KR20180037449A - Brain tumor animal model and the Use thereof - Google Patents

Abstract

The present invention relates to a brain tumor animal model which overexpresses the CreER^T2 gene and cancer gene in the cerebrum, and to a screening method for brain tumor prevention and treatment agent using the same. In an animal transformed with a recombinant vector, since the CreER^T2 and cancer gene are overexpressed in the cerebrum and not expressed in other organs, the CreER^T2 and cancer gene can be used as a brain tumor animal model, wherein the animal model can be used for various purposes such as identification of disease pathology, search for therapeutic substances, development of diagnostic methods and the like to be usefully used for the development of a brain tumor prevention and treatment agent.

Description

Brain Tumor Animal Model and Its Use {

The present invention relates to a brain tumor animal model that overexpresses the CreER ^T2 gene and cancer gene in the cerebrum, and a method for screening a brain tumor prevention and treatment agent using the same.

Brain tumors are classified according to the type of cells that are thought to originate from the tumor. Diffuse, fibrous astrocytoma is the most common type of primary brain tumor in adults. These tumors are histopathologically classified as grade 3 malignancies: World Health Organization (WHO) grade Ⅱ astrocytoma, WHO grade Ⅲ undifferentiated astrocytoma and WHO grade IV polymorphic subspecies (GBM). WHO grade Ⅱ astrocytoma is the most helpless diffuse astrocytoma spectrum. The astrocytomas exhibit a remarkable tendency to penetrate the surrounding brain, which confounded the therapeutic approach in local control. These invasive abilities are often evident not only in low grade but also in high grade tumors. Glioblastoma multiforme (GBM) is the most malignant stage of astrocytoma with a survival time of less than 2 years in most patients. Histologically, these tumors are characterized by dense cytoplasm, hyperproliferative index, endothelial proliferation and focal necrosis. The high proliferation properties of these lesions are likely to result from mitotic effects. One of the characteristics of GBM is endothelial proliferation. There is a biological subset of astrocytomas, which show the clinical heterogeneity observed in these tumors. These subsets include brain stem gliomas, which are often in the form of pediatric diffuse, fibrous astrocytomas followed by a malignant course. Brain stem GBMs share genetic characteristics with their adult GBMs affecting younger patients. Polymorphic yellow astrocytoma (PXA) is a low-grade, low-grade astrocytoma that mainly affects adolescents. These tumors have a unique histologic appearance and are typically slow-growing tumors that can be treated with surgical treatment. However, some PXAs can recur as GBM. The pilocytic astrocytoma is the most common astrocytoma of childhood and is clinically and histopathologically different from diffuse, fibrous astrocytoma affecting adults. Shape cell astrocytoma does not have the same genomic changes as diffuse, fibrous astrocytoma. Subependymal Giant Cell Astrocytoma (SEGA) is a low-grade astrocytic tumor around the ventricle that is usually associated with tuberous sclerosis (TS) and is a histologically so-called "candle- Quot; candle-guttering ". Similar to other tumor lesions in TS, they grow slowly and may be more similar to harmartomas than true neoplasms. (DCAI) and connective tissue forming infant ganglioneuromas (DIGG) in infancy are large, usually cystic, benign astrocytomas affecting one or two year old infants. Hypoglycemia and rare astrocytomas (mixed gliomas) are brain tumors that are most closely related clinically and biologically to diffuse, fibrous astrocytomas in general. However, these tumors are less common than astrocytomas and generally have better predictability than diffuse astrocytomas. Hypoglycemia and rare astrocytoma can progress to WHO grade III malignant hyperstimulation or malignant rare astrocytoma, or WHO grade IV GBM. Thus, genetic changes that cause oligodendroglioma tumors constitute a different pathway for GBM. Cerebrospinal fluid cytokines range from aggressive deep mesenteric tumors in infants to benign spinal cord tumors in adults. Conversion of CTC to GBM is rare. Choroidal neoplasia is a group of tumors predominantly occurring in the ventricular system and may range from an aggressive supratentorial ventricular tumor of the infant to a benign cerebellopontine angle tumor of the adult. Corticoid tumors have often been reported in patients with Li-Fraumeni syndrome and Von Hippel-Lindau disease. Mesoblastoma is a highly malignant primary tumor that occurs mainly in the infant's larynx and posterior fossa. Meningioma is a common intracranial tumor that occurs in the meninges and compresses the underlying brain. Meningiomas are usually benign, but some may have a "local" recurrence of "atypical" meningiomas, and some meningiomas are frankly malignant and may invade or migrate into the brain. Dysplasia and malignant meningiomas are not as common as benign meningiomas. Schwannoma is a benign tumor arising in the peripheral nerve. Schizophrenia can occur in the anterior cranial nerve, especially in the anterior part of the eighth cranial nerve (anterior schwannoma, cranial nerve), and is present as cerebellopontine angle masses. The angiomyocyte is a tumor of unspecific origin, consisting of endothelial cells, pericytes and so-called stromal cells. These benign tumors occur most frequently in the cerebellum and spinal cord of adolescents. Multiple angiomyocytoma is a characteristic of von Hippel-Linda disease (VHL). Aneurysmal tumors (HPCs) are locally aggressive tumors of the brain that can be metastasized. Tissue formation of the meningiomas (HPC) has been discussed for a long time, and some authors classify it as a separate entity and the remaining authors classify it as a subtype of meningiomas. The symptoms of both primary and metastatic brain tumors mainly depend on the location in the brain and the size of the tumor. Since each area of the brain is responsible for a specific function, the symptoms are quite different. Tumors in the frontal lobe of the brain can cause helplessness and paralysis, mood disturbance, difficulty thinking, confusion and disorientation, and a wide range of emotional mood changes. Pseudotumor tumors can cause seizures, numbness or numbness, difficulty in writing, inability to perform simple mathematical problems, difficulty with certain movements, and loss of touch sensation. Tumors in the occipital area can cause vision loss, visual hallucinations and seizures in half of each field of view. Temporal lobe tumors can cause paralysis, perception and spatial impairment and receptive aphasia. If the tumor occurs in the cerebellum, the person may have ataxia, loss of coordination, headache, and vomiting. Tumors in the hypothalamus can cause emotional changes and can cause subjective changes in both hot and cold. Hypothalamic tumors can also affect infant growth and nutrition. With the exception of the cerebellum, tumors on one side of the brain cause symptoms and disorders on the other side of the body.

