KR101960414B1 - Transgenic cloned porcine Models for diabetes disease using human DPP-4 and the use thereof - Google Patents

Abstract

The present invention relates to a transgenic pig expressing human DPP-4 (human dipeptidyl peptidase-4, hDPP-4) gene, and more particularly to a vector for producing transgenic pigs for a diabetic disease model, And a method for producing the same. The transgenic pig according to the present invention can be effectively used for the development of a therapeutic agent for type 2 diabetes and evaluation of drug efficacy.

Description

Transgenic Cloned Porcine Models for Diabetes Mellitus using DPP-4 and the Use of the Human DPP-4 Gene

The present invention relates to a transgenic pig for a diabetic disease model using human DPP-4 (human dipeptidyl peptidase-4, hDPP-4) gene and its use. More specifically, the present invention relates to a method for the treatment and prophylaxis of a type 2 diabetes therapeutic drug targeting DPP-4 by overexpressing the hDPP-4 gene, which is a target protein of a drug used as a therapeutic agent for type 2 diabetes, Pigs and their production methods.

Diabetes is becoming a serious health hazard worldwide. According to the World Health Organization (WHO), more than 1.1 million people die annually from diabetes and there is a continuing trend. According to US statistics, the number of diabetic patients is estimated to reach about 12 million in 2002 and to about 17 million in 2020. In Korea, the number of diabetic patients is reported to be more than 4 million in 2003, which is already 8.3% of Korea's population and it is expected to increase to 6.2 million by 2020.

Recently, a very characteristic trend is that type 2 diabetes, which occurs in older adults, is associated with childhood obesity, and obesity-related type 2 diabetes frequently occurs in adolescents including children. Considering the fact that obesity and diabetes increase in adolescents, it can be thought that it is closely related to the increase of metabolic syndrome which is increasing recently. Metabolic syndrome is a general term for symptoms such as insulin resistance, hyperlipidemia, and hypertension, and is recognized as a risk factor for cardiovascular disease, which is a major cause of death due to diabetes.

To date, various GM animal production techniques require genetically modified recombinant technology and embryo manipulation techniques. In particular, various gene recombination techniques have been utilized depending on the expression pattern of the target gene. For example, recombinant techniques have been developed for 'Overexpression' animals with increased expression of target genes and 'Conditional expression animals' that can artificially control the expression of foreign genes .

Disease model animals facilitate the development of therapeutic agents by understanding the pathogenesis process as well as the function of the genes responsible for the disease. These animals are also used to test the effects, toxicity, and side effects of new therapeutic agents. In the meantime, GM animals have been developed in order to study incurable diseases in which the development of production techniques has not yet developed drugs or treatment methods. In addition, existing GM rodents have been produced mainly as disease models, but in order to compensate for the disadvantages of many limitations such as anatomy and physiological differences, researches are actively conducted to develop GM disease as a disease model.

In particular, GM animals developed to study diabetes can lower the level of disease research and identify the cause of the disease at the gene or protein level. In addition, it is possible to trace the treatment process in real time after the treatment with a new drug or the like, thereby minimizing side effects. Therefore, GM animal models are expected to contribute to the development of the pharmaceutical industry in the future.

Diabetes mellitus is caused by a variety of pathogenic mechanisms. Therefore, the treatment method must be varied. In addition, there is a need for a new treatment method because many of the conventional treatments do not have satisfactory effects. Research on diabetes mellitus has been actively conducted with the development of type 2 diabetes mellitus, which accounts for more than 90% of diabetic patients. Although insulin therapy is effective, drug therapy tends to be preferred over insulin therapy because of the hassle of daily injections, but sulfone urea has limited efficacy in some cases, and buguanide is a serious The indication is more limited because it can cause side effects. Therefore, it is required to develop drugs that can complement or surpass existing drugs. In response to this demand, the inventors of the present invention have found that an insulin sensitizer (pirogliride, linogliride, 2,4-diamino-5-cyan-bromopyridine, incretin, repaglinide, nateglinide), troglitazone, (Such as pirogliride, linogliride, dichloroacetate, insulin lispro and insulin aspart), glucose synthesis inhibitors (lipolysis inhibitors, carnitine transferase inhibitors, beta oxidation inhibitors), insulin antagonists (somatostatin, glucagon derivatives) , A drug that slows the absorption of carbohydrates (dietary fiber, alpha glucosidase inhibitors), and amylin analogs (pramlintide). Some of these are currently on the market, but many are still at an experimental stage or toxicology stage that is still too low for human use.

The effect of inscretin means that the insulin secretion stimulation is more powerful than when the same amount of sugar is administered intravenously when the sugar is orally administered. This concept was already proposed by pioneering researchers in the 1960s. Subsequent studies have shown that the incretin effect occurs in a blood-glucose-dependent manner and that this effect is reduced in diabetic patients compared to healthy individuals. The discovery of the glucone-dependent insulinotropic peptide (GIP), glucagon like peptide-1 (GTP) and the physiological degradative enzyme DPP-4 (dipeptidyl peptidase-4) 1 agonists and DPP-4 inhibitors. Although the GLP-1 agonist initially appeared on the market, it was limited in its prescription and use due to the relatively high price and disadvantages of the route of administration (subcutaneous injection), while the DPP-4 inhibitor shares several advantages of the GLP-1 agonist However, thanks to the relatively cheap price and the advantage of oral administration, it became an easy choice for clinicians. The DPP-4 inhibitor is a total of six types of drugs, such as sitagliptin, vildagliptin, saxagliptin, linagliptin, alogliptin and gemigliptin were developed. Such research and development, however, fails to provide sufficient experimental evidence of the efficacy and safety of these new drugs and of objects similar to humans. Therefore, the development of a model of diabetes mellitus would be the most important evidence for the identification of transgenic products related to the validation of these new drugs.

As a background of the present invention, Korean Patent Publication No. 10-2012-0050485 discloses a transformed pig for the treatment of diabetes.

It is an object of the present invention to provide a transgenic pig expressing human-derived DPP-4 (human dipeptidyl peptidase-4, hDPP-4) gene.

It is another object of the present invention to provide a transgenic pig for a diabetic disease model for identifying the action mechanism of a drug for treating diabetes using the transgenic pig.

Still another object of the present invention is to provide a composition for gene introduction comprising a primer.

Still another object of the present invention is to provide an expression vector purified product SEQ ID NO: 5 for introducing hDPP-4 gene having a nucleotide sequence of SEQ ID NO: 1 and containing (chicken) CAG promoter and HA-tag inserted at its C-terminal .

It is still another object of the present invention to provide embryos of transgenic animals containing human-derived DPP-4 (human dipeptidyl peptidase-4, hDPP-4) gene.

It is another object of the present invention to provide a method for producing the transgenic pig described above.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a transgenic pig expressing human-derived DPP-4 (human dipeptidyl peptidase-4, hDPP-4) gene.

According to one embodiment of the present invention, it may be a model of diabetes mellitus.

