KR100734815B1 - 24 Transgenic dementia mice expressing htau24 and the method for preparing thereof - Google Patents

Abstract

Transgenic dementia mice expressing the htau24 gene and a preparation method thereof are provided to induce dementia symptoms in mice by overexpression of NSE(Neuron-Specific Enolase)/htau24(human tau) gene, and abnormal hyperphosphorylation of tau protein and tau-binding proteins, so that the mice are useful for development of dementia-treating agent or early diagnosis of dementia. The transgenic dementia mouse comprises a NSE promoter of SEQ ID NO:5, htau24 gene of SEQ ID NO:6 and a termination factor sequentially in its chromosome, and expresses the htau24 gene so as to show dementia symptoms which induces abnormal behavior, hyperphosphorylation of tau and tau-binding proteins and PHFs increase. The preparation method of the transgenic dementia mouse comprises the steps of: (1) linking the htau24 gene of SEQ ID NO:6 to the NSE promoter of SEQ ID NO:5 to prepare a recombinant fusion gene; (2) micro-injecting the recombinant fusion gene into a mouse embryo; and (3) implanting the mouse embryo into a surrogate mother and producing a baby mouse, wherein the embryo of step(3) is deposited under the number of KCTC 10906BP.

Description

Transgenic dementia mice expressing htau24 and the method for preparing according to the present invention.

1 is a result of performing Southern blot in order to confirm the insertion of the pNSE / htau24 gene in the transgenic mouse inserted htau24 gene according to the present invention. Here, A represents the structure of the recombinant fusion gene pNSE / htau24 plasmid, and B and C represent the identification result of the insert gene, respectively.

Figure 2 shows the results of the Water Maze Tests of transgenic demented mice and age-matched control mice inserted with NSE / htau24 genes according to the present invention, respectively.

3 is a result of Western blot and electron microscopy confirming the overexpression of tau, the overphosphorylation of tau binding protein, and the production of PHFs in transgenic demented mouse brain tissues in which the NSE / htau24 gene is inserted according to the present invention.

4 is a result of Western blot analysis of the hyperphosphorylation of tau binding proteins of transgenic dementia mice in which NSE / htau24 genes are inserted according to the present invention.

Figure 5 is the result of Western blot confirmed the inhibition of tau binding protein phosphorylation by lithium administration in the brain tissue of the transgenic demented mouse inserted with the NSE / htau24 gene according to the present invention.

The present invention relates to a transgenic dementia mouse expressing the APP / htau24 gene, and more particularly, by connecting a promoter of the brain-specific Neuron-Specific Enolase (NSE) gene to a htau24 dementia causing gene derived from humans. It relates to a transgenic dementia mouse produced by microinjecting a hybrid gene, NSE / htau24, into a fertilized egg of a mouse and implanting the fertilized egg into an oviduct of a surrogate mother mouse.

Dementia is a disorder accompanied by general disorders of systemic function, such as memory impairment and loss of judgment. There are many forms of dementia, about 50% of which are dementia of Alzheimer type, 20 to 30% of dementia of Vascular type, alcoholic dementia and Parkinson's dementia. It is known that ˜20% develop both Alzheimer's and vascular dementia.

Alzheimer's disease (AD), the most important form of dementia, is an elderly dementia disease that occurs rarely before age 50 but gradually increases after age 60. With the increase of the elderly population due to the improvement of quality of life, the onset population is rapidly increasing not only in Korea but also in the world. The number of Alzheimer's disease patients registered in Korea over 65 years in 1995 was 241,000, accounting for 8.3% of the total elderly population, and it was predicted to be 619,000 in 2020. [Health and Social Research Institute (1995), Ministry of Health and Welfare Daily (4747) December 28, 1998]. Alzheimer's disease can be treated only limitedly when diagnosed early, so it is urgently needed to develop a method and treatment that can accurately diagnose it early, but even the cause of this is not known exactly.

The cause of Alzheimer's disease is not yet well understood, but the function of the following two proteins is attracting attention. The first is a neurotoxic protein called amyloid β-39 / 43, which is concentrated at high concentrations to form plaques, which are deposited on nerve cell sites responsible for brain memory and recognition. That damages the DNA of Alzheimer's. The other is a thin, thread-like protein called tau, which twists together to form a tangled tangle, resulting in the death of neuronal cells as the protein is ideally overphosphorylated.

Among them, tau protein, which is attracting attention recently as a key cause of AD disease, is PHF produced by tau hyperphosphorylation. These PHFs are known to play an important role in neuron degeneration by forming Neuron Fibrillary Tangles (NFT).

