KR20190049610A - Production of transgenic dogs that overexpress peroxisome proliferator-activated receptor-alpha in a muscle specific manner - Google Patents

Abstract

The present invention relates to a transgenic dog that overexpresses peroxisome proliferator-activated receptor alpha (PPARα) in a muscle-specific manner, a production method thereof, and a use thereof. Specifically, the present invention relates to the use of a transgenic dog that overexpresses PPARα proteins specifically in muscle tissue as an obesity-resistant animal model with increased fatty acid oxidation and energy expenditure.

Description

Production of transgenic dogs overexpressing peroxisome proliferator-activated receptor-alpha in a muscle specific manner < RTI ID = 0.0 > (PPARa) < / RTI &

More particularly, the present invention relates to a transgenic canine overexpressing peroxisome proliferator activated receptor alpha (PPARα), and a method for the study of metabolic diseases It is used as a model.

As the organism is deteriorated in vital function due to various internal and external factors, the intracellular homeostasis is lost, and the metabolism efficiency of the sugar and the fat is also lowered. In addition, hypertension due to changes in dietary habits increases cardiovascular diseases such as obesity, hyperlipidemia, metabolic dysfunction such as diabetes and hypertension and atherosclerosis, and these diseases are caused by multiple causes including genetic factors and nutritional factors Lt; / RTI >

Peroxisome is one of the intracellular organelles associated with these metabolic dysfunctions and has been found to play an important role in the metabolism of oxygen, glucose, lipids, and hormones through a number of studies. It has also been reported that peroxisome has a wide effect on the regulation of cell proliferation and differentiation and the regulation of the sphingomyelitis mediators.

A number of studies have shown that nuclear hormone receptors called peroxisome proliferator-activated receptors (PPARs) are a good target for pharmacological approaches to disease caused by metabolic dysfunction. PPAR is one of the 48 nuclear receptors, which binds with ligand to regulate the expression of downstream related genes. The PPARs identified so far are the three isoforms of PPARα, PPARδ and PPARγ (J Biochem. Biophys Res Commun., 224, 431, 1996). ≪ RTI ID = 0.0 > Steroid < / RTI > Biochem. Molec. Biol., 51, 157, 1994; Gene Expression, 4, 281,1995;

PPARa is mainly found in the blood vessel wall, liver, heart, muscle, kidney, and brown adipose tissue and is found in fibroblasts such as fenofibrate, bezafibrate, ciprofibrate, gemfibrozil, (fibrate) is an agonist of PPARα, which is known to prevent or delay the onset of arteriosclerosis in rats and humans, and has an anti-obesity effect by promoting fat oxidation. In addition, PPARa plays an important role in accelerating the differentiation and proliferation of keratinocyte differentiation, promoting the formation of skin barrier through lipid metabolism, inhibiting inflammation, and restoring the wound in the epidermis of the skin, and inhibiting the formation of inflammatory mediators by ultraviolet The effect of inhibiting erythema formation was also reported.

PPARδ, also called PPARβ, is distributed in many tissues and is found especially in skin, brain, and adipose tissue. PPARδ is involved in cholesterol reverse transport, myelination, and wound repair, and acts as an important regulator of fatty acid metabolism and energy homeostasis.

PPARγ is most commonly found in adipose tissue and is found in vascular endothelium, macrophages, and pancreatic β-cells, but is found less in tissues where PPARα is found, such as liver, heart, and skeletal muscle. PPARγ regulates the differentiation of adipocytes and plays a crucial role in systemic lipid homeostasis.

Since PPAR plays an important role in lipid metabolism, many studies are underway to develop therapeutic agents for metabolic diseases targeting PPAR. In particular, there is a report that PPARa regulates lipid metabolism, inflammation and arteriosclerosis. Recently, a lot of studies on PPARa as a target protein for improving and treating inflammation, arteriosclerosis, obesity and cancer have been conducted.

