma transgene may be introduced into mammals by microscopic injection of constructs into the nucleus of the modified mammalian embryo (s), and the cells of the mammalian (s) in which one or more copies of the construct are developed Or the like.

After introduction of the transgene construct into the fertilized egg, the embryos are re-implanted into the in vitro fertilized or surrogate host, or both, for various periods of time. Incubation in vitro into mature phase is within the scope of the present invention. One common method is to incubate the embryos in vitro for about 1 to 7 days, depending on the species, and then reimplant them into the surrogate host.

The progeny of the transgenically engineered embryo can be tested for the presence of the construct by Southern blot analysis of the tissue segment. One or more copies of the exogenously cloned construct are integrated into the same genome as the knockout embryo Once stable, it is possible to establish a permanent knockout mammalian line with transgenically added constructs.

Knockout defended mammalian offspring can be analyzed for the integration of the construct into the postnatal offspring genome. Preferably, the assay is accomplished by hybridizing a probe corresponding to the DNA sequence encoding the desired recombinant protein product or segment thereof on the chromosomal material from the offspring. These mammalian offspring, which appear to contain at least one copy of the construct in their genome, are mature.

For the purposes of the present invention, the conjugate is substantially in the form of diploid cells that can develop into complete organisms. In general, the conjugate will consist of eggs containing naturally occurring or artificially formed nuclei by fusion of two haploid nuclei from the gonads or gonads. Thus, the gonadal nuclei should be naturally compatible, that is, to induce viable zygotes capable of differentiation and development into functional organisms. In general, an electrophysiologic conjugate is preferred. When an isomeric conjugate is obtained, the number of chromosomes should not be altered by more than one to about the number of the organisms of genital origin. In addition to similar biological considerations, physical considerations may also apply to genetic material that forms part of the nucleus or junction nucleus of the conjugate (I.e., volume) of exogenous genetic material that can be added. Unless any genetic material is removed, the amount of exogenous genetic material that can be added is limited to an amount that can be absorbed unless physically disrupted. Generally, the volume of exogenous genetic material that is inserted will not exceed about 10 picoliters. The physical effect of the addition should not be large enough to physically destroy the conjugate. Since the genetic material, including the exogenous genetic material of the resulting conjugate, must be biologically capable of initiating and maintaining differentiation and development into a functional organism, the biological limitations of the number and diversity of the DNA sequence will depend on the function of the particular zygotic and exogenous genetic material And will be readily apparent to those skilled in the art.

The number of transgene construct copies added into the conjugate will depend on the total amount of exogenous genetic material added and will be an amount that allows genetic transformation to occur. Theoretically, one copy is required; In general, however, a large number of copies are used, such as, for example, 1,000-20,000 copies of transgene constructs to ensure that one copy is functional.

In the present invention, it would be advantageous to have one or more functional copies of each inserted exogenous DNA sequence to increase the phenotypic expression of exogenous DNA sequences.

Techniques that enable the addition of exogenous genetic material into the nuclear genetic material can be used as long as they do not destroy cells, nuclear membranes, or other existing cellular or dielectric constructs. Exogenous genetic material is preferentially inserted into the nuclear genetic material by microscopic injection.

Microscopic injections of cells and cell structures are known and used in the art.

Reimplantation is achieved using standard methods. Generally, the surrogate host is anesthetized and the embryo is inserted into the fallopian tubes. The number of embryos transplanted into a particular host will vary from species to species, but will generally be comparable to the number of offspring of naturally occurring species.

The knockout offspring of the surrogate host are screened for the presence and / or expression of the metastatic gene by a suitable method. Screening is accomplished by Southern blot or Northern blot analysis using a probe complementary to at least a portion of the transgene. Western blot analysis using an antibody against a protein encoded by a transgene gene is alternatively or additionally used in screening methods for the presence of a transgene product. Generally, DNA is prepared from end tissues and analyzed by Southern analysis or PCR for the transgene. Alternatively, tissues or cells that are considered to express the transgene at the highest level, even if any tissue or cell is used for this assay, are tested for the presence and expression of the transgene using Southern analysis or PCR.