According to one embodiment of the present invention, a promoter and a gene construct operably linked to the nucleotide sequence of SEQ ID NO: 1 may be introduced.

According to one embodiment of the present invention, the promoter may be provided with a transgenic pig including a CAG promoter.

According to one embodiment of the present invention, the human-derived DPP-4 gene can be expressed in the liver tissue, intestine tissue or ear tissues of a pig.

According to one embodiment of the present invention, the human-derived DPP-4 gene can be amplified by a primer set consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4.

According to one embodiment of the present invention, the human-derived DPP-4 gene can be expressed in the cell membrane.

According to one embodiment of the present invention, the transgenic pig may be prepared by introducing the gene construct into the pig embryo nucleus by microinjection.

According to another aspect of the present invention, there is provided a transformed porcine embryo comprising a human-derived DPP-4 gene.

According to another aspect of the present invention, there is provided an embryo of a transgenic animal comprising a human-derived DPP-4 (human dipeptidyl peptidase-4, hDPP-4) gene.

According to another aspect of the present invention, there is provided a method of producing a gene construct, comprising: a) constructing a promoter and a genetic construct operably linked to the nucleotide sequence of SEQ ID NO: 1; b) microinjecting the gene construct into a porcine embryo; And c) implanting the embryo into the uterus of a surrogate mother to obtain a transformed porcine.

According to an embodiment of the present invention, the gene construct of step b) is a pCAGEN expression vector, and the expression vector is cleaved by using HincII and HindIII restriction enzymes, and then only a linear expression vector is selectively recovered, To 1% or less of agarose gel, recovering and purifying the DNA fragment, and performing gene microinjection.

According to an embodiment of the present invention, there is provided a method for preparing a transgenic pig having a concentration of the injected genetic construct of 5 to 20 ng / μl in step b).

According to another aspect of the present invention, there is provided a method for preventing or treating diabetes mellitus comprising the steps of: a) administering a candidate substance for prevention and treatment of diabetes mellitus to the transformed pig; b) measuring the level of insulin expression in cells or tissues of said transformed pig after administration of the candidate substance; And c) screening candidate substances that increase or decrease the level of insulin expression compared to a control group to which the candidate substance is not administered.

The transgenic pig according to one embodiment of the present invention can be used for physiological phenotyping analysis in which hDPP-4 is overexpressed.

The transgenic pig according to one embodiment of the present invention can be used for the development of a therapeutic agent for type 2 diabetes targeting hDPP-4 and for evaluating drug efficacy.

Transgenic pigs according to one embodiment of the present invention can be used for the pharmacological validation of already developed drugs as well as new drugs to be developed as hDPP-4 inhibitors.

Expression of the protein can be rapidly analyzed using a primer and a vector inserted with an HA-tag according to an embodiment of the present invention.

FIG. 1 schematically shows a process of producing a diabetic model transgenic pig according to an embodiment of the present invention.
Figure 2 shows the cloned hDPP-4 gene sequence (SEQ ID NO: 1) according to one embodiment of the present invention.
Figure 3 shows the hDPP-4 protein sequence (SEQ ID NO: 2) according to one embodiment of the present invention.
FIG. 4 shows a tertiary structure of hDPP-4 of HA-tag according to an embodiment of the present invention, and a yellow line indicates HA-tag.
FIG. 5 is a schematic diagram illustrating the construction of an hDPP-4 expression vector according to an embodiment of the present invention.
FIG. 6 shows the expression of the hDPP-4 expression vector according to an embodiment of the present invention in cells.
FIG. 7 is a schematic diagram of pCAGEN-hDPP-4-HA expression vector in which DPP-4 gene is expressed in embryos (lines 2 to 5) after injection of DPP-4 gene according to an embodiment of the present invention by PCR.
FIG. 8 shows PCR confirmation of the expression of DPP-4 gene in embryos (lines 2 to 5) after DPP-4 gene injection according to an embodiment of the present invention. On the other hand, DPP-4 gene was not expressed in the control (lines 6 and 7).
FIG. 9 is a graph showing the results of the assay of the transgenic plants by the PCR method in the analysis of gene expression in the embryo produced by the microinjection method in the embryo for the production of the transgenic animal according to the one embodiment of the present invention.
10 is an ultrasound diagnostic image of a 40th day of gestation and a 114th day of gestation according to an embodiment of the present invention.
FIG. 11 is a view for confirming the expression site of DPP-4 according to an embodiment of the present invention. The transformed porcine ear cells were collected, and the somatic cells isolated from the ear cells were subcultured to obtain the membrane portion and cytoplasm And the expression was confirmed by an immunological method.
FIG. 12 shows genomic analysis of transgenic piglets and the location of foreign gene insertion. Genomic DNA was isolated from transgenic piglet-derived somatic cells, and the region containing the foreign gene was analyzed through NGS. As a result, hDPP -4 gene was inserted into the cells.

Certain terms are hereby defined for convenience in order to facilitate a better understanding of the present invention. Unless otherwise defined herein, scientific and technical terms used in the present invention shall have the meanings commonly understood by one of ordinary skill in the art. Also, unless the context clearly indicates otherwise, the singular form of the term also includes plural forms thereof, and plural forms of the term should be understood as including its singular form.

As used herein, the term " transgenic pig " refers to a pig that has been partially transformed by recombination of an exogenously introduced gene and artificially introducing it into the chromosome of the pig. The transgenic pigs of the present invention can be expressed in various tissues of pigs into which DPP-4, which is an introduced gene, has been transformed, and the tissue is restricted to the tail, ear, liver, heart, stomach, small intestine, But is suitable for the ear, liver, small intestine, and colon tissue.

As used herein, the term " primer " refers to a short sequence that serves as a starting point for template strand replication as a base sequence having a short free 3 'hydroxyl group. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. PCR amplification can be used to predict the sagittal specific abdominal obesity through the production of the desired product. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art.

As used herein, the term " vector " refers to an expression vector capable of expressing a gene of interest in a cell into which the vector has been introduced, including a gene construct comprising an essential regulatory element operably linked to the expression of the gene insert introduced into the vector It says. Preferably, the present invention can produce a recombinant vector comprising a human-derived DDP-4 fusion gene, and introducing the recombinant vector into the fertilized egg to produce a transformed embryo. Transgenic pigs can be produced.

The term " promoter " used in the present invention may be any promoter sequence for producing an expression vector ordinarily used in the art. Examples of the promoter that can be used include CMV promoter, CAG promoter, and the like, but the promoter sequences usable in the present invention are not limited by the above examples. Such a promoter may be a specific promoter for expression in a tissue-specific manner if necessary.

As used herein, the term " microinjection " refers to injecting a very small amount of macromolecular material, which can be accomplished by extending the glass capillary and connecting it to a micropipette, a microsyringe, or using a micro-manipulator under a microscope But are not limited thereto.

