KR101890978B1 - Transgenic cloned porcine Models for alzheimer's disease and the Use thereof - Google Patents
Transgenic cloned porcine Models for alzheimer's disease and the Use thereof Download PDFInfo
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Abstract
The present invention relates to a transgenic pig for an Alzheimer's model overexpressing a mutant human amyloid precursor protein (APP), a method for producing the same, and a method for producing the same. More particularly, the present invention relates to an APP695sw (synapsin 1) promoter linked to a human synapsin 1 promoter Lt; RTI ID = 0.0 > of Alzheimer ' s disease. ≪ / RTI >
Description
The present invention relates to a transgenic pig for an Alzheimer's disease model overexpressing a mutant human amyloid precursor protein (APP), a method for producing the same, and a use thereof, and more particularly, to a transgenic pig for the
Dementia is a disease with general impairment of systemic functions such as memory impairment and loss of judgment. About 50% of Alzheimer's type is dementia of Alzheimer type, about 30% is dementia of Vascular type, Alcoholic dementia and Parkinson's disease, and about 20% It is known that Alzheimer's disease and Alzheimer's disease are both caused by Alzheimer's disease.
Alzheimer disease (AD), which is the most important form of dementia, is an elderly dementia disease in which symptoms do not occur before age 50 but gradually increases after age 60, and the development of medical technology and quality of life The number of elderly people is increasing rapidly as the number of elderly people is increasing. The number of Alzheimer's patients registered in Korea over 65 years of age in 1995 was 241,000, accounting for 8.3% of the total population of the elderly, and it was predicted to reach 619,000 by 2020 [Health and Social Research Institute (1995) 4763) December 28, 1998]. Since Alzheimer's disease can be treated only with limited early diagnosis, it is urgently required to develop a method and a treatment method capable of accurately diagnosing the Alzheimer's disease at an early stage. However, even the cause of the onset is not clear
In this context, the use of animal models to discover new therapies for neurodegenerative diseases is an essential element in discovering new therapeutic targets and performing drug testing at preclinical stages.
The primary concern of neuroscientists is the choice of the most appropriate animal model useful for achieving the research objectives. Researchers are based on mechanisms that are not essential to their patients, but they often face choices between animal models based on known pathogenetic mechanisms, although models with underlying pathologic features of the disease and not all clinical features are reproduced. Therefore, researchers are challenging the development of animal models that are compatible with pathogenesis, symptoms, treatment, and physiological basis. Studies of these animal models will play an important role in accurately identifying the characteristics of dementia, the temporal and spatial changes of abnormal brain cells, and the mechanism of brain dysfunction, and verifying the effectiveness of various new therapeutic targets and new therapies.
Until now, most of the disease models for drug therapy and mechanism studies of degenerative brain diseases have been using rodents, but the pathological patterns and symptoms of animal disease models are much different from those observed in humans. Based on the results from rodent disease models There have been many problems in clinical trials.
That is, a model animal that expresses all the symptoms of human Parkinson's disease (PD) and Alzheimer's disease (AD) enough to be used as an effective disease model has not yet been established.
Therefore, a clear distinction between dimenzon, reproduction, lifespan, and behavior with humans has raised the need for animal models of disease using more human species, and in the case of primates, the cost and difficulty of rarity and breeding management Therefore, there is a growing demand for biomedical research as a new model animal for pigs that can study incurable diseases at a relatively low cost and at a relatively low cost.
Pigs have been recognized for similarity with human anatomy physiologically and have already been used for the pathological mechanism and treatment of various diseases. Especially, when pigs are valued as economic animals for a long time, It can avoid ethical problems and has a stable breeding system, so it is easy to maintain and manage when developing animal models.
Thus, the present inventors have used amyloid precursor protein (APP) as a means of applying a
It is an object of the present invention to provide a pig for Alzheimer's disease model and a method for producing the same.
Another object of the present invention is to provide a recombinant vector, a cell transformed with the recombinant vector, and a nuclear transfer embryo necessary for the method for producing a pig for the Alzheimer's disease model.
It is still another object of the present invention to provide a method for screening for a preventive and therapeutic agent for Alzheimer's disease using the pig for Alzheimer's disease model.
In order to solve the above problems, the present invention provides a pig model useful for the study of the progression and pathological mechanism of Alzheimer's disease and its use.
The present invention, in one embodiment,
There is provided a recombinant vector for producing an animal model of Alzheimer's disease containing a
In this case, the animal model of Alzheimer's disease is most preferably a pig, and the APP695sw mutation is preferably a double mutation in which Lys is substituted with Asn at position 670 of
In a preferred embodiment of the present invention, the nucleic acid encoding the APP695sw and hSYN1 is represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
In another embodiment,
The present invention provides a pig for an Alzheimer's disease model characterized by overexpressing an amyloid precursor protein (APP), which is transformed by a vector containing an APP695sw mutant gene linked to a
The overexpression of the amyloid precursor protein occurs in the cerebrum, midbrain, and cerebellum of the pig, most commonly in the midbrain.
In particular, the transformation is characterized by somatic cell nuclear transfer (SCNT).
Therefore, in another embodiment of the present invention,
There is provided a method for producing a pig for an Alzheimer's disease model characterized in that a nucleus of a nuclear donor cell into which the recombination vector described above is introduced is transplanted into an enucleated oocyte to produce a live egg.
At this time, the nuclear donor cells use porcine somatic cells or stem cell-derived cells, and it is more preferable to use stem cells.
The stem cells may be embryonic or adult stem cells and the adult stem cells may be derived from a tissue selected from the group consisting of bone marrow, umbilical cord blood, blood, fat, skin, gastrointestinal tract, placenta and uterus. In one embodiment of the present invention, adipose-derived stem cells, most preferably adipose-derived mesenchymal stem cells, are used.
Therefore, the method for producing a pig for Alzheimer's disease model of the present invention may include the following steps as a more specific example.
(a) a step of preparing a nuclear donor cell comprising culturing somatic cells or stem cells isolated from a tissue of a pig;
(b) introducing a recombinant vector containing the
(c) removing the nucleus from the oocyte of the pig to produce a enucleated oocyte;
(d) injecting and fusing nuclear donor cells of step (b) into the enucleated oocytes of step (c);
(e) activating the oocyte fused in step (d); And
(f) transplanting the activated oocyte into the fallopian tube of the surrogate pig.
The present invention includes all of the transformation vectors, cells, and nuclear transfer embryos necessary for modeling of Alzheimer's disease, which can be obtained in the process of the above-described method for producing pigs for the Alzheimer's disease model.
That is, according to another embodiment of the present invention,
It is possible to provide a transformed cell for producing an animal model of Alzheimer's disease, into which a recombinant vector containing a
It is also possible to provide porcine nuclear transfer embryos formed by transplanting the nuclei of porcine derived somatic or stem cells transformed with a recombinant vector containing the
The present invention also provides various uses of the pig for the Alzheimer's disease model.