Under these circumstances, in order to find new therapeutic agents for brain tumors, 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 determining the mechanisms of brain tumors and verifying the effectiveness of various new therapeutic targets and new therapies.

Until now, most of the disease models for pharmacotherapy and mechanism studies have been using rodents, but the pathology patterns and symptoms of animal disease models are much different from those observed in humans, so clinical trials based on results from rodent disease models The number of animals that can be used as an effective brain tumor 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.

The present inventors have confirmed that the CreER ^T2 gene and the oncogene is overexpressed in the brain in the established recombinant vector, wherein the recombinant vector introduced into an animal model including a CreER ^T2 gene and the oncogene, as well as this, a brain tumor animal models The present invention has been completed.

It is an object of the present invention to provide a recombinant vector for constructing a brain tumor animal model comprising CreER ^T2 gene and cancer gene.

Another object of the present invention is to provide a brain tumor animal model into which said recombinant vector has been introduced.

It is another object of the present invention to provide a method of screening for a preventive and therapeutic agent for brain tumors.

In order to achieve the above object, the present invention provides a recombinant vector for constructing a brain tumor animal model comprising CreER ^T2 gene and cancer gene.

The present invention also provides a transformed cell for producing a brain tumor animal model into which the recombinant vector is introduced.

In addition, the present invention provides a nuclear transfer embryo formed by transplanting the nucleus of somatic cells transformed with the recombinant vector into an enucleated oocyte.

In addition, the present invention provides a method for producing a brain tumor animal model, characterized in that the core of the nuclear donor cell into which the recombinant vector is introduced is transplanted into the enucleated oocyte to produce a live egg.

In addition, the present invention provides a brain tumor animal model characterized by overexpressing CreER ^T2 and cancer gene transformed by the recombinant vector.

Also, the present invention provides a method for treating a brain tumor, comprising the steps of: 1) administering a tumor tumor prophylactic or therapeutic agent candidate substance to a brain tumor animal model; And 2) judging the brain tissue of the animal model as a brain tumor prevention or treatment agent when the expression of the cancer gene is inhibited compared with a control group in which the candidate agent is not administered after administration of the candidate substance, Screening method.

In an animal transformed with a recombinant vector containing CreER ^T2 gene and cancer gene according to the present invention, CreER ^T2 and cancer gene are overexpressed in the cerebral cells and not expressed in other organs, and therefore, they can be used as a brain tumor animal model . In addition, the animal model can be used for various purposes such as identification of a disease mechanism, search for a therapeutic substance, development of a diagnostic method, and so on, so that it can be usefully used for development of a brain tumor prevention and therapeutic agent.

Figure 1 is a schematic diagram of an astrocyte-specific CreER ^T2 -mediated recombination system.
Fig. 2 shows the result of (a) classification of the donor cell line transformed with the LCMV-EGFP ^LoxP plasmid using the fluorescence activated cell sorting method (FACS) and (b) observation of the expression of EGFP using the fluorescence microscope Fig.
FIG. 3 is a graph showing the results of PCR for confirming the introduction of a system vector into a donor cell line. FIG.
FIG. 4 is a diagram showing the results (a and b) of evaluation of the SCNT fold development and the result (c) summarizing the results.
Fig. 5 shows five transgenic piglets born from embryo-transferred surrogate mothers.
Figure 6 shows the expression of pGFAP-CreER ^T2 ; EGFP ^LoxP gene was introduced by PCR.
FIG. 7 shows the result of performing PCR using F and R primers. FIG.
FIG. 8 is a graph showing the result of observing immunofluorescence (IF) staining in order to confirm whether CreER ^T2 gene controlled by GFAP promoter is expressed in GFAP-positive glial cells.
FIG. 9 is a graph showing the result of observing immunofluorescence (IF) staining for confirming the expression of an EGFP transgene in brain tissue. FIG.
Fig. 10 shows the results of a comparison of porcine astrocytic-specific pGFAP-CreER ^T2 ; ^{FIG. 8} is a ^graph showing the results of PCR analysis performed using the FR 'set in order to confirm whether the EGFP ^LoxP recombination system is operated by the TM treatment.

The present invention provides a recombinant vector for constructing a brain tumor animal model comprising CreERT2 gene and oncogene.

Hereinafter, the present invention will be described in detail. More specifically, in one embodiment of the present invention, pGFAP-CreER ^T2 ; EGFP ^LoxP recombination system.

The recombination system comprises two vectors, one is the GFAP promoter -CreER ^T2 gene vector and the other is the LoxP-EGFP gene vector.

The CreER ^T2 gene may be represented by the nucleotide sequence of SEQ ID NO: 1, and variants capable of functionally equivalent to the nucleotide are included in the scope of the present invention.