According to one aspect of the present invention, a transgenic pig expressing a human-derived DPP-4 gene (human dipeptidyl peptidase-4, hDPP-4) gene can be provided. Human-derived DPP-4 (Dipeptidyl peptidase-4, DPP-4 inhibitor) is known as adenosine deaminase complex protein or CD26 (differentiated cluster 26) , The main role of DPP-4 is known to act as an enzyme that degrades incretins in the intestine, similar to Glucogan like peptide-1 (GLP-1).

According to one embodiment of the present invention, a transgenic pig, which is a diabetes model, can be provided.

The term 'diabetes mellitus' is a disease including diabetes and includes chronic complications due to diabetes. But are not limited to, ischemic heart disease including diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, angina pectoris and myocardial infarction, stroke and gastric ulceration or necrosis, abnormal immune function, imbalance of fat and / or protein metabolism .

A transgenic pig for a diabetic disease model is provided for identifying the action mechanism of a drug for treating diabetes using the transgenic pig. As described above, DPP-4 is an enzyme that decomposes incretin. When the DPP-4 gene is inhibited, the incretin concentration is relatively increased. As a result, the insulin secretion is increased and the glucagon secretion is decreased in the pancreas, , Which slows the rate of digestion and absorption of food, resulting in a slower rise in blood sugar. On the other hand, when the DPP-4 gene is overexpressed, the incretin concentration is lowered, resulting in decreased insulin secretion and increased secretion of glucagon, leading to hyperglycemia. Thus, the present invention provides a transgenic pig for a diabetic model exhibiting a form similar to that of a diabetic disease.

According to one embodiment of the present invention, a transgenic pig in which a promoter and a gene construct operably linked to the nucleotide sequence of SEQ ID NO: 1 are introduced can be provided.

According to an embodiment of the present invention, the promoter may include a CAG promoter, CMV promoter, EF1? Promoter and SR? Promoter for systemic expression, and a tissue specific promoter typified by an albumin promoter. According to one embodiment of the present invention, the promoter may be provided with a transgenic pig including a CAG promoter.

According to one embodiment of the present invention, the human-derived DPP-4 gene may be, but not limited to, a transgenic pig expressed in the liver tissue, intestine tissue or ear tissue of a pig.

According to one embodiment of the present invention, a human derived DPP-4 (human dipeptidyl peptidase-4, hDPP-4) gene is provided with a transgenic pig comprising the nucleotide sequence of SEQ ID NO: 1 (see FIG.

According to another aspect of the present invention, there is provided a composition for introducing hDPP-4 gene comprising a primer set consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4.

According to one embodiment of the present invention, the transgenic pig is characterized in that the human-derived DPP-4 gene can be expressed in cytoplasm and cell membrane, and in particular, is expressed in an extracellular membrane.

According to still another aspect of the present invention, there is provided an expression vector for introducing hDPP-4 gene having a nucleotide sequence of SEQ ID NO: 5 and comprising a CAG promoter and HA-tag inserted at its C-terminal (see FIG. 5) . The electrophoresis image of FIG. 5 shows that the forward and reverse hDPP-4 expression vectors described in FIG. 5 are properly inserted. Also, it can be seen that forward and reverse expression vectors are properly expressed in COS7 (see FIG. 6). For example, in order to construct a tissue-specific expression vector of hDPP-4, the present invention includes a liver-specific albumin promoter, but the present invention is not limited thereto, and promoters of tissues in which each expression is required may be included. The HA-tag was inserted at the 3 'end of hDPP-4 to distinguish it from the endogenous DPP-4 (endogenous pig DPP-4) that was produced endogenously during the expression of the expression vector. Yellow).

According to one embodiment of the present invention, the above tissue is provided with an expression vector for introducing hDPP-4 gene, which is a liver or a foreign tissue.

According to another aspect of the present invention, there is provided an embryo of a transgenic animal comprising a human-derived DPP-4 (human dipeptidyl peptidase-4, hDPP-4) gene.

The fertilized egg is not limited thereto, but is an embryo for the production of the transgenic pig.

According to another aspect of the present invention, there is provided a method of producing a gene construct, comprising: a) constructing a promoter and a genetic construct operably linked to the nucleotide sequence of SEQ ID NO: 1; b) microinjecting the gene construct into a porcine embryo; And c) impregnating the embryo into the uterus of a surrogate mother to obtain a transformed porcine.

The pig embryos were harvested by treatment with hormones and natural mating, followed by gonadal sterilization by surgical methods. The recovered embryos were centrifuged at 15,000 rpm for 10 minutes in order to observe the precursor nucleus, and the gene (DPP-4) for producing a diabetic disease transgenic animal was injected (see FIG. 1). In the present invention, the existing disease model animal has been studied in mice, but mouse-based verification has overcome limitations to be applied to humans and overcomes them by using pigs more similar to humans.

DPP-4 gene was expressed in 3 out of 4 blastocytes (line 2-5) injected with DPP-4 gene (3/4 BL: 75%). However, expression of the DPP-4 gene was not confirmed in the two unit blastocysts (lines 6 and 7) used as the control group (see FIG. 8).

According to an embodiment of the present invention, the gene construct of step b) is a pCAGEN expression vector, and the expression vector is cleaved by using HincII and HindIII restriction enzymes, and then only a linear expression vector is selectively recovered, To 1% or less agarose gel, DNA fragments were recovered and purified, and gene microinjection was performed.

The expression vector of step b) is a pCAGEN expression vector. The expression vector is cleaved by using HincII and HindIII restriction enzymes, and then only the linear expression vector is selectively recovered and the expression vector is subjected to electrophoresis on an agarose gel of 0.1 to 1% And recovering and purifying the DNA fragment after the migration, and performing microinjection of the gene. The agarose gel may be at a concentration of 0.5%, although it is not limited thereto.

According to an embodiment of the present invention, the concentration of the injected gene construct in step b) may be 5-20 ng / μl. The concentration of the introduced gene is not particularly limited but is preferably 10 ng / μl.

According to another aspect of the present invention, there is provided a method for the prevention and treatment of diabetes mellitus comprising the steps of: a) administering a prophylactic and therapeutic agent candidate for diabetes mellitus to the transgenic pig of claim 1; b) measuring the level of insulin expression in cells or tissues of said transformed pig after administration of the candidate substance; And c) screening candidate substances that increase or decrease the level of insulin expression compared to a control group to which the candidate substance is not administered.

In the above screening method, the candidate substances of step a) include peptides, proteins, nonpeptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts or plasma, Or a well-known compound.

The candidate material may form a salt. The salt of the candidate substance may be a salt such as a physiologically acceptable acid (e.g., mineral acid) or a base (e.g., an organic acid), among which physiologically acceptable acid addition salts are preferable, but not limited thereto. Such salts include, for example, salts of inorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid or sulfuric acid) or organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, , Malic acid, oxalic acid, benzoic acid, methanesulfonic acid or benzenesulfonic acid) and the like can be used.