Alternative or additional methods for assessing the presence of a transgene include, but are not limited to, biochemical assays such as enzymatic and / or immunological assays, histological staining for specific marker or enzyme activities, flow cytometric analysis, And the like. In addition, blood analysis is useful not only for detecting the presence of transgene products in the blood, but for evaluating the effects of transgene on the level of various types of blood cells and other blood components.

Retroviral infection can also be used to introduce a transgene into a non-human animal. Developmental non-human embryos can be cultured in vitro in blastocyst stage. During this time, the haploid may be a target for retroviral infection (Jaenich, R. (1976) PNAS 73: 1260-1264). Effective infections of the bladder are obtained by enzymatic treatment to remove the zona pellucida (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986). The viral vector system used to introduce the transgene is generally a replication-defective retrovirus that carries a transgene (Jahner et al. (1985) PNAS 82: 6927-6931; Van der Putten et al. 82: 6148-6152). Transfection is easily and efficaciously obtained by culturing the cells on monolayers of virus-producing cells (Van der Putten, supra; Stewart et al. (1987) EMBO J. 6: 383-388).

Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the recipient (Jahner et al. (1982) Nature 298: 623-628). Since integration occurs only within a subset of the cells that form the transgenic non-human animal, most crude castings will be mosaic of the transgene. The casting also typically includes a variety of retroviral infections of the metastatic gene at other locations within the genome to be isolated within the offspring. Moreover, it is possible to introduce a transgene into germ cells by intrauterine retroviral infection of the gestational middle embryo (Jahner et al. (1982) Homology).

A third type of target cell for transgene transfer is embryonic stem cells (ES). ES cells are obtained from pre-implantation embryos cultured in vitro and fused to embryos (Evans et al. (1981) Nature 292: 154-156; Bradley et al. (1984) Nature 309: 255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322: 445-448). The transgene can be effectively introduced into ES cells by DNA transfection or by retrovirus-mediated transduction. These transformed ES cells are then combined with blastocysts from non-human animals. The ES cells then migrate to the embryo and contribute to the germ cells of the resulting chimeric animal. Jaenisch, R. (1988) Science 240: 1468-1474.

Unless specifically stated otherwise in the practice of the present invention, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology will be within the skill of those skilled in the art. For example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice, Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press, and D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and

See Physical Analysis of DNA Methods in Enzymology, Academic Press.

Hereinafter, the present invention will be described.

In the present invention, the 14-3-3 sigma gene-deficient mice produced by the present invention showed a higher sensitivity for sepsis-related response to lipopolysaccharide (LPS), which is a cause of sepsis, than normal mice.

Hereinafter, the present invention will be described in detail.

14-3-3 sigma wild type (14-3-3 s ^{+ / +} ) And 14-3-3 sigma null (14-3-3 s ^{- / -} Sensitivity analysis of mouse septic shock

Lipopolysaccharide (LPS), which induces a septic shock, was injected into 14-3-3 s ^{+ / +} and 14-3-3 s ^{- / -} mice in the amounts of 3 mg / kg and 5 mg / The survival rate of septic shock was analyzed at time intervals. Six mice were used for each group. The 14-3-3 s ^{+ / +} mouse Survival was 83.4% at 5 days after LPS injection of 3 mg / kg, but survival rate of 83.4% at 14 days after injection was 14.3-3 s ^{- / -} mice. Showed 0% survival rate (Figure 1). When LPS was injected at a dose of 5 mg / kg, the survival rate of 14-3-3 s ^{+ / +} mice was 66.7% after 1 day, 33.3% after 4 days, and 14-3-3 s ^{- / -} mice , The survival rate was 16.7% after 1 day of injection and 0% after 2 days (FIG. 1). These results suggest that 14-3-3 s gene deficient mice have a very low survival rate for septic shock.