As an example, there is provided a method of screening for a preventive and therapeutic agent for Alzheimer's disease, comprising:
1) administering a candidate substance for preventing and treating Alzheimer's disease to a pig for Alzheimer's disease model of claim 4; And
2) analyzing the brain tissue of the pig after administration of the candidate substance, as compared with a control group to which the candidate substance is not administered.
At this time, the brain tissue analysis was performed by confirming at least one of the enlargement of the ventricle, the atrophy of the hippocampus, and the overexpression of the amyloid beta protein, and at least one of the results of enlargement of the ventricle, atrophy of the hippocampus and overexpression of amyloid beta protein Is selected as a preventive and therapeutic agent for Alzheimer's disease.
Thus, the present invention provides animal models of Alzheimer's disease using pigs, which are physiologically similar to humans, and their various uses in Alzheimer's disease.
The pig for Alzheimer's disease model of the present invention is an animal model that is genetically similar to human, useful for genetic analysis, and useful for studying precise cause and mechanism of disease, as well as for evaluation of toxicity and safety. , The search for therapeutic substances, the development of diagnostic methods, and the like, and therefore, it may be very useful as an animal model of Alzheimer's disease.
Figure 1 shows the double mutant position of the human APP695 gene of the present invention
2 is a schematic diagram of a recombinant vector comprising an APP695sw mutant gene (SYN-mtAPP) linked to the
Fig. 3 shows PCR results of expression of amyloid beta precursor protein in fat-derived stem cells of pigs transformed with a recombinant vector.
4 is a photomicrograph of the undifferentiated state and the differentiation state of mesenchymal stem cells expressing SYN-mtAPP into neurons.
Figure 5 shows the results of ELISA analysis on the expression level of APP with and without SYN-mtAPP transfection and differentiation.
FIG. 6 is a schematic diagram illustrating a process according to a somatic cell nuclear transfer process according to the present invention.
FIG. 7 shows results of Southern blot analysis of APP overexpression in the transformed pig nerve cells of the present invention.
FIG. 8 shows the results of ELISA analysis of APP expression in the cerebellum, midbrain, and midbrain of the transformed pigs of the present invention.
FIG. 9 shows the results of 18F FDG PET / CT imaging of a pig for Alzheimer's disease model of the present invention.
FIG. 10 is a photograph of 18F MRI images of a pig for Alzheimer's disease model of the present invention.
FIG. 11 is a schematic diagram of the entire process for the method for producing pigs for the Alzheimer's disease model of the present invention.
12 is a schematic diagram of various experimental methods for behavioral monitoring of pigs for the Alzheimer's disease model of the present invention.
Representative terms used in the present invention are as follows.
"Disease model animal" refers to an animal with a disease that closely resembles a human disease. Diseases in the study of human disease The significance of model animals is due to their physiological or genetic similarities between humans and animals. Biomedical disease models in disease studies Animals provide research materials for various causes of diseases, pathogenesis and diagnosis, and research on disease-related animals to identify disease-related genes and understand the interactions between genes And the basic efficacy and toxicity test of the developed new drug candidates will provide basic data for judging the possibility of practical use.
"Animal" or "laboratory animal" means any mammal other than a human. The animal includes animals of all ages, including embryos, fetuses, neonates and adults. Animals for use in the present invention can be used, for example, from commercial sources. Such animals include, but are not limited to, laboratory animals or other animals, rabbits, rodents (such as mice, rats, hamsters, gerbils and guinea pigs), cows, sheep, pigs, goats, horses, (Eg, chickens, turkeys, ducks, geese), primates (eg, chimpanzees, monkeys, rhesus monkeys). The most preferred animal is a pig.
'Nuclear transplantation' refers to a genetic manipulation technique that allows an enucleated oocyte to artificially bind other cells or nuclei to the same trait. "Nuclear oocyte" refers to an oocyte into which nuclear donor cells are introduced or fused.
'Cloning' is a genetic manipulation technique for creating a new individual having the same gene set as an individual. In particular, in the present invention, a somatic cell, an embryo cell, a fetal cell and / or an adult cell of a pig is substantially And has the same nuclear DNA sequence. The present invention utilizes a technique of replicating pigs using nuclear transfer technology. In particular, somatic cell nuclear transfer technology is a technology that can produce offspring without passing through the meiosis and hemispheric retention cells that are common in the reproductive process. As a technology, transplanted somatic cells are transplanted into the nucleus- And the embryo is transplanted in vivo to generate a new individual.
'Nuclear donor cells' refers to nuclei of cells or cells that deliver nuclei to recipient oocytes, which are nuclear receptors. The term " oocyte " preferably refers to a mature oocyte that has reached the middle stage of the second meiosis. In the present invention, porcine somatic cells or stem cells can be used as the nuclear donor cells.
The term "somatic cell" refers to a cell other than a germ cell that constitutes a multicellular organism, a cell that is specialized for a specific purpose and does not become any other cell, and a cell having several different functions Cells that have the ability to differentiate.
"Stem cell" refers to a cell that can develop into any tissue. There are two basic features: self-renewal, which creates itself by repeated division, and the ability to differentiate into cells with specific functions depending on the environment.
'Mesenchymal stem cells' are a kind of undifferentiated adult stem cells isolated from human or mammalian tissues and can be derived from various tissues. In adult stem cells, hematopoietic stem cells are mainly non-adherent, whereas mesenchymal stem cells are mainly adherent cells. Particularly, it is possible to provide a stem cell, a stem cell, a stem cell, a stem cell, a stem cell, a stem cell, a stem cell, a stem cell, a stem cell, a stem cell, Placenta-derived mesenchymal stem cells. Techniques for separating stem cells from each tissue are well known in the art.
'Cultivation' is the cultivation of living organisms or parts of organisms (organ, tissue, cells, etc.) under moderately artificially controlled environmental conditions. In this case, external conditions such as temperature, humidity, light, gas phase composition The most important direct effect on the organism cultivated is the culture medium, which is a direct environment for the organism and a supply point for various nutrients necessary for survival and growth.
"In vitro culture" means a series of laboratory processes in which cells are cultured in an incubator in a laboratory in a manner similar to the environment in the body, in a manner different from that in which the cells grow.
The term "medium" or "medium composition" refers to a mixture for growth and proliferation of cells, etc., which is essential for growth and proliferation of cells such as sugar, amino acids, various nutrients, serum, growth factors and minerals.
The term "differentiation" refers to a phenomenon in which cells and tissues specialize in the structure or function of one another while the cells are growing and proliferating, that is, the form or function is changed in order to perform the task given to each of them . In general, a relatively simple system is separated into two or more qualitatively different systems.