Examples of the method of administering the candidate substance include oral administration, intravenous injection, swabbing, subcutaneous administration, intradermal administration or intraperitoneal administration, but the present invention is not limited thereto. It can be chosen appropriately. The dose of the candidate substance can be appropriately selected in accordance with the administration method, properties of the candidate substance, and the like.

In the screening method, the level of insulin expression is measured in the cells or tissues of the transformed pigs of steps b) and c), and a candidate substance having increased or decreased the insulin expression level is selected by comparing with the control group to which the candidate substance is not administered Can further measure the level of expression of insulin, as well as anatomical or pathological indicators of abdominal fat loss, weight loss, increased liver tissue, and liver fat cells that are correlated with diabetes or abnormal glucose metabolism However, the present invention is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Example

1. Human DPP4 gene cloning

       MRNA was isolated from Human HEK293 (Kidney-derived) cells in order to clone the gene based on the DPP4 (human DPP4, hDPP4) gene sequence derived from the NCBI GeneBank registered human. cDNA was synthesized using hDPP-4 mRNA as a template and the gene was amplified by PCR method to obtain a gene by cloning into pCR2.1-TA vector. As a result of gene sequence analysis, it was confirmed that the sequence was the same as human DPP4 (accession number: NM_001935) registered in GeneBank (FIG. 2 and FIG. 3).

2. Expression vector construction for systemic expression

After constructing hDPP-4 expression vector for systemic expression and expressing it in the cell, primers (SEQ ID NOS: 3 and 4) in which HA-tag was inserted at the C-terminal of the protein for rapid protein expression analysis were synthesized, The amplified hDPP-4 gene was cloned into the pCR2.1-TA vector, and the mutation was not inserted in the whole nucleotide sequence analysis.

The hDPP-4 gene was digested with restriction enzymes from the plasmid in which the DNA base sequence was confirmed, inserted into the pCAGEN expression vector using the CAG promoter, and the insertion site was confirmed (FIG. 5).

In order to confirm whether the constructed expression vector expresses the gene, COS7 cells were transfected (using Lipofectamine 2000) and confirmed for expression of hDPP-4 by Western blot (Fig. 6). The pCAGEN expression vector (FIG. 7) was digested with HincII and HindIII restriction enzymes, and then the DNA fragment was recovered by electrophoresis on a 0.5% agarose gel to selectively recover only the linear expression vector, Respectively.

3. Gene microinjection and embryo transfer

Pig embryos were collected from 1 st stage of embryos by surgical methods such as motility, hormone treatment and natural mating. The hDPP-4 gene for the production of transgenic animals was injected into the recovered oocyte precursor, and 684 embryos were collected from 733 oocytes oocyte in a total of 27 eggs (blank seeds) 478 hDPP-4 injected embryos were transplanted.

DPP-4 Diabetic Disease Model In order to produce pigs, gene expression analysis was performed by PCR using microinjection method in embryos for the production of transgenic animals. A total of 34 colonies were collected and 29 genes were injected. All of them were transplanted into the fallopian tubes.

 Diagnosis was made to check the progress of the pregnancy on the 40th and 114th days after the embryo implantation. The arrows in Fig. 11 indicate portions of the implanted fetus, respectively (see Fig. 10).

result

Screening of transgenic plants by PCR method

Three out of 15 surrogate mothers were pregnant, but one was still in mid-pregnancy and one of them gave birth to four, but no transgenic pigs were produced. Finally, 6 dogs (2 females, 3 males, 1 females) were produced in one dog. Genomic DNA was isolated from the ear cells of the piglets, and PCR was performed to confirm the transformation. One out of five surviving pigs was found to be a transformant (16.7%, see FIG. 9).

Transgenic pig ear cell harvesting and ear cell culture

In order to analyze the characteristics of cultured cells, the membrane and cytoplasmic parts in which DPP-4 was present were separated and analyzed by immunohistochemistry. As a result, (See Fig. 11).

Genomic analysis of transgenic piglets and location of foreign gene insertion

Genomic DNA was isolated from transgenic piglet-derived somatic cells, and genomic DNA isolated was analyzed by next-generation sequencing to analyze the region containing the foreign gene. As a result, the hDPP-4 gene was detected in the hemoglobin subunit beta gene region (See Fig. 12).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