In order to identify the cause of such dementia and develop a therapeutic substance, it is essential to develop a dementia animal model that can be actually tested before the clinical trial. For this purpose, dementia mice under the control of Thy 1,2 and htau40 and htau24 have been developed and used, but dementia mice induced with dementia due to the insertion of the htau24 gene under the control of NSE as described above have not yet been developed and NFT Failed to express production.

Therefore, the present inventors have been able to develop a dementia mouse incorporating htau24 gene, which is a dementia-inducing gene, as a result of studying the technical reality as described above and developing an animal model for diagnosing and treating Alzheimer's dementia disease. In other words, APP / htau24 gene, which is expressed in the animal and increases the superphosphorylation of tau, one of the toxic proteins that cause dementia, is coupled to the NSE promoter and implanted into a fertilized egg of a mouse. One transgenic dementia mouse was found to exhibit the pathological features observed in patients with dementia and completed the present invention.

It is therefore an object of the present invention to provide a transgenic dementia mouse expressing the NSE / htau24 gene.

The above-described features of the present invention and other features can be well seen by those skilled in the art by the following detailed description of the present invention.

In order to achieve the above object, in the present invention, a promoter of the brain-specific Neuron-Specific Enolase (NSE) gene and a human htau24 dementia gene are linked to produce a fusion gene NSE / htau24, and then the fusion gene is mouse. It is characterized by producing a transformed demented mouse by micro-injecting the fertilized egg of and implanting it into the fallopian tube of the surrogate mother mouse.

The present invention is a transgenic dementia mouse, in which the NSE / htau24 gene is inserted into its chromosome, and this gene is functionally linked to a brain-specific NSE promoter, whereby the htau24 gene is expressed at a level causing dementia, As a result, it provides a transgenic dementia mouse as a dementia model animal in which behavioral abnormalities are expressed.

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

The tau proteins present in normal brains are usually phosphorylated at normal levels, but in Alzheimer's brains, NFTs composed of PHFs are produced in large quantities during the ideal activation of tau and tau-binding proteins. Alzheimer's disease is known to be caused by DNA damage caused by long-term accumulation of NFTs in high concentrations in neurons responsible for memory or cognition in the brain region. In the brains of Alzheimer's patients, ideal hyperphosphorylation with increased production by the tau gene promotes the expression of behavioral abnormalities, resulting in GSK3β and its associated protein dysregulation. NFTs are toxic proteins that damage the neurons responsible for memory and cognition in the brain and cause memory disorders.

In the present invention, the fusion gene NSE / htau24 in which the NSE / htau24 gene and the NSE promoter are coupled to each other may be introduced into various animals except humans. Such animals include a mouse or a rat, but rats are difficult to make model animals, and therefore, it is preferable to use a mouse.

In the present invention, the fusion gene in which the NSE / htau24 gene and the NSE promoter are combined may be inserted into an animal chromosome according to various transformation methods well known in the art. Examples of such transformation methods include a method of microinjecting a fusion gene into a fertilized egg of an animal or a method using a retroviral vector. Alternatively, a method of implanting a fertilized egg transplanted with the recombinant fusion gene into a fertilized egg may be used in another surrogate mother mouse. However, from the viewpoint of the safety of the experimenter, the method of microinjecting the fusion gene into the fertilized egg is more preferable.

In the following preparation example of the present invention, the recombinant fusion gene NSE / htau24, in which the human NSE / htau24 gene and the NSE promoter, respectively, are combined as described above is microinjected into the fertilized egg of BDF1 mouse, and the fertilized egg injected with the gene as described above. Transgenic demented mouse pups were produced by transplantation into ICR mice.

As described above, the transgenic dementia mouse into which the fusion gene in which the NSE / htau24 gene and the NSE promoter are functionally inserted is a dementia-induced mouse model that exhibits various pathologies caused by Alzheimer's disease because the htau24 gene is expressed by the NSE promoter. In other words, as a result of measuring the phenotype of Alzheimer's disease by breeding transgenic dementia mice expressing the prepared NSE / htau24 gene, ideal phosphorylation of tau and tau-binding proteins as a pathological phenomenon characteristic of dementia patients. PHFs were identified, and abnormal water function was confirmed through the Water Maze Test. Through this, the transgenic dementia mouse according to the present invention was found to exhibit a behavioral disorder very similar to the dementia patients.

On the other hand, when the transgenic demented mouse according to the present invention is crossed with normal mice, the NSE / htau24 gene is transferred to the next generation. In other words, the NSE / htau24 gene can be inherited by crossing.