As used herein, the term " functionally equivalent " means that at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% Means a nucleotide sequence capable of encoding a protein having substantially the same physiological activity as a protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 and having a sequence homology of 95% or more. For example, although some base sequences have been modified by deletion, substitution, or insertion, they include variants that can function in a functionally equivalent manner to the nucleic acid molecule encoding the fusion protein .

The oncogene refers to a genetic material for producing cancer cells, that is, a gene having an ability to induce cancer, and is also referred to as a carcinogenic gene or a tumor gene. The cancer gene may be selected from the group consisting of sis, EGFR, FGF family (bFGF, aFGF, int2 / FGF3, hst1 / K-fgf / FGF4, FGF5, hst2 / FGF6, KGF, AIGF, erbB1, erbB2 / neu (FGFR2 / K-sam / bek, KGFR, FGFR1 / N-sam / flg / Cek1 / bF, FGF3, trkA, trkB, trkC, ret kit, PDGFR ?, flt1, flt3, flk1 / kdr, flk2, FGFR family (H-ras, K-ras, fck, fck, blk, csk, fps, fs, mas, gsp, gip, ras family (src, yes, fyn, fgr, lyn, lck, (Myb, A-rac), mos, raf family (raf, A-raf1, B-raf, rel, ski, sno), myc family (eta, ets2, erg, elk1, Spi1 / PU.1, fli1, myb, B-myb), the fos family (fos, fosB, fra1, fra2), jun family (jun, junB, junD, jif1) May be at least one selected from among GABPa, elf1, SAP1), db1, ect2, bcl1, bcl2, bcl3, bcl6, bcr, pml, pbx1 / prf, PIK3CA, all1 / mll, aml1, dec, ews, tls / , Preferably EGFR, H-ras, Myc, PIK3CA, but is not limited thereto.

The recombinant vector comprises a GFAP promoter, which is isolated from the promoter sequence of GFAP (glial fibrous acid protein) gene, one of the marker genes of astrocytic lineage.

Compared to the 2.2 kb human GFAP promoter sequence established and used in many previous studies, the GFAP promoter sequence of the pig has a conserved sequence at a fairly high rate. According to previous reports, the sequence of -1,600 bp at -1,400 bp for GFAP mRNA transcription initiation has been found to play an important role, and the similarity of this part is very high. This implies the possibility of inducing nonspecific expression when a human GFAP promoter sequence is applied to a pig model. Therefore, it is expected that the application of the mini pig-native GFAP promoter sequence in the mini-pig model can realize a more precise stellate cell-specific recombination system.

In one embodiment of the present invention, a porcine GFAP promoter containing a porcine GFAP promoter is used in the expression vector, and the porcine GFAP promoter may be represented by SEQ ID NO: 2.

In addition, the recombinant vector of the present invention may further comprise a reporter gene, which is located next to the reporter gene, LoxP which can be removed through CreER ^T2 mediated recombination. The reporter gene includes a green fluorescent protein (GFP) gene, an enhanced green fluorescent protein (EGFP) gene, a red fluorescence (RFF) gene, a luciferase gene, The gene may be a beta-galactosidase (EBG) gene, a chloramphenicol gene, a chloramphenicol acetyltransferase (CAT) gene or an alkaline phosphatase gene, preferably an enhanced green fluorescent protein ; EGFP) gene, but is not limited thereto.

As used herein, the term "vector" or "expression vector" refers to a plasmid, virus vector, or vector that can be inserted into a nucleic acid encoding a structural gene and capable of expressing the nucleic acid in a host cell Or other media. 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 lentivirus is preferable. The main advantage of lentiviral vectors for gene therapy is that they transfer large amounts of genes into the cloned cells, integrate the genes transferred into the cellular DNA precisely, and do not cause subsequent infection after gene transfection.

The recombinant vector of the present invention 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 does not.

The present invention also provides a transformed cell for producing a brain tumor animal model into which the recombinant vector is introduced.

As used herein, the term " brain tumor " refers to all pre-cancerous and cancer cells that exhibit all neoplastic cell growth and proliferation whether malignant or benign.

The recombinant vector 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.

In addition, the present invention provides a porcine nuclear transfer embryo formed by transplanting the nucleus of a porcine-derived somatic cell transformed with the recombinant vector into an enucleated oocyte.

As used herein, the term " nuclear transfer " refers to a genetic engineering technique that allows an enucleated oocyte to artificially bind other cells or nuclei to the same trait. 'Nuclear transfer embryos' refers to oocytes into which nuclear donor cells are introduced or fused.

As used herein, the term " cloning " is a genetic engineering technique for creating new individuals with the same set of genes as an individual. In particular, in the present invention, somatic cells, embryonic cells, fetal cells and / Quot; nucleotide sequence " of the " nucleotide " 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.

As used herein, the term " nuclear donor cell " refers to the nucleus of a cell or cell that delivers nuclei to a nuclear receptor, a recipient oocyte. 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.

As used herein, the term " somatic cell " refers to a cell other than a germ cell among cells constituting a multicellular organism, Lt; RTI ID = 0.0 > cell < / RTI > having different functions.

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.

The present invention also provides a brain tumor animal model characterized by overexpressing CreER ^T2 and an cancer gene transformed by said combination vector.

The transfection may be carried out by somatic cell nuclear transfer (SCNT), and expression of the recombinant vector may occur in the brain tissue, preferably in the cerebrum. At this time, the expression of the recombinant vector can be induced by treatment with tamoxifen.