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ggaaacagca 600 gtgacaaaaa ttaagccata aacgaaagtt tgactaagtt atattaatta gggttggtaa 660 atctcgtgcc agccaccgcg gtcatacgat taacccaagc taacaggagt acggcgtaaa 720 acgtgttaaa gcaccatacc aaatagggtt aaattctaac taagctgtaa aaagccatga 780 ttaaaataaa aataaatgac gaaagtgacc ctacaatagc cgacgcacta tagctaagac 840 ccaaactggg attagatacc ccactatgct tagccctaaa cacagataat tacataaaca 900 aaattattcg ccagagtact actagcaaca gcttaaaact caaaggactt ggcggtgctt 960 tatatccttc tagaggagcc tgttctataa tcgataaacc ccgataaacc tcaccaattc 1020 ttgctaatac agtctatata ccgccatctt cagcaaaccc taaaaaggaa aaaaagtaag 1080 cgtaattatg atacataaaa acgttaggtc aaggtgtaac ctatgaaatg ggaagaaatg 1140 ggctacattc tctacaccaa gagaatcaag cacgaaagtt attatgaaac caataaccaa 1200 aggaggattt agcagtaaac taagaataga gtgcttagtt gaattaggcc atgaagcacg 1260 cacacaccgc ccgtcaccct cctcaaatag attcagtgca tctaacccta tttaaacgca 1320 ctagctacat gagaggagac aagtcgtaac aaggtaagca tactggaaag tgtgcttgga 1380 taaatcaaga tatagcttaa acaaagcatc cagtttacac ctagaagact tcattcatta 1440 tgaatatctt gaactagacc tagcccaaag ataccctctc gactaaacaa ccaagataga 1500 ataaaacaaa acatttaatc ccaatttaaa gtataggaga tagaaatcta agtacggcgc 1560 tatagagaaa gtaccgcaag ggaacgatga aagaaaaaaa ctaaaagtat aaaaaagcaa 1620 agattacccc ttgtaccttt tgcataatga attaactagt ataagactta acaaaatgaa 1680 ttttagctaa gcagcccgaa accagacgag ctactcacaa acagtttacc aagaactaac 1740 tcatctatgt ggcaaaatag tgagaagatt tgtaagtaga ggtgacatgc ctaacgagcc 1800 tggtgatagc tggttgtcca gaaaatgaat ctaagttcag ctttaaagat accaaaaatt 1860 caaataaacc ccactgtagc tttaaaagtt agtctaaaaa ggtacagcct tttagaaacg 1920 gatacaacct tgactagaga gtaaaattta acactaccat agtaggccta aaagcagcca 1980 tcaattaaga aagcgttaaa gctcaacaac aaaaattaaa tagattccaa caacaaatga 2040 ttaactccta gccccaatac tggactaatc tattatagaa tagaagcaat aatgttaata 2100 tgagtaacaa gaaaaatttt ctccttgcat aagtctaagt cagtgcctga taatactctg 2160 accactaaca gtcaataaaa ataatccaac aataaacaat ttattgatta tactgttaac 2220 ccaacacagg agtgcatcta aggaaagatt aaaagaagta aaaggaactc ggcaaacaca 2280 aaccccgcct gtttaccaaa aacatcacct ccagcattcc cagtattgga ggcattgcct 2340 gcccagtgac aactgtttaa cggccgcggt atcctgaccg tgcaaaggta gcataatcat 2400 ttgttctcta aataaggact tgtatgaatg gccgcacgag ggttttactg tctcttactt 2460 ccaatcagtg aaattgacct tcccgtgaag aggcgggaat gcacaaataa gacgagaaga 2520 ccctatggag ctttaactaa ccaacccaaa gagaatagat ttaaccatta aggaataaca 2580 acaatctcca tgagttggta gtttcggttg gggtgacctc ggagaataaa aaatcctccg 2640 agcgatttta aagactagac ccacaagtca aatcactcta tcgctcattg atccaaaaac 2700 ttgatcaacg gaacaagtta ccctagggat aacagcgcaa tcctattcaa gagtccatat 2760 cgacaatagg gtttacgacc tcgatgttgg atcaggacat cctgatggtg caaccgctat 2820 caaaggttcg tttgttcaac gattaaagtc ctacgtgatc tgagttcaga ccggagtaat 2880 ccaggtcggt ttctatctat tacgtatttc tcccagtacg aaaggacaag agaaataagg 2940 ccaactttaa atcaagcgcc ttaagacaac caatgataac atctcaactg acaacacaaa 3000 accctgccct agaacagggc ttagttaagg tggcagagcc cggtaattgc ataaaactta 3060 aacttttata tccagagatt caaatcctct ccttaacaaa atgttcataa ttaacatctt 3120 aatactaatt attcccatcc tattggccgt agcattcctt acgttagtgg aacgaaaagt 3180 tctaggctat atacaactcc gaaaaggtcc aaatgtcgta ggtccatatg gcctacttca 3240 acccatcgcc gatgcaatca aacttttcat taaagaacca ctacgacccg ctacatcttc 3300 agcctcaata tttatcctag cacctatcat agctttaggc ctagccttaa ccatgtgaat 3360 tcccctacca ataccctatc ctcttatcaa cataaaccta ggagtcctat ttatactagc 3420 catatcaagc ctagccgtat actccattct ctgatcaggc tgagcttcca actcaaaata 3480 cgcactaatc ggagccctac gagcagtagc acaaacaatc tcatacgaag taacgctagc 3540 aattatcctg ttatcagtgg tcctaataag tgggtccttt accctctcca cattaattac 3600 tacacaagaa caaatatggt taatcctccc agcatggcct ctagcaataa tatgatttat 3660 ctcaacacta gcagaaacaa accgagctcc atttgattta actgaaggag aatcagagct 3720 agtctcgggc ttcaacgtag aatatgcagc aggaccattt gccctcttct tcatagcaga 3780 gtacgcaaat attatcataa taaatatctt tacagcaatt ttattcctag gaacatccca 3840 caatccacac ataccagaac tctacacaat caattttacc attaaatccc tactgctcac 3900 aatatccttc ctatgaatcc gagcatccta ccctcgattt cgctatgacc aactaataca 3960 cttactatga aaaaattttc tacctctgac actagccctg tgcatgtgac acgtatccct 4020 acccatcctt acatcaggca tcccaccaca aacataagaa atatgtctga caaaagagtt 4080 actttgatag agtaaataat agaggttcaa accctcttat ttctagaact ataggaatcg 4140 aacctactcc taagaatcca aaactcttcg tgctcccaat tacaccaaat tctattagta 4200 aggtcagcta attaagctat cgggcccata ccccgaaaat gttggtttat atccttcccg 4260 tactaataaa cccaattatc tttattatta ttctactaac cattatacta ggaactatta 4320 ttgtcataat cagttctcac tgactacttg tctgaatcgg gtttgaaata aatatactcg 4380 ccatcatccc catcataata aaaaatcaca acccacgagc tacagaagca tcaactaaat 4440 attttttgac tcaatcaaca gcctcaatac tactaataat agccgtcatc attaacgtaa 4500 tattctcagg ccaatgaacc gtaataaaac tatttagccc aatagcctca atacttataa 4560 cgatagccct agctataaaa ctaggaatag ccccatttca cttctgagtc ccagaagtaa 4620 cacagggcat ccccctatcc tcaggcctta tcctactgac atgacaaaaa ctagcaccta 4680 tatctgtact ttaccaaatc ttcccatcaa ttaacctaaa cttaattcta accctatcag 4740 ttttatcaat cctaattgga ggctgagggg gactaaacca aacacaactc cgaaaaatca 4800 tagcctactc atcaatcgct catataggct gaataacagc agtactacca tataacccca 4860 ccataacatt gctaaactta attatctata tcattataac ttccaccata tttaccatat 4920 ttatagccaa ttccaccacc actaccctgt cattatcaca cacatgaaat aaaacaccca 4980 ttataaccgt cctaattctt gccactctcc tatccatagg aggactccct cccctatctg 5040 ggtttatacc aaaatgaata atcatccaag agataacaaa aaataacagc atcattctac 5100 ccactttcat agcaatcaca gctctactaa acttatattt ttatatacga ctcacgtatt 5160 ctaccacact aacaatattt ccctccacaa acaacataaa aataaaatga caatttcccc 5220 ttatgaaaaa aataactttt ctaccaacaa tagtcgtatt atctaccata atactaccac 5280 tcacgccaat actatcagtg ttagaatagg aatttaggtt aaacagacca agagccttca 5340 aagccctaag caagtacaat ttacttaatt cctgataagg attgcaagac tacaccttac 5400 atcaattgaa tgcaaatcaa ccactttaat taagctaaat cctcactaga ctggtgggct 5460 ccacccccac gaaactttag ttaacagcta aacaccctag ctaactggct tcaatctact 5520 tctcccgccg caagaaaaaa aaggcgggag aagccccggc agaattgaag ctgcttctct 5580 gaatttgcaa ttcaacgtgt aaattcacca cagggcttgg taaaaagagg agtcaaacct 5640 ctatctttag atttacagtc taatgctttg ctcagccatt ttacccatgt tcattaaccg 5700 ctgactattc tcaaccagcc ataaagatat tggtaccctt tatctactat ttgatgcttg 5760 ggccggtata gtaggaacag ctctaagcct tctaattcgc gctgaattag gccaacccgg 5820 aactctgctc ggagacgacc aaatctacaa cgcagttgta accgcacacg catttgtaat 