PPARα increases the expression of several catabolic enzymes related to ω-oxidation in mitochondria and β-oxidation in mitochondria and peroxisomes, thereby increasing the expression of catabolic enzymes in the liver and muscle It is known to promote the oxidation of fatty acids. Recent studies have shown that the expression of genes involved in fatty acid oxidation and energy consumption is increased when artificially activating PPARα in adipocytes and muscle cells. In particular, experimental obesity rats have been shown to reduce lipid accumulation as a result of ligand or agonist treatment of PPARα (Wang Y. X. et al., Cell, 113: 159, 2003).

In addition, Wy14,643, the most well-known ligand of PPARα, decreases triglyceride (TG) and blood cholesterol levels in human metabolism, while high density lipoprotein (HDL) (Peter Zahradka, Cardiovascular Drug Reviews, 25: 99, 2007; Caroline Duval et al., Biochemica et Biophysica Acta, 1771: 961, 2007). In addition, ligands of PPARα are known to stimulate the ABCA1 pathway, thereby reducing the formation of dysfunction while promoting cholesterol removal from blood vessels (Chinetti et al., Nat Med., 7: 53-58, 2001). In addition, fibrate, a specific ligand for PPARα, is known to be involved in glucose homeostasis by lowering fasting insulin and glucose concentrations in hyperinsulinemic patients with metabolic syndrome. Apo AI and A-II, which are related to PPARα lipoprotein metabolism, are the main proteins of HDL, and they are effective in preventing vascular diseases by transferring excess cholesterol in tissues to liver. PPARα Apo AI and A-II gene expression is increased (Stael B. et al., Circulation, 98: 2088-2093, 1998). It is known that increasing the HDL concentration reduces the incidence of arteriosclerosis.

In addition, PPARα KO mice have been reported to exhibit obesity and hypertriglyceridemia without increasing the daily caloric intake. This effect is mainly explained by a decrease in fatty acid uptake by the liver and inhibition of fatty acid oxidation (J. Biol. Chem., 1998, 273, 29577-29585). These studies have shown that PPARα is widely involved in lipolysis and is an important target protein for metabolic diseases caused by obesity.

In order to produce an animal model that can be used for the study of metabolic diseases, the present inventors have applied PPARα, which is considered to be an important target protein of metabolic diseases, to transplant a nucleus of a somatic cell transformed to overexpress it, And overexpressed dog model, and completed the present invention.

U.S. Published Patent Application No. 2004-0038249 A1 U.S. Published Patent Application No. 2006-0242716 A1

1. PPARα Protein Expression Was Increased by Four Weeks of Intermittent Hypoxic Training via AMPKα2-Dependent Manner in Mouse Skeletal Muscle. [PLoS One. 2015 Apr 29; 10 (4): e0122593] 2. Fatty acid homeostasis and induction of lipid regulatory genes in skeletal muscles of peroxisome proliferator-activated receptor (PPAR) alpha knock-out mice. Evidence for compensatory regulation by PPAR delta. [J Biol Chem. 2002 Jul 19; 277 (29): 26089-26097)

It is an object of the present invention to provide a dog for a muscle-specific PPAR? Over-expression model and a method for producing the same.

Another object of the present invention is to provide a method for overexpressing muscle-specific PPAR? Using the muscle-specific expression system necessary for the method for producing the above-mentioned muscle-specific PPAR? Overexpression model.

Another object of the present invention is to provide a muscle specific expression system necessary for the method for producing the above-mentioned muscle specific PPAR? Overexpression model, a cell transformed with the muscle specific expression system, and a nuclear transfer embryo.

It is another object of the present invention to provide a variety of uses for the above-described muscle-specific PPAR < alpha > overexpression model.

In order to solve the above problems, the present invention provides a dog for a muscle-specific PPAR? Over-expression model and a method for producing the same.

The present invention, in one embodiment,

A human myoglobin promoter and a PPAR alpha gene that overexpress the peroxisome proliferator activated receptor alpha (PPAR alpha) in a muscle-specific manner to provide a recombinant vector for constructing a muscle-specific PPAR alpha overexpressing animal model containing the PPAR alpha gene.