The brain tumor animal model can be obtained by transplanting the nucleus of the nuclear donor cell into which the recombinant vector has been introduced into the enucleated oocyte to produce a live egg.

As used herein, the term " animal model " refers to an animal having a disease in a form very similar to that of a human. 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.

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.

As used herein, the term "cultivation" refers to cultivating an organism or a part of an organism (organ, tissue, cell, etc.) under an appropriately artificially controlled environmental condition. In this case, the temperature, humidity, light, (Carbon dioxide and oxygen partial pressure), and other important factors directly affect the organism to be cultivated is the medium (incubator), which is a direct environment for the organism and at the same time a supply point for various nutrients to be.

As used herein, the term " in vitro culture " refers to a series of laboratory procedures in which cells are cultured in an incubator in a laboratory in a manner similar to the environment in the body, in a manner distinct from the growth of cells.

As used herein, the term "medium" or "medium composition" refers to a medium which is capable of growing and growing cells in vitro, including essential elements for growth and proliferation of cells such as sugars, amino acids, various nutrients, serum, Refers to a mixture for growth.

As used herein, the term " differentiation " refers to a phenomenon in which the structure or function of one another is specialized during the growth of cells through proliferation and proliferation, that is, I mean that function changes. In contrast, relatively simple systems are separated into two or more qualitatively different sub systems.

As used herein, the term " fusion " refers to the association of the nuclear donor cell with the lipid membrane portion 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.

As used herein, the term " 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.

The recombinant gene comprising the CreER ^T2 gene and the cancer gene according to the present invention is highly expressed in the cerebral cells and highly expressed in the cerebral cells and the recombinant gene is not expressed in other organs, , The manufactured animal model can be used for various purposes such as identification of disease mechanism, search for therapeutic substance, development of diagnostic method, and thus, it can be usefully used for development of brain tumor prevention and treatment agent.

Also, the present invention provides a method for treating a brain tumor, comprising the steps of: 1) administering a tumor tumor prophylactic or therapeutic agent candidate substance to a brain tumor animal model; And 2) judging the brain tissue of the animal model as a brain tumor prevention or treatment agent when the cancer gene expression is suppressed as compared with a control group in which the candidate agent is not administered after administration of the candidate substance, and screening for a brain tumor prevention or treatment agent ≪ / RTI >

The term "candidate substance " used in the present invention means a substance to be tested as a brain tumor prevention and treatment agent, and includes any molecule such as an extract, protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, . Such candidate materials also include synthetic materials as well as natural materials.

As used herein, the term "prevention" means any action that inhibits or delays brain tumors by administration of a candidate agent compared to a control without administration of the candidate agent, The administration of the candidate substance means any action that improves or alters the brain tumor symptom of the animal model.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

Example  One. Astrocytes - specific pGFAP - Creer ^T2 ; EGFP ^LoxP  Preparation of Donor TG Pig Fibroblast with Recombination System

1-1. Lent  Virus production

To produce lentivirus, 293 FT cells were plated in complete DMEM medium containing 100 mm plates of 10% fetal bovine serum (Serena) and 2 mM L-glutamine (Lonza, Basel, Switzerland) and allowed to attach for 12 hours Respectively. Transfection was performed according to the manufacturer's instructions with 4 μg transfer vector plasmid, 6 μg third generation packaging plasmid and 24 μl polyexpress agent (Excellgene Inc. Rockville, USA) in serum-free DMEM medium (Invitrogen) . After 6 hours of transformation, the cells were washed with phosphate buffered saline (PBS) and added to fresh complete DMEM medium containing L-glutamine and 10% FBS and incubated for 42 hours. The medium containing the lentivirus was then collected after 48 hours of transformation and filtered through a 0.45 micron syringe. Each lentivirus was enriched using a Lenti-X Concentrator (Clontech Laboratories, Inc.) according to the manufacturer's instructions.

1-2. Preparation of recombinant vector

Stromal cell-specific GFAP-CreER ^T2 using a porcine GFAP promoter sequence; An EGFP LoxP recombination system was devised and the vector used in the present invention is schematically shown in Fig.

As shown in Figure 1, the system consists of a plasmid vector construct comprising a lentiviral vector of the CreER ^T2 transgene controlled by the porcine GFAP promoter and an expression EGFP transgene con- nected by two LoxP sites The two vectors were introduced into nuclear donor fibroblast cells. At this time, as the porcine GFAP promoter, the promoter represented by the nucleotide sequence of SEQ ID NO: 2 was used.

First, the pigs -GFAP-CreER ^T2 gene construct in order to produce a lentiviral vector containing the root trucks, pCDH-CMV-MCS-EF1 -puro lentiviral vector of the CMV promoter, the two restriction enzymes, SnaBI and NheI and, pGL3- Pig GFAP promoter sequences (from -1,817 to 17 upstream from the GFAP transcription start site) truncated from EcoRI (Blunted by Klenow fragment) and NheI from the base-porcine GFAP promoter plasmid were removed and ligated into the vector. The CreER ^T2 gene cleaved by EcoRI from the pCre-ER ^T2 plasmid was then inserted into the same restriction enzyme site of pCDH-pGFAP-MCS-EF1-puro.

To prepare the CMV-LoxP-EGFP-pA-LoxP construct, LCMV: ECFP (LoxP) MCS (# 31377, Addgene) was modified. EGFP-C2 (# 6083-1, CLONTECH Laboratories, Inc.) digested with the same restriction enzyme was inserted into the ECFP sequence removed by Nco1 and BsrG1.