5880 aatcttcttc atagtaatac caatcataat tggaggattc ggtaactgac ttgttcccct 5940 aataattggt gctcccgata tagcatttcc ccgaataaat aatataagct tctgactcct 6000 ccctccctca ttcctactac tcctcgcatc ctctatagtt gaagctgggg caggaacagg 6060 ctgaaccgtg taccctccct tagcaggcaa cctagcccat gcaggagctt cagtagatct 6120 aaccattttc tctttacact tagcaggagt ttcctcaatt ttaggagcca tcaacttcat 6180 tacaacaatt atcaacataa agccccccgc aatgtcacaa taccaaaccc ctctgttcgt 6240 atgatccgta ataattaccg ccgtactact actactctcg ctccctgtat tagcagccgg 6300 catcacaatg ctattaacag accggaacct aaatacaacc ttcttcgacc cggcaggagg 6360 aggagaccct attctatatc aacacttatt ctgattcttt ggacaccccg aagtctatat 6420 tttaatctta cctgggtttg gaataatctc tcatatcgtg acctactact caggaaaaaa 6480 agaaccattc ggatatatgg gaatagtttg ggctataatg tcaatcggat ttctaggttt 6540 catcgtatga gcccaccata tattcactgt cggaatagac gtcgacacac gagcctactt 6600 cacatcagcc actataatta ttgctattcc aaccggggta aaagtcttca gctgattggc 6660 aacacttcat ggaggtaata tcaaatggtc tcctgctata atgtgagccc taggctttat 6720 tttcttattt acagtagggg gtttaactgg aattgtctta gccaactctt ccctcgatat 6780 tgttcttcac gacacatact acgttgtcgc acatttccac tatgttttat caataggagc 6840 tgtatttgct attatagggg gatttgttca ttgattccca ctattctcag gttatactct 6900 caacgataca tgagccaaaa tccacttcgc aattatattt gtaggcgtca atataacctt 6960 cttcccacaa cactttctag gactatctgg catgcctcga cgatactccg actacccaga 7020 tgcatacaca atatgaaata ctatctcatc aataggctca ttcatttccc taacagcagt 7080 tatactaata gttttcatca tctgagaagc atttgcatct aaacgagaag tcttgactgt 7140 agacttaacc acgacaaatc tagaatgatt aaacggatgc cctccaccat atcacacatt 7200 tgaagaaccc acctatgtta acctaaaata agaaaggaag gaatcgaacc ccctactatt 7260 ggtttcaagc caacatcata acctctatgt ctctctcaat aaacgaggtg ttagtaaaac 7320 attatataat tttgtcaaag ttaagttaca agtgaaagtc ctgtacacct catatggcat 7380 atcccataca actaggattc caagatgcaa catcaccaat catagaagaa ctacttcact 7440 ttcatgacca cacgctaata attgtcttct taattagctc attagtactt tacattattt 7500 cactaatact aacgacaaag ctgacccata caagcacgat agatgcacaa gaagtagaga 7560 caatctgaac cattctgccc gccatcatct taattctaat tgctcttcct tctttacgaa 7620 ttctatacat aatagatgaa atcaataacc catctcttac agtaaaaacc ataggacatc 7680 agtgatactg aagctatgag tatacagatt atgaggactt aagcttcgac tcctacataa 7740 ttccaacatc agaattaaag ccaggggagc tacgactatt agaagtcgat aatcgagttg 7800 tactaccaat agaaataaca atccgaatgt tagtctcctc tgaagacgta ttacactcat 7860 gagctgtgcc ctctctagga ctaaaaacag acgcaatccc aggccgtcta aaccaaacaa 7920 cccttatatc gtcccgtcca ggcttatatt acggtcaatg ctcagaaatt tgcgggtcaa 7980 accacagttt catgcccatt gtccttgagt tagtcccact aaagtacttt gaaaaatgat 8040 ctgcgtcaat attataaaat cactaagaag ctatatagca ctaacctttt aagttagaga 8100 ttgagagcca tatactctcc ttggtgacat gccgcaacta gacacgtcaa catgactgac 8160 aatgatctta tcaatattct tgaccctttt tatcatcttt caactaaaag tttcaaaaca 8220 caacttttat cacaatccag aactgacacc aacaaaaata ttaaaacaaa acaccccttg 8280 agaaacaaaa tgaacgaaaa tttatttacc tcttttatta cccctgtaat tttaggtctc 8340 cctctcgtaa cccttatcgt actattccca agcctactat tcccaacatc aaaccgacta 8400 gtaagcaatc gctttgtaac cctccaacaa tgaatacttc aacttgtatc aaaacaaata 8460 atgagtatcc acaattctaa aggacaaaca tgaacattaa tattaatatc tctgatccta 8520 tttattggat caacaaacct actaggccta ttaccccatt cattcacacc aacaacacaa 8580 ctatcaataa acctaggcat agccatcccc ctgtgagcag gagccgtaat tacaggattc 8640 cgcaataaaa ctaaagcatc acttgcccat ttcttaccac aaggaacacc cactccacta 8700 atcccaatac tagtaattat tgaaactatc agccttttta ttcaacctat agccctcgcc 8760 gtgcggttaa cagctaacat cactgcagga cacctattaa ttcacctaat cggaggagct 8820 acacttgcac taataagcat tagcactaca acagctctaa ttacattcac cattctaatc 8880 ctactaacaa ttctagagtt tgcagtagct ataatccaag cctatgtatt cactctccta 8940 gtcagcctat atctgcatga caacacataa tgacacacca aactcatgct tatcatatag 9000 taaacccaag cccttgacct cttacaggag ctttgtctgc cctcttaata acatccggcc 9060 taaccatgtg atttcacttt aactcaatga ccctgctaat aattggccta acaacaaata 9120 tactaacaat ataccaatga tgacgagatg ttatccgaga aagcaccttc caagggcacc 9180 ataccccagc tgtccaaaaa ggcctccgtt atggaataat tctttttatt atctccgaag 9240 tactattctt taccggattt ttctgagctt tctaccactc aagcctcgcc cccacccctg 9300 aactaggcgg ctgctgaccc ccaacaggca ttcacccact aaacccccta gaagtcccac 9360 tgctcaacac ctctgtccta ttggcttccg gagtttctat tacctgagcc catcatagtt 9420 taatagaagg ggaccgaaag catatattac aagccctatt tatcaccatc acattaggag 9480 tctacttcac actactacaa gcctcagaat actatgaagc accttttact atctccgacg 9540 gagtttacgg ctcaactttt tttgtagcca caggcttcca cggcctccac gtcatcattg 9600 ggtccacctt cttaattgtc tgcttcttcc gccaattaaa atttcatttt acttctaacc 9660 accacttcgg ctttgaagcc gctgcctgat actgacattt cgtagacgta gtctgacttt 9720 tcctctatgt ttctatctat tgatgaggct cctattcttt tagtattaac tagtacagct 9780 gacttccaat cagctagttt cggtctagtc cgaaaaagaa taataaattt aatactagcc 9840 ctcctgacca attttacact agccacccta ctcgtcatca tcgcattctg acttccccaa 9900 ctaaatgtat actctgagaa aacaagccca tacgaatgtg gatttgaccc cataggatca 9960 gcccgccttc ccttctctat aaaattcttt ctggtagcca tcacattcct cttatttgac 10020 ctagaaattg cactcctcct accactgcca tgagcctcac aaacagcaaa tctaaacaca 10080 atgcttacca tagccctctt cctaattatc ctcctagctg taagcctagc ctatgagtga 10140 actcaaaaag gactagaatg aaccgaatat ggtacttagt ttaaaataaa ataaatgatt 10200 tcgactcatt agattatgat ttaattcata attaccaaat gtctatagta tacataaaca 10260 ttataatagc attcacagta tctcttgtag gactactaat ataccgatcc cacctaatat 10320 cctcccttct atgcttagaa ggaataatgc tatccctatt cgttatagca gccctaacaa 10380 tcctcaactc acattttaca ttagctagca taatacctat tatcctacta gtcttcgcag 10440 cctgtgaagc agccctaggt ctatctctac tagtaatagt atcaaataca tatggtactg 10500 attatgtaca aaacctcaac ttactccaat gctaaaatac attattccaa caattatact 10560 tataccccta acctggttat caaaaaataa tataatttgg gttaactcca cagcacacag 10620 ccttctaatt agctttacaa gcctcctcct cataaaccag tttggcgaca acagccttaa 10680 tttttcacta ctatttttct ccgactccct atccactcca ctactaattt taaccatatg 10740 gctcctccct ctaatactaa tagctagcca acatcatcta tcaaaagaaa acctaacccg 10800 aaaaaaacta tttattacta tgctgatctc actacaacta ttcctaatta taacctttac 10860 cgccatggaa ctaatcttat tttatattct atttgaagca acactagtcc caacactcat 10920 tattattacc cgatgaggaa accaaacaga acgcctaaac gccggactct atttcctatt 10980 ctatacacta gctggctccc tacccctatt agtcgcacta atttatatcc aaaacacagt 11040 aggatcccta aatttcctaa tattacagta ctgagtacaa cctgttcata actcttgatc 11100 taatgtcttc atatgactag catgtataat agctttcata gtaaaaatac cactatatgg 11160 cctccacctt tgactaccta