Here, the vector may be a viral vector, and the viral vector may be at least one vector selected from the group consisting of a retrovirus vector, an adenovirus vector, an adeno-associated viral vector, a herpes virus vector, an avipox virus vector, .

In another embodiment of the present invention,

 A PPAR alpha overexpression model characterized by overexpressing the PPAR alpha protein in a muscle-specific manner, transformed by a vector containing the human myoglobin promoter and the PPAR alpha gene.

The overexpression of PPAR [alpha] protein is expressed in cells constituting dog muscle tissue or muscle tissue.

The dog for the muscle specific PPAR [alpha] over-expression model may be transformed with the vector described above. In particular, the transformed cell is a genomic human myoglobin promoter and a PPARa gene inserted therein, and the muscle for the muscle-specific PPAR [alpha] over-expression model expresses the nucleus of the transformed cell with somatic cell nuclear transfer technology , SCNT). ≪ / RTI >

The dog for the muscle-specific PPAR [alpha] over-expression model is characterized by being an obesity-resistant model for obesity-resistant models.

Therefore, in another embodiment of the present invention,

There is provided a method for producing a muscle-specific PPAR? Overexpression model, characterized in that the core of the nuclear donor cell into which the recombination vector described above is introduced is transplanted into the enucleated oocyte to produce a human.

At this time, the nuclear donor cells may be dog embryonic cells, somatic cells, or stem cells. Preferably, it may be a fetal-derived cell and an adult fibroblast, a cumulus cell. In one embodiment of the present invention, fibroblasts isolated from dog fetuses and adults are used.

Therefore, the method for producing a dog for the muscle-specific PPAR? Overexpression model of the present invention may include the following steps as a more specific example.

(a) preparing a nuclear donor cell comprising culturing somatic cells or stem cells isolated from the dog;

(b) introducing into the nuclear donor cell a recombinant vector containing a human myoglobin promoter (hMb p ) and a PPAR alpha gene;

(c) removing the nucleus from the eggs to prepare a enucleated oocyte;

(d) injecting and fusing nuclear donor cells of step (b) into the enucleated oocytes of step (c);

(e) activating the oocyte fused in step (d); And

(f) transplanting the activated oocyte into a surrogate mother's fallopian tube.

The present invention includes all the transformation vectors, cells, and nuclear transfer embryos necessary for the production of a muscle-specific PPAR? Overexpression model which can be obtained in the course of the method for producing the above-described muscle specific PPAR? Overexpression model.

That is, according to another embodiment of the present invention,

A transgenic cell for constructing a muscle specific PPAR alpha overexpressing animal model into which a recombinant vector containing a PPAR alpha gene and a human myoglobin promoter has been introduced can be provided,

It is also possible to provide a nucleus-transferred oocyte formed by transplanting the nucleus of a somatic or stem cell derived from a canine-derived somatic cell transformed with a recombinant vector containing a PPAR alpha gene and a human myoglobin promoter into an enucleated oocyte.

The present invention also provides a variety of uses for the above-described muscle-specific PPAR [alpha] over-expression model.

As an example, there is provided a method of screening for a drug for maintenance or an increase in obesity resistance, comprising:

1) administering a candidate substance capable of maintaining or increasing obesity resistance in a muscle-specific PPAR? Overexpression model of the present invention;

2) After administering the candidate substance, analysis of the muscle tissue of the dog by comparing the obesity resistance with the control group to which the candidate substance is not administered.

Thus, the present invention provides muscle-specific PPAR alpha overexpressing animal models and their various uses that are resistant to metabolic diseases in metabolic diseases (e.g., obesity, etc.).

The dog specific for the muscle-specific PPAR? Overexpression model of the present invention is an dog model having improved physical function and obesity resistance as the expression of genes involved in fatty acid oxidation and energy consumption is increased by muscle-specifically overexpressed PPAR? (Including obesity resistance research), exercise / muscle strength improvement research (including muscle atrophy), and anti-aging research.