1-3. Donor cell line  Establish

First, Yucatane miniature swine fibroblasts are transformed with LCMV-EGFP (LoxP) -ESD using an electroporation tool (Neon Transfection System, Invitrogen) according to the manufacturer's instructions. After 3 to 4 weeks, EGFP-positive cells were isolated using FACS Aria II (BD biosciences, San Jose, USA).

To introduce the porcine GFAP-CreER ^T2 construct, EGFP-positive cells were infected with enriched lentiviral cells of pCDH-pGFAP-CreER ^T2 -EF1-puro. Three days after infection, infected cells were cultured in complete medium and puromycin (2.0 ug / ml) was added for 5 days.

In order to confirm whether the donor cell was properly established, the transformed LCMV-EGFP (LoxP) plasmid vector was classified using the fluorescence activated cell sorter (FACS) method, and the result is shown in FIG. 2A . In addition, the expression of EGFP was observed using a fluorescence microscope, and the results are shown in FIG. 2B.

Thereafter, three cell colonies obtained after antibiotic selection were determined as the final donor cells. PCR was used to confirm the introduction of each transgene into the genome. The results are shown in FIG.

As shown in Fig. 3, CreER ^T2 and EGFP appeared, confirming that a recombinant gene was introduced into donor cells.

Example  2. GFAP - CreER; EGFP ^LoxP  TG using cells Replica of pig  production

2-1. Primary culture

Ear tissues separated from dead pigs were washed with dPBS + (dPBS with 1x anti-anti), then sterilized with ethanol and washed with dPBS +. The skin tissue was detached from the sterilized ear tissue and the tissue was chopped with a razor blade. Clipped skin tissue was incubated with trypsin for 3 hours and then added to cell culture medium (2x anti-anti + 10% FBS + DMEM) for trypsin neutralization. The tissue treated with trypsin was cultured in a 100 mm culture dish and adherent cells were subcultured. The initial culture was carried out in a humid incubator (Astec, Fukuoka, Japan) under 5% CO ₂ at 39 ° C.

2-2. Oocyte collection and in vitro maturation, IVM )

Porcine ovaries were harvested from slaughterhouses and then porcine follicular fluid (pFF) and cumulus-ovum complexes (COCs) were obtained by inhalation in 3-6 mm ovarian follicles. The medium used during IVM was TCM 199 (Gibco, Life Technologies, Melbourne, Australia), 0.6 mM cysteine, 0.91 mM sodium pyruvate, 10 ng / ml epithelial cell growth factor, 75 g / ml kanamycin, 1 g / ml insulin, 10% (v / v) and Pff. Porcine COCs were co-cultured in 50-60 cells per well in 500 ml IVM medium in 4-well plates (Nunc, Roskilde, Denmark). IVM was performed at 39 ° C and 5% CO ₂ using a humid incubator (Astec, Fukuoka, Japan). Maturation was carried out for 22 hours in IVM medium containing 10 IU / ml horse chorionic gonadotropin (eCG) and 10 IU / ml human CG. The cells were then transferred to hormone-free IVM medium and incubated for 18 hours. The mature COCs were then stripped off using a soft pipette in 0.1% hyaluronidase and TLH-PVS medium. Peeled oocytes were obtained through the above process and were then used in the experimental procedure.

2-3. Somatic cell Nuclear transfer ( SCNT ) And in vitro culture IVC )

(Normal cell line), Cloud # 5 pGFAP-CreER ^T2, EGFP ^LoxP (vector-inserted cell line), and TG donor cell line were subjected to somatic cell nuclear transfer (SCNT) And a recombinant-TG cell line were used as donor cells.

Specifically, after 40 hours of IVM, medium defect II oocytes were selected for the enucleation process. Middle-stage II-stage oocytes were washed three times with 0.2% bovine serum albumin (TLH-BSA) containing calcium-free TLH supplemented with 5 μg / ml cytochalasin B (CB) and washed three times with a brown rice manipulator (Humagen, Charlottesville, Va., USA) was used for enucleation. After enucleation, fetal fibroblasts treated with trypsin were transferred into the inner nuclear layer of the enucleated oocytes. Then, using two pulses of 180 V / mm DC for 60 μs in a 260 mM mannitol solution containing 0.1 mM CaCl ₂ and 0.05 mM MgCl ₂ using a cell fusion generator (LF201; Nepa Gene, Chiba, Japan) Respectively. After electrical fusion, SNCT embryos were incubated with 6 μl of 6-dimethylaminopurine (6 DMAP) for 30 min with 30 μl of porcine ligation medium (PZM) supplemented with 5 μg / ml CB for 4 h. The embryo was then transferred to the PZM drop for IVC. On the second day after fusion, embryo cleavage was evaluated (1 cell, 2 cell, 4 cell, 8 cell, piece) and quantification of blastocyst formation (initial blastocyst, expanded blastocyst and hatching blastocyst) 4A to 4B, and the results are summarized in FIG. 4C.

As shown in Figure 4c, Cloud male # 5 (normal cell line), Cloud # 5 pGFAP-CreER ^T2 ; The fusion rates (71.8 ± 6.1, 74.2 ± 2.6, and 76.7 ± 2.9) and truncation rates (73.8 ± 4.0, 63.6 ± 2.2, and 62.6 ± 13.7) of EGFP ^LoxP (vector insertion cell line) and clone-TG cell line were not significantly different . The blastocyst formation rate was not significantly different between the normal cell line (Cloud male # 5) and vector insertion cell line (Cloud # 5 pGFAP-CreERT2; EGFPLoxP) (19.5 ± 1.2, 21.0 ± 1.6) (21.0 ± 1.6, 39.2 ± 5.0), which is significantly higher than that of vector-injected cell lines.