aagctcacgt agaagccccc atcgcaggct ccatagtcct 11220 tgcagcagtt ctgctaaaac taggggggta cggtatgcta cgaatcacac taattctaaa 11280 ccctatgacc gactttatag catacccatt cattatactc tccctatgag gcataattat 11340 aaccagctca atctgcctcc gtcaaacgga cctaaaatca ctcatcgcat actcctctgt 11400 aagccacata gcactcgtta tcgtagccat ccttatccag acaccttgaa gctacatagg 11460 agcaaccgcc cttatgattg cccacggcct cacatcctcc atacttttct gtctagcaaa 11520 ctcaaactac gaacgaatcc acagccgaac cataattcta gctcgaggcc tacaaacgct 11580 ccttccacta atagccacct gatgactact agcaagtcta accaacttag ctctaccccc 11640 aacaatcaac ttaattggag aactatttgt agtaatgtca accttttcat gatctaacat 11700 tacaattatt ctaataggag taaatatagt aatcaccgcc ctatattctc tatacatgct 11760 aattataacc caacgaggaa aatataccta ccacattaat aatatctcgc cttcctttac 11820 cctacccta aaaaattatt ctaggacctc tatactgtaa atatagttta acaaaaacat tagattgtga 11940 atctaacaat agaaactcat taccttctta tttaccgaaa aagtatgcaa gaactgctaa 12000 ttctatgctc ccatatctaa tagtatggct ttttcgaact tttaaaggat agtagtttat 12060 ccgttggtct taggaaccaa aaaattggtg caactccaaa taaaagtaat aaacatattc 12120 tcctcactct cactagttac tttactctta ctaactacac ccattataat aataagcttt 12180 aacacctaca aaccttccaa ctacccactc tacgtaaaaa cagctatctc atacgccttc 12240 attaccagca taattcccac aataatattt atccactcag gccaagaact aattatttca 12300 aactgacact gactaaccat ccaaactctt aaattatccc tcagctttaa aatagactat 12360 ttctcaataa tatttatccc agtagcacta ttcgtcacat gatctattat agaattctca 12420 atatgatata tatactcaga ccccaatatt aacaaattct tcaaatatct actcctattc 12480 ctcattacta tgctcatcct tgtaaccgca aacaacctct tccagctatt cattggctga 12540 gaaggcgtcg gaatcatatc atttctactc atcggatgat gatacggacg agcagatgca 12600 aacacagcag ccctacaagc aatcttatat aaccgcatcg gcgacattgg tttcatttta 12660 gcaatagcat ggttcctaac aaatctcaat acctgagacc tccaacagat cttcatacta 12720 aacccaagcg actcaaacat acccttgatt ggactagcat tagctgcaac cggaaaatcc 12780 gcccaatttg gcctccaccc gtgacttccc tctgcaatag aaggcccaac tcccgtctca 12840 gcactactcc attcaagcac aatagtggta gcaggtatct tcctactaat ccgtttctat 12900 cccctcacag aaaacaataa atacatccaa tctattacat tatgcttagg agccattacc 12960 acactattta cagcaatatg cgccctcacc caaaatgaca ttaaaaaaat catcgccttc 13020 tccacatcca gtcaactggg ccttataata gtaactattg gcattaacca accttaccta 13080 gctttcctcc acatctgtac ccacgccttt ttcaaagcta tactattcat atgctccggt 13140 tccattattc acagcctaaa cgacgaacaa gatattcgaa aaataggagg cctatttaaa 13200 gccatgccat tcaccacaac agccctcatt gttggcagtc tcgcactaac aggaataccc 13260 ttcctcacag gattctactc caaagaccta atcatcgaag ccgccaacac gtcttatacc 13320 aacgcctgag cccttctaat aacattaatt gccacctctt tcacagctat ttacagcacc 13380 cgtattattt ttttcgcact tctaggacaa ccccgattcc ctaccctagt taatattaac 13440 gaaaacaacc cccttctgat caactctatc aaacgcttac taattggaag cctcttcgca 13500 ggatacatca tttccaacaa tattcctcca acaacaattc cccaaataac tatgccctac 13560 tacctaaaaa caacagccct aattgttaca atcctaggct tcatcttagc cctagaaatc 13620 agtaatataa ctaaaaatct aaaatatcac tacccctcaa acgccttcaa gttctcaacc 13680 ttgctagggt atttccccac aattatacat cgcctagctc catacataaa tttatcaata 13740 agccaaaaat cagcatcctc ccttctagac ctaatctgac tagaagccat cctaccaaaa 13800 accatctcac tcgcccaaat aaaagcatct accctggtca caaaccaaaa aggcctgatc 13860 aaactatatt tcctctcctt cttaatcaca atccttatca gcataatctt atttaatttc 13920 cacgagtaat ttctataata accacaacac caattaataa agaccaccca gttacaataa 13980 ctaatcaggt accataactg tataaagccg caatccctat ggcctcttca ctaaagaacc 14040 cagaatcccc tgtatcataa atcacccaat cccctaaacc attaaactca aacacaacct 14100 caacttcttt atcctttaat acataataga ccataaagaa ctccatcaac aagccagtaa 14160 caaatgcccc taaaacagcc ttattagaaa gccaaatttc aggatactgt tctgtagcca 14220 tagccgttgt ataaccaaaa actaccatca tacctcccaa ataaattaaa aagaccatca 14280 accccaaaaa ggatccacca aaattcaata caattccaca gccaacccca ccactcacaa 14340 ttaaccctaa ccccccataa ataggtgaag gtttcgaaga aaaccccaca aaacctatca 14400 cgaaaataac gcttagaata aatacaatgt atagtatcat tattcttaca tggaatctaa 14460 ccatgactaa tgatatgaaa aaccatcgtt gtcattcaac tacaagaaca ctaatgacta 14520 acattcgaaa gtcccaccca ctaataaaaa ttgtaaacaa tgcattcatc gaccttccag 14580 ccccatcaaa catttcatca tgatgaaatt tcggttccct cctgggaatc tgcctaatcc 14640 tacaaatcct cacaggccta ttcctagcaa tacactacac atccgacaca acaacagcat 14700 tctcctctgt tacccatatc tgccgagacg tgaactacgg ctgaatcatc cgatacatac 14760 acgcaaacgg agcttcaatg ttttttatct gcttatatat gcacgtagga cgaggcttat 14820 attacgggtc ttacactttt ctagaaacat gaaatattgg agtaatcctt ctgctcacag 14880 taatagccac agcatttata ggatacgtcc taccatgagg acaaatatca ttctgaggag 14940 caacagtcat caccaacctc ttatcagcaa tcccatacat cggcacaaat ttagtcgaat 15000 gaatctgagg cggattctca gtagacaaag caacccttac ccgattcttc gctttccatt 15060 ttatccttcc atttatcatc atagcaattg ccatagtcca cctactattc ctccacgaaa 15120 caggctccaa caacccaaca ggaatttcct cagacgtaga caaaatccca ttccacccct 15180 actataccat taaggacatc ttaggggccc tcttactaat tctagctcta atactactag 15240 tactattcgc acccgacctc ctcggagacc cagataacta caccccagcc aatccactca 15300 acacaccccc tcacatcaaa cccgagtgat acttcttatt tgcatacgca atcttacgat 15360 caatccccaa caaactagga ggagtactag ccctagcctt ctctatccta attcttgctc 15420 taatccccct actacacacc tccaaacaac gaagcataat attccgacca ctcagccaat 15480 gcctattctg agccctagta gcagacctat tgacactcac atgaattgga ggacaaccag 15540 tcgaacaccc atatatcacc atcggacaac tagcatctgt cctatacttt ctcctcatcc 15600 tagtgctaat accaacggcc ggcacaatcg aaaacaaatt actaaaatga agacaggtct 15660 ttgtagtaca tctaatatac tggtcttgta aaccagagaa ggagaacaac taacctccct 15720 aagactcaag gaagaaactg cagtctcacc atcaaccccc aaagctgaag ttctatttaa 15780 actattccct gaacactatt aatatagttc cataaataca aagagcctta tcagtattaa 15840 atttatcaaa aatcccaata actcaacaca gaatttgcac cctaaccaaa tattacaaac 15900 accactagct aacataacac gcccatacac agaccacaga atgaattacc tacgcaaggg 15960 gtaatgtaca taacattaat gtaataaaga cataatatgt atatagtaca ttaaattata 16020 tgccccatgc atataagcaa gcacatgacc tctataggca gtacataata catataatta 16080 ttgactgtac ataatacatt atgtcaaatt cattcttgat agtatatcta ttatatattc 16140 cttaccatta gatcacgagc ttaattacca tgccgcgtga aaccagcaac ccgctaggca 16200 gggatccctc ttctcgctcc gggcccataa accgtggggg tcgctatcca atgaatttta 16260 ccaggcatct ggttctttct tcagggccat ctcatctaaa acggtccatt ctttcctctt 16320 aaataagaca tctcgatgg 16339 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 2 acccccaaag ctgaagttct at 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 3 cttgatgcca gctcctctta gt 22