'Fusion' refers to the coupling of the nuclear donor cell with the lipid membrane part of the recipient oocyte. For example, a lipid membrane can be a plasma membrane or a nuclear membrane of a cell. Fusion can occur by applying electrical stimulation when the nuclear donor cell and the recipient oocyte are adjacent to each other or when the nuclear donor cell is located in the perivitelline space of the recipient oocyte.
'Activation' refers to stimulation of cells to divide before, during, and after nuclear transfer.
"Living offspring" refers to an animal that can survive outside the uterus. Preferably, it refers to an animal that can survive for 1 second, 1 minute, 1 hour, 1 day, 1 week, 1 month, 6 months, or 1 year. The animal does not require an intrauterine environment for survival.
&Quot; Treatment " refers to an approach to obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction &Quot; Treatment " refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented. Palliating the disease may reduce the extent of the disease state and / or undesirable clinical symptoms and / or delay or slow the time course of the progression compared to when the disease is not treated, It means to lose.
By "about" is meant a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length of 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.
Throughout this specification, the words " comprises "and" comprising ", when not explicitly required in the context, include a stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.
Hereinafter, the present invention will be described in detail.
The present invention relates to an animal model of Alzheimer's disease.
Animal model of Alzheimer's disease
Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. It begins with the accumulation of neurotoxic substances such as amyloid, which is abnormally clustered protein, in the temporal lobes, The amount of amyloid accumulation inside and outside the nerve cell of the cortex of the cervix increases and the disease progresses. The cause of Alzheimer's disease has not yet been clearly identified, but the following two proteins are attracting attention. One is a neurotoxic protein called amyloid β-1/42, which is concentrated at a high concentration to form a plague and is immersed in the neural cell site responsible for brain memory and recognition Causing damage to the DNA of the neurons and causing Alzheimer's. The other is a tau-like thin protein, which is twisted together to form a tangle of tangles that ideally hyperphosphorylate proteins, resulting in the death of neuronal cells
The main symptom starts with memory impairment, resulting in language impairment and loss of ability to think or reason. Eventually, the patient becomes unable to move and becomes very weak, resulting in complications such as pneumonia, urinary tract infections, and pressure ulcers, leading to death. There are no clear remedies or remedies available to cure this disease.
Until the early 2000s, many animal models of Alzheimer 's were produced using mice, and most of them were transgenic mouse models modified with amyloid protein, which is the main cause of Alzheimer' s disease. In addition, tau protein-modified mouse models and GRK5 (G protein-coupled receptor kinase 5) knockout mouse models are used.
[Table 1] Alzheimer's mouse model
Model animals for conventional degenerative brain disease studies can be classified as rodent models with neurotoxic agents or transgenic techniques, and other animal species have many limitations. Thus, the existing Alzheimer's animal model is limited to mice, and the need for the production of AD disease pig models is rapidly increasing for various reasons, such as genetic similarity with humans and fertility, and the Alzheimer's pig model has not been developed to date.
As a model animal, pigs have a longer survival period than conventional rodent models and are known to have a similar biological mechanism to humans, and are thus attracting attention as a next-generation disease model animal species that can overcome the limitations of the prior art.
Therefore, the present inventors have developed a more reliable animal model of Alzheimer's disease by utilizing the large animal pig, confirming the induction of Alzheimer's disease through the production and action of a disease model that exceeds the rodent model, and developed an animal model of Alzheimer's disease.
The present invention, in one aspect, relates to a transgenic animal model, preferably a pig model, having a mutation associated with genetic Alzheimer ' s disease and over-expressing an amyloid precursor protein (APP).
That is, it relates to an animal model in which amyloid is accumulated, much like amyloidosis found in Alzheimer's patients, and relates to recombinant pigs, preferably mini pigs, that overexpress amyloid precursor protein (APP) .
Pigs are anatomically similar in size to organs and are suitable as heterogeneous organs, as well as physiologically and genetically similar to humans. In addition, it has high breeding capacity, which makes it suitable for the development of biological materials for production and treatment as well as an animal model that is good for toxicity and safety evaluation.
The pigs to be used in the present invention can be any known kind which can be appropriately selected and used by a person skilled in the art.
Pigs (Scientific name: Susscrofadomestica) belong to the subspecies, descent descent, wildflower subspecies, right titles (including cattle, chlorine) and wild boar, and the number of chromosomes is 2n = 38. Originally, pigs have attracted much attention as experimental animals as well as ovarian animals raised with cattle, with a similar interest in physiological anatomical findings and a high interest in animal welfare in recent years. Pigs are attracting much attention as physiological anatomies. Pigs are widely used throughout the study because they are similar to humans in many physiological and anatomical points.
Particularly, in the present invention, a small pig (typical example: Pittman, Moore) having a maximum body weight of 60-70 kg when maturing is used, or a small money (adult: 30-40 kg) A yukatan mini pig, a micro pig, or the like may be used. In recent years, there has been production of a near-mini-pig (NIH system) not only having a small body shape but also having a characteristic. In one embodiment of the present invention, mini pigs were used.
The Alzheimer's disease porcine model of the present invention is characterized in that it is transformed to overexpress an amyloid precursor protein (APP).
At this time, the APP gene can be transformed by any known method, but somatic cell nuclear transfer (SCNT) is preferably used. That is, a cloned animal model of Alzheimer's disease according to the present invention is produced using a somatic cell nuclear transfer (SCNT) method using a transgenic cell line overexpressing a specific gene APP.
Therefore, for example, a method for producing a pig for an Alzheimer's disease model of the present invention through a somatic cell nuclear transfer method,
(a) a step of preparing a nuclear donor cell comprising culturing somatic cells or stem cells isolated from a tissue of a pig;
(b) introducing into the nuclear donor cell a recombinant vector containing an APP695sw mutant gene linked to a
(c) removing the nucleus from the oocyte of the pig to produce a enucleated oocyte;
(d) injecting and fusing nuclear donor cells of step (b) into the enucleated oocytes of step (c);
(e) activating the oocyte fused in step (d); and
(f) transplanting the activated oocyte into the tubal duct of the surrogate mother. A schematic diagram of such a process is shown in Fig.
The conventional technology for each step can be understood with reference to a conventional method for producing a cloned animal using a somatic cell nuclear transfer technique known in the art.
Hereinafter, major constitutions used in the production of pigs for the Alzheimer's disease model of the present invention will be described in detail.
Recombinant vector
The present invention provides a vector for inducing Alzheimer's disease that over-expresses amyloid precursor protein (APP).