14-3-3 sigma wild type (14-3-3 s ^{+ / +} ) And 14-3-3 sigma null (14-3-3 s ^{- / -} ) Expression levels of inflammatory cytokines in tissues by septic shock in mice

Lipopolysaccharide (LPS), which induces septic shock, was injected into 14-3-3 s ^{+ / +} and 14-3-3 s ^{- / -} mice in the amounts of 1 mg / kg and 3 mg / kg, MRNA levels of the inflammatory cytokines interleukin1-beta (IL-1beta), IL-6 and Cycloxygenase-2 (COX-2) were compared. The expression of inflammatory cytokines was significantly higher in 14-3-3 s ^{- / -} mice compared to 14-3-3 s ^{+ / +} mice (FIG. 2). In particular, the difference in the expression of COX-2 was remarkable. Also, the same difference was confirmed in the results of comparative analysis of expression of COX-2 from the mouse spleen of the same condition by tissue immunostaining (FIG. 2).

14-3-3 sigma wild type (14-3-3 s ^{+ / +} ) And 14-3-3 sigma null (14-3-3 s ^{- / -} ) Analysis of inflammatory cytokine production in serum by septic shock in mice

Lipopolysaccharide (LPS), which induces a septic shock, was injected into the 14-3-3 s ^{+ / +} and 14-3-3 s ^{- / -} mice in a dose of 3 mg / kg, and after 0, 2, (IL-1beta), IL-6 and Cycloxygenase-2 (COX-2) were measured by ELISA assay. The production of inflammatory cytokines in 14-3-3 s ^{- / -} mice was significantly higher than that of 14-3-3 s ^{+ / +} mice (FIG. 3).

These results indicate that 14-3-3 s ^{- / -} mice are very sensitive to septic shock induced by LPS. In other words, if the expression of the 14-3-3 s gene is not expressed, gene expression may cause problems in the biological control system to cope with the inflammatory reaction such as septic shock and may play an important role in inducing sepsis.

The significance of the present invention is that the 14-3-3 s gene plays a key role in the innate immune response in which the inflammatory response is appropriately controlled, using the in vivo animal model, and ultimately, Suggesting that the 14-3-3 s ^{- / -} mouse animal model could be used as a new in vivo system for exploring and identifying anti-septic and anti-inflammatory agents.

As can be seen from the present invention, the present invention has developed an animal study model in which 14-3-3 sigma deficiency is a key gene for sepsis, enabling in vivo investigation of inflammatory diseases, The efficiency of excavation became possible in vivo.

Figure 1 shows a comparison of the sensitivity (survival rate) of 14-3-3 s ^{+ / +} and 14-3-3 s ^{- / -} mice to septic shock induced by LPS,
FIG. 2 is a graph comparing the sensitivity of 14-3-3 s ^{+ / +} and 14-3-3 s ^{- / -} mice to LPS-induced septic shock (inflammatory cytokine expression levels in tissues (lungs & spleen))
Figure 3 is a comparative analysis of the sensitivity (inflammatory cytokine production level) of 14-3-3 s ^{+ / +} and 14-3-3 s ^{- / -} mice to septic shock induced by LPS

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Example  1: Production of 14-3-3 sigma gene-deficient mice

14-3-3 sigma gene deficient mice were obtained from Chen Ling, Dongmei Zuo, Bin Xue, et al.

Genes Dev . 2010 24: 947-956. Therefore, the basic contents are described in detail in the paper.

In summary, genomic BAC DNA clones with 14-3-3s locus were identified and secured in the Ensembl database. Since the 14-3-3s gene consists of one exon, a 10.4 kb genomic DNA fragment having about 7.5 kb of the exon and about 2.9 kb of the 3 'toward the 5' end is inserted into the pLMJ235 vector. The loxP sequence was cloned before about 2.2 kb of the 14-3-3s exon. A neomycin-resistant (neo ^R ) cassette with both loxP and frt sequences was cloned approximately 160 bp downstream of the 14-3-3s exon. The vector was linearized with NotI and gene targeting was performed according to standard techniques using embryonic stem cell line J1. Embryonic stem cells containing the correct insertion were identified by Southern blotting with a 3 'foreign probe. The probe used was Sfn3-F1, 5'-atccctttactctctctagttgct-3'; And Sfn3-R1, 5'-ccagtcttccacgttgacctgc-3 'primers. The embryonic stem cell clones were used to produce chimeras and germ-line transmission mice were obtained. The mice thus obtained were crossed with beta-actin Cre transgenic mice expressing Cre recombinase in all cells including germ cells to prepare 14-3-3 sigma gene-deficient mice.