Brief Description of the Drawings Fig. 1 is a schematic diagram showing one embodiment of a recombinant vector comprising a PPAR alpha gene and a human myoglobin promoter.
FIG. 2 is a fluorescence microscopic photograph of GFP expression of a fibroblast transformed with a recombinant vector.
Figure 3 shows PCR results of PuroR and GFP expression of fibroblasts transfected with recombinant vectors.
Figure 4 is a schematic representation of a somatic cell nuclear transfer technique.
Fig. 5 shows primers for PCR for confirming expression of PPAR [alpha] in dogs produced through somatic cell nuclear transfer technology.
FIG. 6 shows the number of dogs generated through the somatic cell nuclear transfer technique.
Figure 7 is an image of dogs generated through somatic cell nuclear transfer technology.
FIG. 8 shows PCR results of PPARa expression in dogs produced by somatic cell nuclear transfer technology.
9 is a graph showing changes in the body weight of dogs produced through the somatic cell nuclear transfer technique.
FIG. 10 shows the result of Southern blotting of long-gene insertion in transgenic plants (N, non-cloned dog, P, vector, transgenic cloned dogs).
Fig. 11 shows the serum lipase concentrations in the transgenic animals of 2 month old, 6 month old and 2 years old, and the control group corresponding to each age.
FIG. 12 is a graph comparing weight gain amounts, showing the weight gain of (A) all three transgenic dogs and (B) only two transgenic dogs (AL1 and AL2 fed 6 weeks of all feeds) .

Representative terms used in the present invention are as follows.

&Quot; Muscle specific expression system " is a term collectively referred to as a system in which an arbitrary gene or protein is expressed in cells constituting muscle tissue or muscle tissue. The muscle specific expression system may be a regulatory element capable of regulating the expression of an arbitrary gene or protein in a muscle tissue or muscle tissue, for example, a promoter, an enhancer, And the expression system includes a vector system including a nucleic acid sequence encoding an arbitrary gene, a virus system, and the like, but also includes any system capable of expressing an arbitrary gene or protein in a cell .

A " vector " or " expression vector " means a plasmid, a transposable element-containing vector, a viral vector, or the like, which is capable of introducing a nucleic acid encoding the structural gene and capable of expressing the nucleic acid in the host cell Or other media. Preferably a vector containing a transfer factor or a viral vector.

"Recombinant vector" refers to a gene construct containing an essential control element operatively linked to the expression of a gene insert, which is capable of expressing a desired protein or RNA of interest in a suitable host cell.

&Quot; Expression control sequence " or " regulatory element " refers to a DNA sequence that controls expression of a nucleic acid sequence operably linked to a particular host cell. Such regulatory sequences include any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. In the present invention, the regulatory factor may be a promoter, an enhancer, or the like.

&Quot; Promoter " means a DNA sequence capable of regulating the transcription of a specific nucleotide sequence into mRNA when linked to a specific sequence. Typically, the promoter is located in the 5 '(ie, upstream) of the desired nucleotide sequence to be transcribed into mRNA, but not in all cases, and includes a region in which the RNA polymerase and other transcription factors for transcription initiation specifically bind to provide. The promoter of the present invention is a constitutive promoter. The term " constitutive " as used in connection with a promoter means that the promoter can direct transcription of operably linked nucleic acid sequences without stimulation (e.g., heat shock, chemicals, etc.). The promoter of the present invention may preferably be a human myoglobin promoter. The myoglobin promoter can be regulated so that a particular sequence linked to the promoter is muscle-specific transcribed.

&Quot; Operably linked " refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or RNA to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA can affect the expression of a nucleic acid sequence that is operably linked. Operational linkage with recombinant vectors can be made using genetic recombination techniques well known in the art.

&Quot; Transformation " means changing the genetic properties of a living organism by exogenously given DNA. Examples of the transformation method include various known methods such as microinjection, electroporation, particle bombardment, sperm-mediated gene transfer, viral infection A method using viral infection, direct muscle injection, insulator, and trnasposon may be appropriately selected and applied. Preferably, in the present invention, an expression vector containing human PPAR alpha can be transformed into a fetal fibroblast through a viral infection method.