In particular, the vector is characterized by comprising the
Therefore, the present invention, in another aspect, relates to a recombinant vector for constructing an animal model of Alzheimer's disease, most preferably a porcine model, comprising an APP695sw mutant gene linked to a
The APPsw gene in the present invention is human Alzheimer and is one of causative genes involved in causing diseases. The APP gene is located on human chromosome 21, and people who inherit the mutant gene of this gene usually develop Alzheimer's disease before the age of 65. As a mutant form of APP, the mutant gene form in which the amino acids at positions 670 and 671 are substituted with AL in KM is referred to as APPsw (APP swedish) gene. That is, APP695sw of the present invention refers to a gene having a mutation of K595N and M596L at
Each of the nucleic acids encoding APP695sw and hSYN1 can be used without limitation as long as it has a nucleotide sequence encoding APP and hSYN1 known in the art.
Also, the nucleic acid encoding APP695sw and hSYN1 can be produced by a recombinant method known in the art. For example, PCR amplification for amplifying a nucleic acid from a genome, chemical synthesis, or a technique for producing a cDNA sequence.
The nucleic acid may also have a base sequence encoding each functional equivalent of APP695sw and hSYNl.
The functional equivalent means a sequence homology of at least 70%, preferably 80%, more preferably 90% or more with the amino acid sequence represented by SEQ ID NO: 1, 2 or 3 as a result of addition, substitution or deletion of amino acids Quot; refers to a polypeptide having substantially the same physiological activity as APP695sw and hSYN1 of the present invention of the present invention. The 'equivalent physiological activity' refers to an activity of inducing differentiation of neural stem cells or neural progenitor cells into dopaminergic neurons of the midbrain characteristics.
At this time, deletion or substitution of the amino acid is preferably located in a region that is not directly related to the physiological activity of the polypeptide of the present invention. In a preferred embodiment of the present invention, the nucleic acid encoding the APP695sw and hSYN1 is represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
The term "vector" or "expression vector" of the present invention refers to a plasmid, a viral vector or other medium known in the art capable of introducing a nucleic acid encoding the structural gene and capable of expressing the nucleic acid in a host cell it means. Preferably a viral vector.
Such viral vectors include, but are not limited to, retroviral vectors, adenovirus vectors, herpes virus vectors, avipoxvirus vectors, lentivirus vectors, and the like. Particularly, a method using a retrovirus is preferable. The retroviral vector is constructed such that all of the viral genes have been removed or altered so that the non-viral proteins are made in cells infected with the viral vector. The main advantage of retroviral vectors for gene therapy is that they transfer a large number of genes into the cloned cells, accurately integrate the genes transferred into the cellular DNA, and do not lead to subsequent infection after gene transfection.
The nucleic acid encoding APP695sw and hSYNl of the present invention may be operably linked to an expression control sequence and inserted into the vector.
&Quot; Operably linked " means that one nucleic acid fragment is associated with another nucleic acid fragment so that its function or expression is affected by other nucleic acid fragments. An expression control sequence also refers to a DNA sequence that controls expression of a nucleic acid sequence operably linked to a particular host cell. Such regulatory sequences include any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. These may be referred to as a DNA construct comprising a nucleic acid encoding APP695sw and hSYN1, or a DNA construct comprising a nucleic acid encoding a fusion gene SYN-APP.
Meanwhile, the recombinant expression vector may include a selection marker, and the selection marker includes an antibiotic resistance gene such as a kanamycin resistance gene and a neomycin resistance gene, a fluorescent protein such as a green fluorescent protein and a red fluorescent protein, It is not limited.
Further, in order to confirm whether the APP695sw gene (APP protein) is overexpressed, the vector of the present invention may further include a tag sequence for protein separation purification or confirmation. Examples of the tag sequence include GFP, Glutathione S-transferase (GST) -tag, HA, His-tag, Myc-tag and T7-tag. However, the tag sequence of the present invention is not limited by these examples. In a preferred embodiment of the present invention, the presence and expression level of TRESK was confirmed using GFP.
Preferably, the vector produced by the above method may be the vector disclosed in Fig.
The recombinant vector comprising the
Vector introduction into nuclear donor cells
The present invention also relates to a method for introducing a recombinant vector comprising APP695sw and hSYN1 into a nuclear donor cell, and a cell into which the recombinant vector has been introduced.
The two genes may be introduced separately or simultaneously, or may be introduced in the form of a fusion gene.
At this time, the nuclear donor cells used in the present invention may be somatic cells or stem cells of pigs.
The somatic cells that can be used in the present invention include, but are not limited to, cumulus cells, epithelial cells, fibroblasts, neurons, keratinocytes, hematopoietic cells, melanocytes, cartilage cells, macrophages, monocytes, B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, fetal derived cells, embryonic cells and embryonic cells. More preferably, it may be a fetal-derived cell, an adult fibroblast, or a cumulus cell. Most preferably, fibroblasts isolated from fetal and adult pigs are used. The characteristics of these cells are that they can obtain a large number of cells at the initial separation, have relatively easy cell culture, and are easy to cultivate and manipulate in vitro.
The somatic cells provided as nuclear donor cells can be obtained from a method for producing a surgical specimen or a specimen for biopsy.
In addition to somatic cells, pig embryo-derived or adult stem cells may be used as the nuclear donor cells that can be used in the present invention.
The term "stem cell" refers to a cell capable of self-replicating and capable of differentiating into two or more cells. Embryonic stem cells or adult stem cells can be used depending on the source. In the present invention, Adult stem cells derived from tissues such as fat, uterus, bone marrow, muscle, placenta, umbilical cord blood, or skin (epithelium) can be used. In particular, mesenchymal stem cells (MSCs) are preferred. The mesenchymal stem cells are generally stromal cells that aid in hematopoiesis and have the ability to differentiate into various mesodermal cells including bone, cartilage, fat and muscle cells. They can be easily proliferated while maintaining undifferentiated state There are features. In one embodiment of the present invention, adipose derived mesenchymal stem cells (MSCs) were used.
Methods for obtaining embryonic stem cells from pigs or for isolating adult stem cells from various tissues of pigs can be performed by conventional methods known in the art. When stem cells are used, the undifferentiated genes, which are considered to be essential for early embryonic development due to the characteristics of the stem cells, can be expressed as an advantage thereof. The recombinant vector of the present invention also has the function of differentiating the stem cells into neuronal cells by overexpressing the amyloid beta precursor protein.
The recombinant vector according to the present invention can be introduced into cells by a method known in the art.
For example, but not by way of limitation, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran The present invention relates to a method for introducing a nucleic acid into a cell, such as, for example, DEAEDextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, Into a cell for the production of a transgenic animal.
Meanwhile, SYN-APP-transformed nuclear donor cells for the production of an Alzheimer's disease pig model according to the present invention can be proliferated and cultured according to a method known in the art.
Suitable media are those which have been developed for the cultivation of animal cells and in particular mammalian cells, or for any use which may be produced in the laboratory together with suitable components necessary for animal cell growth, such as assimilable carbon, nitrogen and / or micronutrients Possible media can be used.