This indicates that SCNT embryo development due to foreign genes in the donor cell is not affected, and that the clone cell line is established and that the recombinant cell line can be used in SCNT donor cells to increase TG embryo production and TG production efficiency.

2-4. Embryo implantation

Non-overfed, natural circulation Landrace x Duroc's young hybrid sows were used as surrogate mothers. First, 1 ml of ketamine (Yuhan 50 mg / ml) and 3 ml of Xylezine (100 mg / ml) were injected into the ear vein of the pig for anesthesia. Thereafter, isoflurane (liquid phase) was used to maintain the respiratory anesthesia state. All surgical instruments were disinfected for surgery. Abdominal laparotomy was performed for exposure of the reproductive tract. Four hours later, SCNT embryos were transferred from the bulbous isthmus junction to the oviduct. Thirty days later the pregnancy was diagnosed by ultrasound.

Embryo transfer was performed three times immediately before the ovulation state of the surrogate mother. One of them was pregnant and gave birth to 5 TG piglets 115 days after pregnancy, which is shown in FIG.

However, the three piglets were stillborn, another died after 22 days, and only one survived. The results from the gDNA of skin cells showed that all five piglets were pGFAP-CreER ^T2 ; The introduction of the EGFP ^LoxP gene was confirmed by PCR, and the results are shown in Fig. After 3 months, 15 mg / kg of TM was orally administered for 5 days and euthanized 7 days later.

As shown in Fig. 6, it was confirmed that all the recombinant genes appeared in piglets # 1 to # 5.

Example  3. Transgenic pig  In the nervous system pGFAP - Creer ^T2 ; EGFP ^LoxP  Whether the system is manifested

To determine whether the transgene controlled by the porcine GFAP promoter sequence was specifically expressed in the astrocytic lineage, two TG pigs spontaneously aborted, four TG pigs containing # 2 and # 3 Each tissue was analyzed by quantitative PCR (qPCR) method. Specifically, qRT-PCR was performed first to determine mRNA levels. Total RNA was isolated from the cells using a trizol reagent (Invitrogen) according to the manufacturer's instructions. RNase-free DNase-treated RNA (1 μg) was used as a template for cDNA synthesis using the RevertAid ™ First strand Cdna synthesis kit (Thermo) according to the manufacturer's instructions. qRT-PCR analysis was performed using Takara SYBR Premix Ex Taq and CFX096 (Bio-Rad). The expression level of the target gene was normalized by 18S rRNA as a control group, and the respective primer sequences are shown in Table 1 below.

Genomic DNA samples from each frozen tissue and cell were isolated using the Wizard® Genomic DNA Purification Kit (Promega, Madison, USA) according to the manufacturer's instructions. For large and coarse tissue, each tissue was homogenized prior to the genomic DNA extraction process using Bullet Blender® (Next Advance Inc. New York, USA) according to the manufacturer's recommended method. Genomic DNA transfected genes were amplified in heat cycler with each primer set (EGFP, CreER ^T2 , 18s) and DNA polymerase (Genecraft, Manchester, UK) .

As shown in Fig. 7, the CreER ^T2 gene showed high expression in the brain as compared with other tissues along with the endogenous GFAP level.

Immunofluorescence (IF) staining was also performed to determine whether the CreER ^T2 gene, which is regulated by the GFAP promoter, is expressed in GFAP-positive glial cells. Specifically, the pig brain tissue contained in the paraffin was cut into 4 μm-thick pieces and GFAP (691102; 1: 500; MP Biomedical ImmunOTM), Cre recombinase (1: 200; Cell Signaling) for 12 hours at 4 ° C, , EGFP (AB6673; 1: 200; Abcam, AB290; 1: 200; Abcam) and as100? (S2644; 1: 200; Sigma). After staining, secondary antibodies conjugated with Alexa Fluor 488 or 594 (A11001, A11005, A11012, or A11055; 1: 500, respectively; Thermo Fisher Scientific Inc.) were incubated for 1 hour at room temperature. The nuclei were stained with 1 μg / mL of DAPI (4 ', 6-diamidino-2-phenyl) for 5 minutes, and then fluorescence images were observed using a confocal laser microscope (LSM5 Pascal, Carl Zeiss) The results are shown in Fig.

As shown in Fig. 8, CreER ^T2 Gene expression was found not only in star-shaped GFAP-positive astrocytes but also in some non-astrocytes.

In addition, fibroblasts from ear tissues of # 2 and # 3 TG pigs were treated with 4OH-TM to confirm whether the GFAP promoter transcribed its transgene in non-astrocytic cells. CreER ^T2 regulated by the GFAP promoter did not work in these cells. In addition, the expression of EGFP transgene in brain tissue was confirmed by IF staining, and the results are shown in Fig.

As shown in FIG. 9, most of the cells expressed EGFP, but GFAP-positive cells showed relatively weaker intensity than the other cells in brain tissue. From the above results, it is presumed that the two transgenic genes of astrocytic-specific recombination system were expressed in TG pigs.