Experimental Example 1: Mouse toxicity test

(Sigma E. coli 055: B5) 0, 1, 3 and 5 mg / kg were intraperitoneally injected into C57 / BL6 mice (n = 5) 00, PM 9:00).

Experimental Example 2: Cytokine analysis in mouse serum

3 mg / kg of LPS was injected into the abdominal cavity of the mice (n = 3), and blood was collected from the heart at 0, 2, 6, 12 hours and serum was isolated. In the serum separation method, the collected blood is allowed to stand at room temperature for about 1 hour, and after hardening, the serum is separated into supernatant by centrifugation at 2000 rpm for 20 minutes. The serum thus obtained was diluted 50-fold and analyzed using a Quantikine Elisa kit (R & D system).

Experimental Example 3: Tissue Immunostaining

The mouse spleen was separated, fixed in 10% formalin solution, embedded in paraffin, and cut to a thickness of 4 mm to attach to the slide. xylene for 3 minutes for 3 minutes to remove paraffin, then treated with 100%, 85%, 70%, 50% alcohol for 3 minutes, and then added to distilled water for hydration. 10 mM Citrate pH 6.0 buffer and boil for 15 minutes using microwave. H ₂ O ₂ for 10 minutes to block the peroxidase and react overnight at 4 ° C using the primary antibody Cox-2 (1200) (Santa Cruz Biotechnology, Cat # SC-1746). After washing with PBS-T solution, the secondary antibody, biotinylated anti-goat (Vector, Cat # BA-9500) was reacted at room temperature for 1 hour, washed with PBS-T solution, . The cells were washed with PBS-T solution and reacted with DAB (Vector, Cat # SK-4100) for 5 minutes. The cells were washed with water, counterstained with hematoxylin, dehydrated with alcohol and xylene, and sealed with canada balsam.

Experimental Example  4: RT - PCR

RNA  extraction

Liver and spleen tissues are separated and placed in a sterilized mulberry bowl and frozen with liquid nitrogen. Earl breaks the tissue and goes to the bowl finely. 500 μl of Trizol reagent was added to the gallin tissue, and the mixture was allowed to stand at room temperature for 3 minutes, and 125 μl of chloroform was added thereto for 15 seconds, followed by centrifugation at 12,000 g for 15 minutes. 100 μl of the supernatant was transferred to a new tube and 100 μl of Isopropyl alcohol was added to precipitate the RNA, which was then washed with 75% ethanol. RNA precipitates were dissolved in DEPC solution and stored at -70 ° C.

cDNA  synthesis

Using 2 ug of extracted RNA, reverse transcription was performed in a volume of 20 μl. (DT) 18 primer was added to each well and reacted at 70 ° C for 10 minutes. The oligonucleotide (dT) was ligated to the RNA and reacted with 2.5 mM dNTP, 50 mM KCl, 10 mM Tris-HCl, 5 mM MgCl 2, Reverse transcriptase was added to the reaction mixture at 42 ° C for 60 minutes and at 70 ° C for 15 minutes to synthesize cDNA.

PCR

2 μl of cDNA and 10 pmol of primer were mixed with water to a volume of 20 μl and amplified using PCR premix (Bioneer). The PCR cycle conditions were denaturation at 94 ° C for 30 min, annealing at 60 ° C for 30 sec, extension at 72 ° C for 1 min, amplification at 35 cycles, and final cycle at 72 ° C for 10 min. Primer sequences were synthesized as follows.

cox-2

Fw: 5'-caa tga gta ccg caa acg ct-3 '

Re: 5'-agg tgc tcg gct tcc agt at -3 '

IL1 beta

Fw: 5'-gtt gac gga ccc caa aag at-3 '

Re: 5'-aag gtc cac ggg aaa gac ac-3 '

IL-6

Fw: 5'-ttg gga ctg atg ctg gtg ac-3 '

Re: 5'-tgc aag tgc atc atc gtt gt-3 '