The medium may be any basic medium suitable for animal cell growth, including but not limited to MEM (Minimal Essential Medium), DMEM (Dulbecco modified Eagle Medium), RPMI (Roswell Park Memorial Institute Medium) and K-SFM (Keratinocyte Serum Free Medium). In addition, there is no limit to the use of any medium used in the industry. Preferably, the cells are cultured in a culture medium such as? -MEM medium (GIBCO), K-SFM medium, DMEM medium (Welgene), MCDB 131 medium (Welgene), IMEM medium (GIBCO), DMEM / F12 medium, PCM medium, M199 / (GIBCO), and MSC expansion medium (Chemicon).
To this base medium may be added an assimilative source of carbon, nitrogen and micronutrients, as a non-limiting example, a serum source, a growth factor, an amino acid, an antibiotic, a vitamin, a reducing agent, and / or a sugar source.
It will be apparent that those skilled in the art can select or combine media most suitable for stem cells from a variety of tissue sources and cultivate them appropriately by known methods.
In addition, it is apparent that culturing can be carried out while adjusting conditions such as a suitable culture environment, time, temperature and the like on the basis of ordinary knowledge in this field.
In one embodiment of the present invention, it was confirmed that the amyloid beta precursor protein was overexpressed in the pig cells transformed with the recombinant vector. Furthermore, when stem cells were used as nuclear donor cells, the neurons expressing the amyloid beta precursor protein Was also observed.
Transgenic pig
The present invention also relates to a method for producing a transgenic pig for an Alzheimer's model that overexpresses a mutant amyloid precursor gene (APP695sw) by transplanting the nucleus of a nuclear donor cell into which the recombinant vector is introduced into an enucleated oocyte, The present invention relates to a pig for a model of Alzheimer's disease.
For the production of the enucleated oocyte, a known denucleation process of the recipient oocyte can be used.
For example, a method of judging the optimal timing of ovulation when the oocyte epithelial cells are confirmed to be over 70% by vaginal cell smear test, a method of determining the optimal period of ovulation using ultrasound imaging A method for confirming the development status in real time, and a method for determining the timely period by measuring plasma progesterone.
As a method for recovering the recipient oocyte, a surgical method including anesthetizing the subject animal and then lapping it can be used. More specifically, recovery of recipient oocytes in vivo can be accomplished by tubal resection, a method known in the art
The enucleation process can be performed using methods known in the art (US Pat. No. 4,994,384, US Pat. No. 5,057,720, US Pat. No. 5,945,577, European Patent Publication No. 0930009A1, Korean Patent No. 342437, Kanda et al., J. Vet. Med. Sci., 57 (4): 641-646, 1995; Willadsen, Nature, 320: 63-65, 1986, Nagashima et al., Mol. Reprod. Dev. Prather et al., Biol. Reprod 41: 414-418, 1989, Prather et al., J. Exp. Zool. 255: 355- 358, 1990; Saito et al., Assis Reprod Tech Andro, 259: 257-266, 1992; Terlouw et al., Theriogenology 37: 309, 1992). Preferably, a cumulus cell of a mature recipient oocyte is removed, and a portion of the zona pellucida of the recipient oocyte is cut using a micro needle to form a incision window, through which the nucleus and cytoplasm of the first polar body, , Removing the cumulus cells of the recipient oocyte, dyeing the oocyte, and removing the nucleus of the first polar body and the oocyte using a micropipette aspiration pipette. More preferably, The Enucleation (Squeezing) method can be used to form an incision in the oocyte.
Nuclear transplantation can be performed by injecting nuclear donor cells between the cytoplasm and the zona pellucida of an enucleated oocyte using an implantable pipette. It is preferable that the enucleated oocytes in which the nuclear donor cells have been microinjected are fused with nuclear donor cells electrically using a cell manipulator. In electrical fusion, the current may be alternating current or direct current, and may be performed one to three times at a voltage of 1.5 to 4.0 kV / cm, for a time of 15 to 45 占 퐏. A schematic diagram of such a process is shown schematically in Fig.
Therefore, the present invention provides, in one embodiment, a method for producing a recombinant vector comprising the step of transplanting the nucleus of a porcine derived somatic cell or stem cell transformed with a recombinant vector comprising an APP695sw mutant gene linked to a
The fused nuclear transfer embryo can be further subjected to an activation step.
Activation of fused nuclear transfer embryos implies a process of resuming the cell cycle that stops in the middle of the second meiosis in vitro maturation. For this purpose, high activity of MPF, CSF, etc., which stop the cell cycle, should be lowered, and low activity of APC which promotes metastasis from the middle stage of the second meiosis should be increased.
In general, in order to enable the nuclear transfer embryos, and to increase the concentration of intracellular Ca 2 + ions it should induce chromosomal condensation and embryonic development, the physical method To this end, chemical methods, or an electrical method is used. Physical methods include mechanical stimulation, heat, and direct current. By chemical means a substance, such as ethanol, inositol triphosphate, Ca + 2 or Sr + 2, Saito collar Shin B, calcium child Ono fore, 6-dimethyl amino purine, cyclohexyl during mid, ball four 12-myristate 13-acetate . ≪ / RTI > As an electrical method, there is a method of performing 1-3 times for 30 to 60 으로 at a DC voltage of 1.5 to 2.5 kV / cm.
Therefore, in another embodiment of the present invention, there is provided a method for producing a transgenic pig for an Alzheimer's model that overexpresses a mutant amyloid precursor protein (APP) comprising the step of transplanting the nuclear transfer embryo into a tubal duct of a surrogate mother to produce a live egg And an overexpression of the APP mutant gene produced thereby. The present invention also provides a pig for Alzheimer ' s disease model.
In one embodiment of the present invention, it was confirmed by Southern blot analysis (FIG. 7) that overexpression of actual amyloid precursor protein in the transgenic pig neurons of the present invention was confirmed, and furthermore, expression in the cerebellum, midbrain and midbrain was confirmed by ELISA analysis (Fig. 8).
Particularly, the pig for the Alzheimer's disease model produced by the method of the present invention exhibits a decrease in brain metabolism in the general region except for the primary somatosensory cortex, which is a symptom in a human AD patient (Fig. 9), ventriculomegaly and atrophy of the cerebral cortex (arrow) (Fig. 10)
Therefore, the pig for Alzheimer's disease model of the present invention exhibits a pathological mechanism very similar to that of human Alzheimer's disease, and is very useful as an animal model for human Alzheimer's treatment research.
Transgenic pigs for Alzheimer's model overexpressing the mutant human amyloid precursor gene according to the present invention are capable of producing live plants through crosses and can later deliver the foreign genes.
Therefore, pigs overexpressing the APP mutant gene of the present invention are advantageous as an animal model of Alzheimer's disease, because they have an advantage of reproducibility.
That is, it is possible to confirm the induction of Alzheimer's by using the mini-pig model of Alzheimer's disease of the present invention, to compare the onset of the Alzheimer's disease model developed by other methods than the administration of the neurotoxic agent, It will be possible to use various methods such as searching for substances and developing diagnostic methods.