Transgenic demented mice according to the present invention exhibit behavioral abnormalities similar to those exhibited by patients with dementia, and are characterized by almost complete dementia symptoms such as ideal phosphorylation of tau and tau-binding proteins. Therefore, in the case of using the expression traits such as the increased expression of the dementia-related substances, it can be very useful for the study of the mechanism of dementia, and particularly useful for the development of a therapeutic or early diagnosis of dementia. The treatment of dementia currently developed or developed at home and abroad is used to treat various dementia treatment effects under conditions similar to those of the human body, such as inhibiting amyloid β-42 accumulation, alleviating behavioral abnormalities due to dementia, and decreasing GSK3β and its binding protein activity. By confirming, the dementia treatment effect of the dementia treatment agent can be tested, and it can also be used as an animal model for studying the cause of dementia disease.

Hereinafter, the structure of the present invention will be described in more detail with reference to Examples and Test Examples, but the scope of the present invention is not limited thereto.

Preparation Example 1 Preparation of Transgenic Dementia Mice Inserted with the NSE / htau24 Gene

① Gene acquisition and recombination

Among the genes used in this experiment, the NSE promoter was obtained from Sutcliffe Ph.D. (Research Institute of Scrippe Clinic) and obtained from human cell lines by RT-PCR method.

Recombination of the gene was performed by combining the NSE promoter of SEQ ID NO: 5, the htau24 gene of SEQ ID NO: 6, and the terminator, followed by specific primers (sense: 5'-GCACT AGTCA GGTGA ACTTT GAACC AGGAT G-3 '(SEQ ID NO: 1)). Antisense: 5′-GCACT AGTCT GATCA CAAAC CCTGC TTG-3 ′ (SEQ ID NO: 2)], followed by 25 cycles of denaturation at 94 ° C. for 30 seconds, annealing at 62 ° C. for 30 seconds, and synthesis at 45 ° C. for 45 seconds. PCR amplification. Cloning into pGEM-T was completed by insertion into a pNSE-splice vector digested with XhoI / EcoRI.

② Fine injection of fusion gene and injection into mouse fertilized egg

Recombinat fusion gene (SEQ ID NO: 7) NSE / htau24 was purified by cleaving a gene region derived from bacteria for pure separation, and only eukaryotic expression sites were harvested and dissolved in water. The purely isolated fusion gene NSE / htau24 was diluted to a concentration of 4 ng / μl and injected into the fertilized eggs of mice (BDF1). Embryos implanted with the gene were implanted into ICR mice of fertility to produce offspring.

③ Identification of Transgenic Mouse

After cutting the tail of the produced offspring to isolate genomic DNA, primers specific for the recombinant fusion gene (Gibco BRL Co. production request) [Sense: 5 '-CTGCAGCTCTTGACTCATCG-3' (SEQ ID NO: 3), antisense : DNA-PCR was performed using 5'-GCACTAGTCTGATCACAAACCCTGCTTG-3 '(SEQ ID NO: 4)] for 25 seconds of denaturation at 94 ° C for 30 seconds, annealing at 62 ° C for 30 seconds, and 45 seconds at 72 ° C. Next, Southern blot hybridization was performed to confirm the insertion of the NSE / htau24 gene, and the results are shown in FIG. 1.

Plasmid pNSE / htau24 contains the htau24 gene located under the control of the NSE gene promoter (FIG. 1A). In addition, DNA-PCR and Southern blot analysis revealed a Southern blot product of 7.5 kb PCR having a size of 1,112 bp in transgenic mice carrying the NSE / htau24 trans gene (# 7424 in FIG. 1B).

In addition, as a result of crossing the mouse with a transgenic mouse identified with the htau24 gene as described above, it was confirmed that the htau24 gene is transferred later.

As described above, a fertilized egg of a mouse transformed by inserting an NSE / htau24 fusion gene into an eukaryotic cell expression site, that is, a fertilized egg of a transgenic demented mouse in which an NSE / htau24 gene is inserted into a chromosome, is a Korea Deposit Research Institute gene. It was deposited with the Bank of Korea (Korean Collection for Type Cultures) on February 2, 2006, and was given accession number KCTC 10906BP.

[Test Example 1] Confirmation of NSE / htau24 gene expression and hyperphosphorylation of tau and tau-binding proteins in tissues of transgenic dementia mice

Each tissue was isolated from the transgenic demented mouse to confirm whether the NSE / htau24 gene was expressed in a tissue-specific manner. To this end, the brain, liver and kidney of the transgenic demented mouse was confirmed by RT-PCR expression in each tissue of the htau24 gene under the control of rat NSE promoter. The RT-PCR is denatured at 94 ° C. for 30 seconds, annealed at 62 ° C. for 30 seconds, and synthesized for 45 seconds at 72 ° C. after 28 cycles, followed by treatment at 72 ° C. for 7 minutes.