Claims (13)

A DPP-4 gene (human dipeptidyl peptidase-4, hDPP-4) and a gene construct operably linked to the nucleotide sequence of SEQ ID NO: 1 are introduced, and the human DPP- A transformed pig which is a diabetic disease model amplified by a primer set consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4.
delete delete The transformed pig according to claim 1, wherein the promoter comprises a CAG promoter.
2. The transgenic pig as claimed in claim 1, wherein the human-derived DPP-4 gene is expressed in a liver tissue, a gut tissue or an ear tissue of a pig.
delete 2. The transgenic pig according to claim 1, wherein said human-derived DPP-4 gene is expressed in a cell membrane.
[Claim 2] The transformed pig according to claim 1, wherein the transgenic pig is prepared by introducing the gene construct by microinjection into the nucleus of a porcine embryo.
A DPP-4 promoter comprising a human-derived DPP-4 gene and a gene construct operably linked to the nucleotide sequence of SEQ ID NO: 1 are introduced, and the human DPP-4 gene comprises a primer set comprising the nucleotide sequences of SEQ ID NOS: 3 and 4 The embryo of a transgenic pig, a diabetic disease model amplified by.
a) constructing a promoter and a genetic construct operably linked to the nucleotide sequence of SEQ ID NO: 1;
b) microinjecting the gene construct into a porcine embryo; And
c) implanting said fertilized eggs into the uterus of a surrogate mother to obtain a transformed pig,
Promoter and the nucleotide sequence of SEQ ID NO: 1 are operatively linked, and the DPP-4 gene derived from a human is transformed into a model of diabetes mellitus which is amplified by a primer set consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4 Method of manufacturing pigs.
11. The method of claim 10,
The gene construct of step b) is a pCAGEN expression vector,
The expression vector was digested with HincII and HindIII restriction enzymes, and then only a linear expression vector was selectively recovered. After electrophoresis on agarose gel of 0.1 to 1%, DNA fragments were recovered and purified, Wherein the method comprises the steps of:
11. The method of claim 10,
wherein the concentration of said injected genetic construct in step b) is 5-20 ng / μl.
a) administering a prophylactic and therapeutic agent candidate for diabetes mellitus to the transgenic pig of claim 1;
b) measuring the level of insulin expression in cells or tissues of said transformed pig after administration of the candidate substance; And
and c) selecting a candidate substance that increases or decreases the level of insulin expression compared to a control group to which the candidate substance is not administered.

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