Therefore, the present invention, from another point of view, includes various uses of pigs for Alzheimer ' s disease models produced by the above method.
For example, an animal model according to the present invention can be used in a research required to treat or prevent Alzheimer's disease, for example, as a method of screening a test drug.
In one embodiment, the screening method of the present invention comprises
1) administering a candidate substance for preventing and treating Alzheimer's disease to a model swine of Alzheimer's disease;
2) analyzing the brain tissue of the pig after the administration of the candidate substance, in comparison with a control group to which the candidate substance is not administered, wherein the brain tissue analysis is performed by measuring the extent of enlargement of the ventricles, atrophy of the hippocampus, Expression or expression of tau protein, and the like.
Preferably, the candidate substance is any one selected from the group consisting of peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and plasma. The compound may be a novel compound or a well-known compound. These candidate substances may form salts.
The method of administering the candidate substance may be suitably selected in accordance with the symptoms of the subject animal, the nature of the candidate substance, etc., for example, by oral administration, intravenous injection, subcutaneous administration, intradermal administration or intraperitoneal administration. The dose of the candidate substance can be appropriately selected in accordance with the method of administration, the nature of the candidate substance, and the like.
As described above, the present invention will be very useful for the development and practical use of Alzheimer's disease treatment technology by providing an animal model of Alzheimer's disease using pigs, which are physiologically similar to humans, in an Alzheimer's disease considered to be an incurable disease.
Example
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
Statistical analysis was performed by t test with Welch's correction using Graphpad prism, and P <0.05 was considered statistically significant.
Example
1: Mutant human
APP
Production of gene nerve-specific expressing cell line
The specific mutation site in the
DNA (approximately 2.0 kb) synthesized from AAAGATG and AACTTG in hAPP695 was obtained to make cells overexpressing mutant hAPP, and mhAPP was cloned into a clone of pLV with a human synapsin promoter for neuron-specific expression Cloning site to construct a pLV vector (Figure 2).
In order to prevent the loss of cells caused by selection of antibiotics, the puromycin gene was removed and the GFP gene (719 bp), which is easily confirmed by eye, was cloned into LV-Synp- mutAPP-GFP. Using the vector thus prepared, a SYN-mhAPP-GFP expressing mini-pig pimple was established by infection with lentivirus.
PCR was performed to confirm expression of amyloid beta precursor protein in the fat-derived stem cells of the pig transformed with the recombinant vector.
As shown in FIG. 3, it was confirmed that the amyloid beta precursor protein was expressed in the fat-derived stem cells of the pig transformed with the vector of the present invention.
Example 2 : AD Neuronal specificity of transformed cell line mhAPP Expression
To differentiate mesenchymal stem cells into neural cells, we examined whether mhAPP protein was detected in differentiated neurons.
First, proteins were extracted from normal adipose stem cells (NTG), mhAPP-expressing transformed adipose stem cells (TG), neural differentiated normal adipose stem cells (DF-NTG) and neurogenic transformed adipose stem cells (DF-TG) Respectively. At this time, protein extraction from the cells was carried out using a Pro-PREPTM kit.
To the cell pellet washed with PBS, 400ul PRO-PREP solution was added and mixed well and cultured at -20 ° C for lysis of cells for 10-20 minutes. After centrifugation at 13,000 rpm and 4 ° C for 5 minutes, the supernatant was transferred to a 1.5 ml tube and protein quantification was performed using the Bradford assay.
The total protein isolated from normal adipose stem cells (NTG), mhAPP-expressing transformed adipose stem cells (TG), neural differentiated normal adipose stem cells (DF-NTG) and neurogenic transformed adipose stem cells (DF-TG) , Each sample was diluted to an appropriate concentration, and then incubated in a microplate equipped with APP antibody for 2 hours at room temperature. Biotinylated human-APP conjugation, streptavidin-HRP solution, and chromogen were sequentially reacted on each plate washed with PBS, and the absorbance value at 450 nm was used to calculate human APP Was measured.
The mhAPP expression patterns of AD transfected cell lines are shown in FIG. 4 and FIG.
First, FIG. 4 shows a microphotograph of the undifferentiated state of the adipose stem cells expressing SYN-mtAPP and the differentiation state into neurons. It was confirmed that the ability of the adipose stem cells expressing SYN-mtAPP to differentiate into neurons was remarkably excellent.
In addition, the results of the ELISA analysis are shown in Fig. 5. The results are shown in Fig. 5. In the case of neurogenic transformed adipose stem cells (DF-TG), normal adipose stem cells (NTG), mhAPP- expressing adipose stem cells (TG) (DF-NTG) cells. The results are shown in Fig.
Example 3: APP Neuronal cell Production and transplantation of specific overexpressed cloned embryos
FIG. 6 shows a simplified schematic diagram of a process according to the somatic cell nuclear transfer according to the present invention.
3-1. Somatic cell nucleus Preparation of recipient oocytes for transplantation
The pig ovary was transferred from the slaughterhouse into physiological saline solution at 28-31 ° C and transferred to the laboratory. Using a 10-ml syringe equipped with an 18-gauge needle from follicles having a diameter of 3-6 mm, the cumulus-oocyte complex , COC) were recovered. In vitro fertilization was induced by in vitro maturation culture for 40 hours in a 5% CO 2 incubator at 39 ℃.
The cells were immersed in 0.1% (v / v) hyaluronidase in TALP (Tyrode's albumin lactate pyruvate) medium and then repeatedly pipetted using a micro-glass pipette to obtain cumulus cells . Each oocyte was then fixed with a holding micropipette and incubated in a TALP medium supplemented with 5 ug / mL of bisbenzimide (Hoechst 33342) and 5 ug / mL of cytochalasin B using a micro manipulator (Nikon-Narishige , Tokyo, Japan).
The first polar and middle-II chromosomes stained with bezuanimide were removed using an aspiration pipette, and enucleated oocytes were placed on PZM (Porcine zygote media) -5 and used for somatic cell nuclear transfer.
3-2. Somatic cell nuclear transfer Preparation of donor cells for
Transgenic cloned embryos were produced by using cells expressing hmtAPP (human mutation amyloid precursor protein) gene specifically in pig brain.
For somatic cell nuclear transfer, hmtAPP cells were thawed, cultured until 100% confluency, treated with trypsin for about 3 minutes, recovered as a single cell, and recovered as a nuclear donor cell. Cloned embryos were prepared using the cells expressing the reporter gene, GFP, as the cells expressing hmtAPP.
3-3. Somatic cell Nuclear transfer technique Used AD Production of cloned embryos
Donor cells expressing a single GFP were injected into the perivitelline space of the prepared enucleated oocytes and the couplets were precipitated in a fusion medium containing 0.26 M mannitol, 0.1 mM MgSO4, 0.5 mM HEPES , And fused using needle-shaped electrodes.