Example  4. 4OH - TM  Post-treatment GFAP - Expressing cell  of mine pGFAP - Creer ^T2 ; EGFP ^LoxP  Recombination

Porcine astrocytes-specific pGFAP-CreER ^T2 ; Genomic DNA PCR analysis was performed by amplifying the recombination sites using pairs of FR primers linked to the two LoxP sites to confirm that the EGFP ^LoxP recombination system was working by TM treatment. As a result, it was not a product of floxed DNA, but PCR products amplifying pre-recombinant DNA were detected. Semi-nested PCR to find CreER ^T2 mediated recombination was performed in a heat cycler using the first pair of primers, F and R, and NeoTherm ^TM DNA polymerase (Genecraft, Manchester, UK) in the first round of PCR do. All PCR products are separated by DNA electrophoresis on a 1% agarose gel and a fraction of the expected product size after being cut without UV exposure. The gel fragments are then displayed using a gel extraction kit (Elpis Biotech Inc. Daejeon, Korea). The second round of PCR was then performed using the FR 'set in which the internal sequence of the first product was amplified and the PCR analysis results are shown in FIG.

As shown in Fig. 10, it was confirmed that a 450 bp PCR product showing post-recombination was detected only in TM-treated pigs' brains.

Based on the above data results, it was confirmed that the CreER ^T2 gene was regulated by the GFAP promoter which operates in the rare place of the brain astrocytes in accordance with 4-OHTM treatment.

<110> HBION CO., LTD. <120> Brain tumor animal model and the Use thereof <130> 1-13P <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 1983 <212> DNA <213> Artificial Sequence <220> <223> Cre-ERT [synthetic construct] <400> 1 atgtccaatt tactgaccgt acaccaaaat ttgcctgcat taccggtcga tgcaacgagt 60 gatgaggttc gcaagaacct gatggacatg ttcagggatc gccaggcgtt ttctgagcat 120 acctggaaaa tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac 180 cggaaatggt ttcccgcaga acctgaagat gttcgcgatt atcttctata tcttcaggcg 240 cgcggtctgg cagtaaaaac tatccagcaa catttgggcc agctaaacat gcttcatcgt 300 cggtccgggc tgccacgacc aagtgacagc aatgctgttt cactggttat gcggcggatc 360 cgaaaagaaa acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact 420 gatttcgacc aggttcgttc actcatggaa aatagcgatc gctgccagga tatacgtaat 480 ctggcatttc tggggattgc ttataacacc ctgttacgta tagccgaaat tgccaggatc 540 agggttaaag atatctcacg tactgacggt gggagaatgt taatccatat tggcagaacg 600 aaaacgctgg ttagcaccgc aggtgtagag aaggcactta gcctgggggt aactaaactg 660 gtcgagcgat ggatttccgt ctctggtgta gctgatgatc cgaataacta cctgttttgc 720 cgggtcagaa aaaatggtgt tgccgcgcca tctgccacca gccagctatc aactcgcgcc 780 ctggaaggga tttttgaagc aactcatcga ttgatttacg gcgctaagga tgactctggt 840 cagagatacc tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg agatatggcc 900 cgcgctggag tttcaatacc ggagatcatg caagctggtg gctggaccaa tgtaaatatt 960 gtcatgaact atatccgtaa cctggatagt gaaacagggg caatggtgcg cctgctggaa 1020 gatggcgatc tcgagccatc tgctggagac atgagagctg ccaacctttg gccaagcccg 1080 ctcatgatca aacgctctaa gaagaacagc ctggccttgt ccctgacggc cgaccagatg 1140 gtcagtgcct tgttggatgc tgagcccccc atactctatt ccgagtatga tcctaccaga 1200 cccttcagtg aagcttcgat gatgggctta ctgaccaacc tggcagacag ggagctggtt 1260 cacatgatca actgggcgaa gagggtgcca ggctttgtgg atttgaccct ccatgatcag 1320 gtccaccttc tagaatgtgc ctggctagag atcctgatga ttggtctcgt ctggcgctcc 1380 atggagcacc cagtgaagct actgtttgct cctaacttgc tcttggacag gaaccaggga 1440 aaatgtgtag agggcatggt ggagatcttc gacatgctgc tggctacatc atctcggttc 1500 cgcatgatga atctgcaggg agaggagttt gtgtgcctca aatctattat tttgcttaat 1560 tctggagtgt acacatttct gtccagcacc ctgaagtctc tggaagagaa ggaccatatc 1620 caccgagtcc tggacaagat cacagacact ttgatccacc tgatggccaa ggcaggcctg 1680 accctgcagc agcagcacca gcggctggcc cagctcctcc tcatcctctc ccacatcagg 1740 cacatgagta acaaaggcat ggagcatctg tacagcatga agtgcaagaa cgtggtgccc 1800 ctctatgacc tgctgctgga ggcggcggac gcccaccgcc tacatgcgcc cactagccgt 1860 ggaggggcat ccgtggagga gacggaccaa agccacttgg ccactgcggg ctctacttca 1920 tcgcattcct tgcaaaagta ttacatcacg ggggaggcag agggtttccc tgccacagct 1980 tga 1983 <210> 2 <211> 1800 <212> DNA <213> Sus scrofa <400> 2 aattcacaag cagcctataa catgcccaaa tgaagggcat ctcccccagc atagaggagg 60 ggcattgctc agacaggagc ggagggaccg agacggctct gagagctggg gagggggcga 120 gtagccaggc cttgtctaca gagccggagt cctggcaatg ctgggccacc ctccaaggcc 180 ttcccctggt gcccactgaa tgactcacct tggcacagac acaatggtca gaggtgggca 240 cagagcctgc tccccactgc accccagcac ccctcagatg ctttccgaga agttctttga 300 gccaggggct tgggctgcat cccgcctaac aggctggcat cttgggataa aaacagcaca 360 gccccctggg ggctgccctc actgtgcgac gccctcacca gcggcagaga acaaggctcc 420 attcagcaag tgcccaagga gagggatcag aggcccaggc atgggcagtg ggggtggagg 480 cagggggagg agggctgccc acttcccaga agtccagggg caccgatgag ccaagagcct 540 gggctgggtg ctgaccctgg gcaaccagat agaaggggtg gatggatctg aaatcttgag 600 gggcagcagg gtccaggttc ctggctccca gtttagaccc accggctcct aagttcccaa 660 acccatccat ctccagaggg ctttcccagc ttctcaacct gatgcgtggg aacttaaagg 720 agataaatct ctagcgtgtg agcgaggtga gggggtggcc agggaaacag cgtgtcacag 780 gaacagagat gggaggtggg acagccagct cattcataac tgctttgtgg agctgtcaag 840 gcctgcctct gggggtggag gtgcagggag gctgagccag gacagcggcg gcatgagcct 900 aatgagactg agagcgtctc agggagagga ccagagagtc agcgcctgcc ccccttgccc 960 tatcaggggg cgcacgttcc ttccgttgtc cccacactgc tggacagagg tctccattga 1020 ccctccggac aaagggctga ttgggattcc agcccacggc cccacagctc caggcggggg 1080 atggggaagg tgggaatgca gacgactggg ttccgagggg agggctccag ggggccattg 1140 agaatgaggc tggcaaaacc cacaacccag gctgttcctt gagggtaggg aacaggtttg 1200 ttcttcaaat tcccagcgtc tagcgggcac tggaaggaga atacggagcg aggaaaggcc 1260 tgggatatgg ggtctaagag ttagaagaca ggggactttc tgctgtggct cagcagatta 1320 tgaaccccaa ctagaatcca cgaggacgtg ggtttgacct ctagcctaac tccgtgggtt 1380 aaggatccgg cgtggccgtg cactgcggtg taggccggca gctgcagctc tgattcaacc 1440 cctagccttg ggaacttcca tatgctgtgg gtgcagccct aaaaagacaa aagaaaaaaa 1500 aaagttagaa gacaggtcag aggtcatcag gtccagccct tccctgaccc acatcacaga 1560 gaagaggtct ccttgcccag ggatcaggtc cccagaccca gcatcagccc cacgaactgc 1620 ccactggttc ctctgtctgc agccaggcct gcctctgggc agtgaccccg atggggtgag 1680 aggcgctctc ctaacggctg ggctgcagcc cagccccacc ccctcaggct atgccagggg 1740 ggcgttgcca ggggcagcct gggccgtgcc aggccggccc actcctccat aaagcctgca 1800                                                                         1800