&Quot; Cell ", " host cell ", " transformed host cell " and the like refer to a progeny or potential progeny of such a cell as well as a specific target cell. Such a offspring, in fact, would not be identical to the parent cell, but is still included within the scope and range of terms used in the present invention, since certain modifications may occur later in time by mutation or environmental effects.

&Quot; Model animal for disease research " refers to an animal having a disease that is very similar to a human disease or resistant to disease. Diseases in the study of human disease The significance of model animals is due to their physiological or genetic similarities between humans and animals. In animal studies, animal models for study of diseases of biomedical diseases provide research materials for various causes of diseases, pathogenesis and diagnosis, and studies on disease-related genes through study of model animals for disease studies, And the basic efficacy and toxicity test of the developed new drug candidates will provide basic data for judging the possibility of practical use.

&Quot; 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 dog.

&Quot; Overexpression " means that any gene or protein is expressed above normal levels. In the present invention, the expression of PPARα protein is expressed in the expression level of normal cells or more. In particular, expression of PPARα protein may be expressed specifically in muscle tissue or muscle tissue. Over-expressing transformants may be transgenic cells, transformed tissues or transgenic animals in which PPAR [alpha] protein is expressed above normal levels. For example, it may be an overexpressed transformant produced by exposing the cell, tissue or animal to a substance comprising the muscle-specific expression system, or by introducing the substance.

&Quot; Nuclear transplantation " refers to a genetic engineering technique that allows an enucleated oocyte to artificially bind other cells or nuclei to have the same trait. &Quot; Nuclear transfer egg " refers to an oocyte into which nuclear donor cells have been introduced or fused.

"Cloning" is a genetic manipulation technique for creating new individuals having the same gene set as an individual. In particular, the present invention provides a genetic manipulation technique in which a somatic cell, an embryo cell, a fetal cell, and / or an adult cell is substantially identical Nuclear DNA sequence. The present invention utilizes a technique of replicating dogs 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.

&Quot; Nuclear donor cell " refers to a nucleus of a cell or a cell that transfers nuclei to a nuclear receptor, a recipient oocyte. &Quot; oocyte " preferably refers to a mature oocyte that has reached the middle stage of the second meiosis. In the present invention, somatic cells or stem cells may be used as nuclear donor cells.

The term " somatic cell " refers to a cell other than a germ cell among the cells constituting the multicellular organism, and is capable of differentiating into differentiated cells that do not become other cells, Lt; / RTI > cells.

&Quot; Stem cell " means a cell capable of developing into any tissue. There are two basic features: self-renewal, which creates itself by repeated division, and the ability to differentiate into cells with specific functions depending on the environment.

"Cultivation" is the cultivation of an organism or part of an organism (organ, tissue, cell, etc.) under moderately artificially controlled environmental conditions. In this case, external conditions such as temperature, humidity, light, gas phase composition The most important direct effect on the organism cultivated is the culture medium, which is a direct environment for the organism and a supply point for various nutrients necessary for survival and growth.

&Quot; In vitro culture " refers to a series of laboratory procedures in which a cell is cultured in an incubator in a laboratory in a manner similar to the environment in the body, in a manner different from that in which the cells grow.

The term "medium" or "medium composition" refers to a mixture for growth and proliferation of cells, etc., which is essential for growth and proliferation of cells such as sugars, amino acids, various nutrients, serum, growth factors and minerals.

&Quot; 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.

"Obesity" refers to an excess of body fat, which means that the body mass index is clinically 25 in Korea and over 30 according to the World Health Organization (WHO). The body mass index (BMI) is calculated as body weight (kg / m ² ) relative to the height of the kidney. Generally, it means that the body weight is higher than the normal value. However, when the body fat percentage of the body composition is high, even if the body weight does not go out, it is diagnosed as obesity. Obesity is also a disease caused by inactivation or abnormalities / disorders of mechanisms or factors associated with fatty acid oxidation, energy consumption and the like.