The single cell-oocyte complex was placed between two opposing electrodes attached to a micro manipulator (Nikon-Narishige, Tokyo, Japan), and the contact surface between the oocyte cytoplasm and the nucleus donor cells was placed in parallel with the electrode, The cells were electrostimulated with an electro-cell fusion apparatus (NEPA GENE Co., Chiba, Japan). After 30 minutes of electrical stimulation, the fusion between the nuclear donor cell and the oocyte cytoplasm was observed under a stereomicroscope.
Fused embryos were selected and placed in a chamber containing active medium supplemented with 0.26 M mannitol, 0.5 mM HEPES, 0.1 mM CaCl 2 , and 0.1 mM MgSO 4 , and the electrodes were connected to each other using a BTX electro-cell Manipulator 2001 machine for 1.5 lt; RTI ID = 0.0 > kV / cm < / RTI > Five to six embryos were cultured in 20 μL of PZM-5 microdrops applied with mineral oil.
3-3. Somatic cell Nuclear transfer Transplantation method to embryo's surrogate mother
A surrogate mother with synchronized estrus is supplied from the farm. The surrogate mothers were anesthetized by administering 1 mg / kg of ketamine and 0.5 mg / kg of xylazine to the pigs. After taking the combination, the patient fell for a while and then the unconscious pig was moved to the operating table, and isopulllan was used to maintain general anesthesia.
After the surgical site was cleaned with soap, the hair was cleanly removed using a razor, and the surgical site was disinfected in the order of povidone, alcohol, povidone, alcohol, and povidone, followed by complete sterilization Surgery was performed.
First, the middle part of the lower abdomen was cut open, the uterus was pulled, and the ovaries and tubal status were visualized. Transplanted embryos were transplanted into the fallopian tubes. Using a Tomcat catheter, the catheter was directly inserted into the fallopian tube with a sharp needle, and the catheter loaded with the transgenic cloned embryos was inserted and the oocyte was transplanted into the surrogate mother. The transplanted embryos of this transgenic embryo thus returned the uterus and sutured the lacerations.
After the suture was completed, the surrogate was disinfected with betadine, then moved to the waiting room after the operation, and the patient was post-operatively cared for. In addition, a wide range of antibiotics were administered to prevent infection after surgery. When the anesthesia was completely broken and the stamina was restored, it was transferred to the pregnant woman who was originally inhabited and went into individual management.
Example
4 :
APP
Neuronal cell
Specially overexpressed mini pig analysis
4-1. Transgenic pig DNA Extraction and PCR analysis
In order to test whether the hmtAPP gene of the cloned pigs produced was inserted into the genome, a genetic test was carried out using a negative control, a cloned pig, and a positive control before hmtAPP was inserted.
Genomic DNA was isolated from tissue fragments and somatic cell donor cells from the blood or tail of the born subject. In order to analyze whether hmtDNA was inserted into the extracted DNA, primers for amplifying GFP and hmtAPP, which are markers in the vector, were respectively prepared. PCR was performed using genomic DNA isolated from the prepared primers.
The amplified PCR products were separated by electrophoresis, and the sequence analysis was performed using an automated DNA sequencer. The presence or absence of hmtAPP introduced exogenously in the genomic DNA of the cloned pig was determined.
4-2. Southern Blat Analysis and ELISA analysis
Southern blot and ELISA were used to confirm insertion of the target gene.
The primers for the synthesis of GFP and hmtAPP genes were tested on the basis of the sequence of the vector constituting the target gene. A probe with 713 nucleotide sequences was synthesized using PCR DIG Probe Synthesis kit (Roche, Mannheim, Germany) and purified by electrophoresis. The DNA fragment was transferred to a positively charged nylon filter and hybridized with the DNA probe was confirmed using a DIG luminescent detection kit (Roche, Mannheim, Germany).
The Southern blot analysis results are shown in Fig.
Through the Southern blot analysis, it was confirmed that the APP gene was inserted into the genomic DNA of the transgenic reproduction pig according to the method of the present invention.
The results of ELISA analysis of APP expression in the cerebellum, midbrain, and cerebrum of transgenic pigs are shown in Fig. Compared with the control group, the expression of APP was further increased in both the cerebellum, midbrain, and midbrain, and especially in the midbrain.
Example 5: APP Genetically Modified Mini Pigs Imaging analysis
: APP Genetic Modification of Mini-pigs in the First Year of Life MRI , PET Photographed Imaging Assessment and analysis
APP gene-regulated mini-pig brain-PET and brain-CT were performed to obtain brain image data of APP gene-regulated mini-pigs.
The brain 18F-FDG PET images were taken using HRRT-PET (Siemens Medical Systems and CTI, Inc) for the study of brain PET images. F-18 FDG (500 μCi / 100 g of body weight) was intravenously injected before general anesthesia, and 30 minutes later, 18F-FDG PET imaging was performed using isoflurane inhalation anesthesia via bronchial intubation. The extent of the disease was determined by comparing the functional evaluation in the cerebral cortex and middle cerebral cortex, which are related to AD, and the morphology of the brain.
In order to establish the evaluation criteria of the pig model to be developed by brain MRI of the APP gene-regulated mini-pig, an image analysis of APP gene-regulated mini-pig was performed using a brain MRI tomograph of a collaborative animal hospital.
Imaging analysis has the advantage of being able to visualize lesions more efficiently than MRI. The xylazine is administered at a dose of 2 mg / kg at a dose of 4 mg / kg of ketamine through the apical meristem of the APP gene control mini pig Isoflurane, an inhalation anesthetic, was maintained during imaging.
MRI images were taken under plain and enhanced conditions, and brain images were acquired through MRI of APP gene-regulated mini-pigs. Using the normal mini pig as a control, the same brain imaging data were obtained.
FIG. 9 shows PET / CT images of 18F FDG of a pig for the Alzheimer's disease model of the present invention. Compared with the control group, a decrease in brain metabolism was observed in the overall area of the AD model pig except for the primary somatosensory cortex. This was similar to the typical FDG imaging findings seen in patients with severe AD.
FIG. 10 shows the results of 18F MRI imaging. In the MRI image, it was confirmed that the AD model pig of the present invention showed enlargement of the ventricles and atrophy of the cerebral cortex (arrow) compared to the control group.
These results show that the transgenic pig of the present invention can be very useful as a human Alzheimer's disease model.
Example 6: Behavioral monitoring
And to develop and standardize the motor rating scale and behavioral rating scale in the control animals and the normal cloned animals and the transformed model animals of the present invention. The following three behavioral experiments were performed (Fig. 12) to confirm the pattern (not shown).
① Observing basic behavior
- CCTV was installed and monitored in the money pig breeding piglets. They were able to observe and understand basic behaviors and tendencies such as daily movement, behavior patterns, and personality, and how they differed from the original behavior patterns of mini pigs and whether they showed typical symptoms of AD.