Claims (12)

A recombinant vector for constructing a brain tumor animal model comprising CreER ^T2 gene and oncogene. 2. The recombinant vector for producing a brain tumor animal model according to claim 1, wherein the CreER ^T2 gene is represented by SEQ ID NO: The cancer gene of claim 1, wherein the oncogene is selected from the group consisting of sis, EGFR, bFGF, aFGF, int2 / FGF3, hst1 / K-fgf / FGF4, FGF5, hst2 / FGF6, KGF, AIGF, erbB1, erbB2 / FGFR1, FGFR1, FGFR1, FGFR1, FGFR1, FGFR1, FGFR1, FGFR1, FGFR1, FGFR1, FGFR1, FGFR1, FGFR1, K-ras, N-ras, mos, raf, ck3, FGFR4, abl, src, yes, fyn, fgr, lyn, lck, hck, blk, csk, fps, fes, mas, gsp, Myb, fos, fosB, fra1, fra2, jun, junB, junD, myb, myb, myb, A-myb, B- b1, b1, b1, b1, b1, b1, b1, b1, b1, b1, b1, b1, b1, b1, b1, b1, b1, b1, b1, b1, aml1, dec, ews, tls / fus, and tel. 2. The recombinant vector according to claim 1, wherein the recombinant vector comprises a GFAP promoter. 5. The recombinant vector for producing a brain tumor animal model according to claim 4, wherein the GFAP promoter is represented by the nucleotide sequence of SEQ ID NO: 2. The recombinant vector according to claim 1, wherein the recombinant vector comprises a green fluorescent protein (GFP) gene, an enhanced green fluorescent protein (EGFP) gene, a red fluorescence (RFF) gene, a luminescent enzyme a gene selected from the group consisting of a beta-galactosidase gene, a beta-galactosidase (EBG) gene, a chloramphenicol gene, a chloramphenicol acetyltransferase (CAT) gene, and an alkaline phosphatase gene A reporter vector for producing a brain tumor animal model, which comprises a reporter gene. A transformed cell for producing a brain tumor animal model, wherein the recombinant vector of any one of claims 1 to 6 is introduced. A nuclear transfer embryo formed by transplanting the nucleus of a somatic cell transformed with the recombinant vector of any one of claims 1 to 6 in an enucleated oocyte. A method for producing a brain tumor animal model, characterized in that the nucleus of a nuclear donor cell into which the recombinant vector of any one of claims 1 to 6 is introduced is transplanted into an enucleated oocyte to produce a live animal. 6. A brain tumor animal model characterized by overexpressing CreER ^T2 and cancer gene transformed by the recombinant vector of any one of claims 1 to 6. 11. The model of brain tumor according to claim 10, wherein said animal is a pig. 1) administering a tumor tumor prophylactic or therapeutic agent candidate agent to the brain tumor animal model of claim 10; And
2) comparing the brain tissue of the animal model with the control group in which the candidate substance is not administered after the administration of the candidate substance, and judging the brain tissue as a brain tumor prevention or treatment agent when the cancer gene expression is inhibited; and .

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