&Quot; Obesity-resistant " refers to a state in which the occurrence or suppression of obesity is inhibited or reduced by increasing or activating mechanisms or factors that inhibit obesity, such as fatty acid oxidation and energy consumption, thereby inhibiting or inhibiting factors and circumstances that may cause obesity.

&Quot; Treatment " means an approach to obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction &Quot; Treatment " refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented. &Quot; Palliating " a disease may reduce the extent of the disease state and / or undesirable clinical symptoms and / or delay or slow the time course of the progression, It means to lose.

&Quot; about " means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprises " and " comprising ", when not explicitly required in the context, include a stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to an animal model of muscle specific PPARa expression.

More specifically, the present invention relates to a muscle specific PPARα over-expressing model, a method for producing the same, and its use.

The muscle-specific PPAR? Overexpression model of the present invention is characterized in that it is used to transform PPAR? Overexpression specifically in cells constituting muscle tissue or muscle tissue.

At this time, the known method can be transformed into the PPAR alpha gene by using any method, but somatic cell nuclear transfer (SCNT) is preferably used. That is, a cloned animal model of PPAR alpha overexpression of the present invention is produced using a somatic cell nuclear transfer (SCNT) method using a transgenic cell line overexpressing a specific gene PPAR alpha.

Therefore, for example, a method of producing a muscle specific PPAR [alpha] over-expression model of the present invention through a somatic cell nuclear transfer method,

(a) preparing a nuclear donor cell comprising culturing somatic cells or stem cells isolated from the dog;

(b) introducing into the nuclear donor cell a recombinant vector containing a human myoglobin promoter (hMb p ) and a PPAR alpha gene;

(c) removing the nucleus from the eggs to prepare a enucleated oocyte;

(d) injecting and fusing nuclear donor cells of step (b) into the enucleated oocytes of step (c);

(e) activating the oocyte fused in step (d); And

(f) transplanting the activated oocyte into a surrogate mother's fallopian tubes.

The conventional technology for each step can be understood with reference to a conventional method for producing a cloned animal using a somatic cell nuclear transfer technique known in the art.

[Muscle specific PPARα expression system]

Muscle-specific PPARα  Expression recombinant vector

The present invention provides a muscle-specific PPAR? Expression vector that specifically overexpresses PPAR? In cells constituting muscle tissue or muscle tissue.

In particular, the vector is characterized by comprising a human myoglobin promoter (hMb p ). The hMb p and PPAR alpha genes can be used in operable linkage.

Therefore, in another aspect of the present invention, a human myoglobin promoter (hMb p ) is directed to a recombinant vector for muscle-specific expression of PPARa operably linked to a PPARa gene.

In the present invention, PPAR? Is one of PPAR, which is a protein encoded by PPAR? (Or PPARA) gene and composed of 468 amino acids, also known as NR1C1. It is expressed mainly in the blood vessel wall, liver, heart, muscle, kidney and brown adipose tissue and has several catabolisms related to ß-oxidation in mitochondria and peroxisome and ω-oxidation in mitochondria It is known that it increases the expression of catabolic enzymes and thus promotes the oxidation of fatty acids in liver and muscle. In particular, there is a report that PPARa regulates lipid metabolism, inflammation and atherosclerosis. Recently, PPARa has been attracting attention as a kind of target protein for improving and treating inflammation, arteriosclerosis, obesity and cancer.

The muscle-specific PPARα expression recombinant vector of the present invention can be used for transformation mutation that knock-in the PPARα gene into the genome of a cell, wherein the knock-in gene expresses a foreign gene ≪ / RTI > to the genome of the host. A somatic cell into which a recombinant vector has been introduced can be used for nuclear transfer into an embryo of an animal and a nuclear transplanted embryo can be implanted to produce a transgenic plant in which the PPAR alpha gene is knocked in.