The results are shown in (FIG. 3A). Β-actin signals and their transcripts (492 bp) as controls were shown to indicate the loading of RNA. Expression of the NSE / htau24 fusion gene was regulated in a brain-specific manner.

The ideal hyperphosphorylation of tau and tau-binding proteins due to overexpression of the Tau protein was confirmed by Western blot. The result is shown in (FIG. 3B).

Overexpression of the Tau protein promoted hyperphosphorylation of tau and tau-binding proteins and production of PHFs. (FIG. 3C, FIG. 3D)

Test Example 2 Measurement of Brain Function Abnormalities

The NSE / htau24 transgenic dementia mouse (18 months old) was subjected to a water maze test (Water Maze Test) to check whether there is any abnormality in brain function compared to normal mice.

The results are shown in FIG. FIG. 2 measures the pattern of escape latency (FIG. 2A), escape distance (FIG. 2B) and swimming velocity (FIG. 2C) across the front platform position in an underwater maze test. Results [mean SD of mean 3 experiments, maze specimens in water and median values and SD of 3 experiments performed n = 3 P <0.01]. Escape delays and escape distances of transgenic demented mice showed a consistent tendency to appear longer than control mice. However, transgenic mice and control mice showed similar levels in swimming speed.

As can be seen from the above results, there was a significant difference in escape delay and escape distance measurements between NSE / htau24 transgenic dementia mice and normal mice. As a result, the transgenic demented mice according to the present invention had abnormal brain function. As a result, it was confirmed that the abnormal behavior appeared.

Experimental Example 3 Hyperphosphorylation Increased in the Brain of Transgenic Dementia Mice

In the brain of transgenic demented mice, it was confirmed by Western blot that the ideal hyperphosphorylation of tau, a toxic protein that causes dementia, increased (FIG. 4).

Nitrocellulose filters, which transferred 40 μg of protein isolated from the brains of transformed and control mice, were treated with anti-human tau, p-tau, GSK3β, p-GSK3β, β-calinin, and p-β. -Incubated with kalinin antibodies and quantified their levels (FIG. 4A). Each figure represents the mean ± SD of three experiments (P <0.028).

Figure 5 after transgenic mice and age standard control mice treated with lithium by concentration, brain protein extracts were insulated with anti-GSK3β and anti-pGSKβ, β-kalinin, tau and p-tau and PS1-CTF, and then Rabbit-HRP visualization to confirm tau binding protein phosphorylation inhibition by Western blot. An increase in control mouse p-GSK3β activity was observed (FIG. 5B). From the above results, it was confirmed that phosphorylation of tau binding protein was more activated in the transgenic dementia mouse of the present invention.

As described above, in the present invention, a transgenic demented mouse was prepared by injecting a fusion gene combining the NSE / htau24 gene, which is a dementia-inducing gene, with the NSE promoter, into a mouse fertilized egg, and the demented mouse of the present invention is a NES / htau24 gene. Is an overdevelopment of dementia patients' behavioral abnormalities, and is particularly effective in treating dementia or early diagnosis, as it has an excellent effect of almost complete dementia symptoms such as abnormal phosphophosphorylation of tau-binding proteins and PHFs as well as tau proteins. It can be usefully used for the development of.

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Claims (6)

A transgenic dementia mouse incorporating a recombinant fusion gene produced by concatenating a sequence of the Neuron-Specific Enolase (NSE) promoter of SEQ ID NO: 5, the htau24 gene of SEQ ID NO: 6, and a terminator in the chromosome. The transgenic dementia mouse according to claim 1, wherein the htau24 gene is expressed so that dementia is induced. The transgenic dementia mouse according to claim 2, wherein the htau24 gene is overexpressed, respectively, resulting in increased behavioral abnormalities, hyperphosphorylation of tau and tau-binding proteins, and increased paired helical filaments (PHFs). (1) preparing a recombinant fusion gene by linking the htau24 gene of SEQ ID NO: 6 with a Neuron-Specific Enolase (NSE) promoter of SEQ ID NO: 5; (2) microinjecting the recombinant fusion gene into a fertilized egg of a mouse; And (3) implanting the fertilized egg into a surrogate mouse to produce a pup; Method of producing a transgenic dementia mouse comprising a. The method of claim 4, wherein the fertilized egg of step (3) is a fertilized egg of Accession No. KCTC 10906BP. The htau24 gene of SEQ ID NO: 6 prepared by the method according to claim 4 or 5 is integrated, and the htau24 gene is a transgenic dementia having a gene linked to the NSE (Neuron-Specific Enolase) promoter of SEQ ID NO: 5. mouse.

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