<110> KNU-Industry Cooperation Foundation          Gachon University of Industry-Academic cooperation Foundation <120> 14-3-3 sigma null mouse, development of in vivo septic shock          research model using the same and use the same <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 747 <212> DNA <213> Mouse <400> 1 atggagagag ccagtctgat ccagaaggcc aagttggctg aacaggccga acggtatgaa 60 gacatggcag ctttcatgaa gagcgccgtg gaaaagggcg aggagctctc ctgcgaggag 120 cgaaacctgc tttccgtagc ttacaagaac gtggtgggcg gccagagagc ggcctggagg 180 gtcctgtcca gcatcgagca gaagagcaac gaggaggggt cagaagagaa gggccccgag 240 gtgaaagagt accgggagaa ggtagagacc gagctcagag gtgtgtgcga caccgtactc 300 ggcctgctgg actcgcacct catcaaaggg gctggagatg cagagagccg cgtcttctac 360 ctgaagatga agggtgacta ctaccgctac ctagccgagg tggccactgg cgatgacaag 420 aagcgcatca tcgattctgc ccggtcagcc taccaggagg ccatggacat cagcaagaag 480 gagatgccgc ctaccaaccc catccgcctg ggcctggccc tgaacttttc agtcttccac 540 tacgagatag ccaacagccc cgaggaggcc atctcgctgg ccaagaccac cttcgacgag 600 gccatggccg acctgcacac cctcagtgag gactcctaca aggacagcac cctcatcatg 660 cagctcctga gagacaacct gacgctgtgg acagccgaca gtgctgggga agagggtggt 720 gaggctccgg aggagcccca gagctga 747

Claims (17)

14-3-3 Method of producing a transgenic mouse model by deleting the sigma gene and studying sepsis. The method according to claim 1, wherein the 14-3-3 sigma gene has the nucleotide sequence of SEQ ID NO: 1. 14-3-3 Transgenic mice for septicemia with sigma gene deletion. 4. The transgenic mouse for sepsis research according to claim 3, wherein the 14-3-3 sigma gene has the nucleotide sequence of SEQ ID NO: 1. Treating a transgenic mouse lacking the 14-3-3 sigma gene as a test substance; And
And measuring the survival rate of the transgenic mouse in the presence of the test substance, wherein when the survival rate of the transgenic mouse is increased in the presence of the test substance as compared with the case where the test substance is not present Wherein the test substance is a candidate substance for treating sepsis. [Claim 6] The method according to claim 5, wherein the candidate substance is an extract or a compound derived from a natural product. 6. The method of claim 5, wherein the method further comprises treating the transgenic mouse with lipopolysaccharide (LPS) prior to candidate treatment. The method according to claim 7, wherein the lipopolysaccharide (LPS) is treated with 3 mg / kg body weight to 5 mg / kg body weight of transgenic mice. A method for screening candidate substances for the treatment of inflammation,
Treating a transgenic mouse lacking the 14-3-3 sigma gene as a test substance; And
Measuring the expression level of an inflammatory cytokine of the transgenic mouse in the presence of the test substance, wherein the inflammatory cytokine of the transgenic mouse in the presence of the test substance as compared to the absence of the test substance Wherein the test substance is a candidate substance for treating inflammation when the expression level is decreased. 10. The method according to claim 9, wherein the candidate substance is an extract or compound derived from a natural product. 10. The method of claim 9, wherein the method further comprises treating the transgenic mouse with lipopolysaccharide (LPS) prior to candidate material treatment. 12. The method of claim 11, wherein the lipopolysaccharide (LPS) is treated with 3 mg / kg body weight to 5 mg / kg body weight of transgenic mice. 10. The use according to claim 9, wherein the inflammatory cytokine is a cytokine selected from the group consisting of IL-1beta, IL-6 and Cycloxygenase-2. A method for screening a substance. [Claim 11] The method according to claim 9, wherein the inflammatory cytokine expression ratio is measured by mRNA or tissue immunostaining. A kit for studying sepsis comprising transgenic mice lacking the 14-3-3 sigma gene. 14-3-3 Kit for the analysis of inflammatory cytokines including transgenic mice deficient in sigma gene. The inflammatory cytokine assay according to claim 16, wherein the inflammatory cytokine is a cytokine selected from the group consisting of interleukin 1-beta (IL-1beta), IL-6 and Cycloxygenase- For the kit.

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