- Disease model After mini-pig was born, it was observed continuously, and the time when abnormal behavior or personality occurred and basic data about mini-pig was built.
- Analysis of the movement through the analytical system by repeatedly recording the usual behavior on the side and sides of the pension company. Especially, it shows the change of the behavior pattern (dietary, negative number of times, sleep time, etc.) The results were as follows: (1) the severity of the disease was assessed by scoring the most severe symptom to 4;
② Open field test
- A mini-pig was placed in a room of 3m × 3m size, and the movement and movement direction, the moving time, and the movement pattern were examined, and the abnormality and the change were observed. In addition, mini-pigs were constructed in such a way that they could not be seen, and whether mini-pigs were more preferred, or how many times they were crossing the block.
- The same experiment was carried out periodically to identify the point at which the abnormality occurred, and after the abnormality was found, the analysis was carried out with an emphasis on grasping the extent.
- We compared the results of the control group with those of the control group to determine the degree of disease in the mini pigs based on whether the results of the test group were significantly different when analyzed based on parameters such as time and distance.
③ Position memory test
- Prepare a plate with a total of four rows and four holes per row, with a total of sixteen holes in it, randomly pick a few holes and put the food in it, then move the mini pig around freely To eat food. The same experiment was repeated several times to make it possible to remember the hole containing the food among the 16 holes. It was judged whether or not the position was memorized by measuring the time from the hole where the food was placed to the time when all the food was found and eaten.
- The memory capacity of the control group and the experimental group was compared and analyzed by observing the change of the time taken through repeated execution.
These observations also show that the transgenic cloned pigs of the present invention exhibit AD typical symptoms as compared to control animals and normal cloned animals and lose their ability to exercise and memory to a similar extent as in the case of serious AD patients in humans I could confirm.
<110> Seoul National University R & DB Foundation Corporation <120> Transgenic cloned porcine Models for alzheimer's disease and the Use thereof <130> CP15-104 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 577 <212> DNA <213> Artificial Sequence <220> <223> human synapsin 1 promotor <400> 1 atcgattgca gggcccacta gtatctgcag agggccctgc gtatgagtgc aagtgggttt 60 taggaccagg atgaggcggg gtgggggtgc ctacctgacg accgaccccg acccactgga 120 caagcaccca acccccattc cccaaattgc gcatccccta tcagagaggg ggaggggaaa 180 caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca ccgcggacag tgccttcgcc 240 cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa ggcgcgctga cgtcactcgc 300 cggtcccccg caaactcccc ttcccggcca ccttggtcgc gtccgcgccg ccgccggccc 360 agccggaccg caccacgcga ggcgcgagat aggggggcac gggcgcgacc atctgcgctg 420 cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc agcggaggag tcgtgtcgtg 480 cctgagagcg cagctgtgct cctgggcacc gcgcagtccg cccccgcggc tcctggccag 540 accaccccta ggaccccctg ccccaagtcg cctcgag 577 <210> 2 <211> 2088 <212> DNA <213> Artificial Sequence <220> <223> Amyloid Precursor protein 695 swedish <400> 2 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt tcgagttcct acaacagcag ccagtacccc tgatgccgtt 900 gacaagtatc tcgagacacc tggggatgag aatgaacatg cccatttcca gaaagccaaa 960 gagaggcttg aggccaagca ccgagagaga atgtcccagg tcatgagaga atgggaagag 1020 gcagaacgtc aagcaaagaa cttgcctaaa gctgataaga aggcagttat ccagcatttc 1080 caggagaaag tggaatcttt ggaacaggaa gcagccaacg agagacagca gctggtggag 1140 acacacatgg ccagagtgga agccatgctc aatgaccgcc gccgcctggc cctggagaac 1200 tacatcaccg ctctgcaggc tgttcctcct cggcctcgtc acgtgttcaa tatgctaaag 1260 aagtatgtcc gcgcagaaca gaaggacaga cagcacaccc taaagcattt cgagcatgtg 1320 cgcatggtgg atcccaagaa agccgctcag atccggtccc aggttatgac acacctccgt 1380 gtgatttatg agcgcatgaa tcagtctctc tccctgctct acaacgtgcc tgcagtggcc 1440 gaggagattc aggatgaagt tgatgagctg cttcagaaag agcaaaacta ttcagatgac 1500 gtcttggcca acatgattag tgaaccaagg atcagttacg gaaacgatgc tctcatgcca 1560 tctttgaccg aaacgaaaac caccgtggag ctccttcccg tgaatggaga gttcagcctg 1620 gacgatctcc agccgtggca ttcttttggg gctgactctg tgccagccaa cacagaaaac 1680 gaagttgagc ctgttgatgc ccgccctgct gccgaccgag gactgaccac tcgaccaggt 1740 tctgggttga caaatatcaa gacggaggag atctctgaag tgaacttgga tgcagaattc 1800 cgacatgact caggatatga agttcatcat caaaaattgg tgttctttgc agaagatgtg 1860 ggttcaaaca aaggtgcaat cattggactc atggtgggcg gtgttgtcat agcgacagtg 1920 atcgtcatca ccttggtgat gctgaagaag aaacagtaca catccattca tcatggtgtg 1980 gtggaggttg acgccgctgt caccccagag gagcgccacc tgtccaagat gcagcagaac 2040 ggctacgaaa atccaaccta caagttcttt gagcagatgc agaactag 2088
Claims (17)
(a) a step of preparing a nuclear donor cell comprising culturing somatic cells or stem cells isolated from a tissue of a pig;
(b) introducing a recombinant vector containing the human synapsin 1 promoter and the APP695sw mutant gene into the nuclear donor cell;
(c) removing the nucleus from the oocyte of the pig to produce a enucleated oocyte;
(d) injecting and fusing nuclear donor cells of step (b) into the enucleated oocytes of step (c);
(e) activating the oocyte fused in step (d); And
(f) transplanting the activated oocyte to the fallopian tubes
Wherein the method comprises the steps of:
1) administering a candidate substance for preventing and treating Alzheimer's disease to a pig for Alzheimer's disease model according to any one of claims 1 to 4;
2) After administration of the candidate substance, the brain tissue of the pig was compared with a control group to which no candidate substance was administered. At this time, the brain tissue analysis revealed that at least one of the enlargement of the ventricle, atrophy of the hippocampus and overexpression of amyloid beta protein Selecting a candidate substance that reduces the amount of the candidate substance; And
3) selecting the candidate substance as an agent for preventing and treating Alzheimer's disease.
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| KR102563238B1 (en) * | 2020-05-06 | 2023-08-04 | 주식회사 옵티팜 | Transgenic cloned porcine for animal models with increased susceptibility to severe acute respiratory syndrome coronavirus 2, and method for producing the same |
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