The nucleic acid encoding the PPAR alpha can be suitably used by those skilled in the art from sequences having a base sequence encoding a non-limiting PPAR alpha known in the art.

The nucleic acid encoding the PPAR alpha can be produced by a gene recombinant method known in the art. For example, there are PCR amplification for amplifying a nucleic acid from a genome, chemical synthesis, or a technique for producing a cDNA sequence.

In addition, the nucleic acid may have a base sequence encoding each functional equivalent of PPARa.

The functional equivalents are those having at least 70%, preferably 80%, more preferably 90% or more sequence homology with the amino acid sequence of wildtype as a result of amino acid addition, substitution or deletion, Quot; refers to a polypeptide that exhibits physiological activity substantially equivalent to that of the polypeptide. Said " equivalent physiological activity " increases the expression of several catabolic enzymes related to? -Oxidation in mitochondria and peroxisomes and? -Oxidation in mitochondria, Thereby inducing the oxidation of fatty acids.

At this time, deletion or substitution of the amino acid is preferably located in a region that is not directly related to the physiological activity of the polypeptide of the present invention.

The muscle specific PPARα expression recombinant vector of the present invention is preferably a plasmid, a viral vector or other medium known in the art capable of expressing a nucleic acid encoding a PPARα gene specifically in cells constituting muscle tissue or muscle tissue, May be 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.

The nucleic acid encoding the PPARa of the present invention can be inserted into a vector operably linked to a regulatory factor that allows it to be expressed specifically in the muscle.

A construct comprising a nucleic acid encoding PPAR alpha 'or a DNA construct comprising a nucleic acid encoding' hMb p -PPAR alpha 'as a whole.

That is, the vector may consist of a human myoglobin promoter (hMb p ); and a behavior containing the PPAR alpha gene.

Meanwhile, the recombinant vector may include a selection marker, and the selection marker includes an antibiotic resistance gene such as a kanamycin resistance gene and a neomycin resistance gene, a fluorescent protein such as a green fluorescent protein and a red fluorescent protein, It does not.

In addition, in order to confirm whether the PPAR alpha protein is over-expressed, the vector of the present invention may further include a tag sequence for protein separation purification or confirmation. Examples of the tag sequence include GFP, Glutathione S-transferase (GST) -tag, HA, His-tag, Myc-tag and T7-tag. However, the tag sequence of the present invention is not limited by these examples. In the preferred embodiment of the present invention, expression and expression level were confirmed using GFP.

Preferably, the vector produced by the above method may be the vector disclosed in Fig.

The recombinant vector comprising the human myoglobin promoter (hMb p ) and the PPARα gene of the present invention overexpresses PPARα protein specifically in cells constituting muscle tissue or muscle tissue to induce fatty acid oxidation and induce obesity resistance do.

Introduction into nuclear donor cells

The present invention also relates to a method for introducing a recombinant vector containing the PPARα into a nuclear donor cell and a cell into which the recombinant vector is introduced.

A recombinant vector consisting of the human myoglobin promoter (hMb p ); and the behavior containing the PPAR alpha gene is introduced into the nuclear donor cell.

In one embodiment, the nuclear donor cell can be a dog embryo cell, a somatic cell, or a stem cell.

In certain embodiments, for example, but not limited to, cumulus cells, epithelial cells, fibroblasts, neurons, keratinocytes, hematopoietic cells, melanocytes, cartilage cells, macrophages, monocytes, muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, fetal derived cells, embryonic cells and embryonic cells. Also, adult stem cells derived from various origins can be used, such as adult stem cells such as fat, uterus, bone marrow, muscle, placenta, cord blood or skin (epithelium). Non-human host embryos can generally be embryos comprising a 2-cell stage, a 4-cell stage, an 8-cell stage, a 16-cell stage, a 32-cell stage, a 64-cell stage, .

More preferably, it may be a fetal-derived cell, an adult fibroblast, or a cumulus cell. Most preferably, fibroblasts isolated from fetuses and adults 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 embryonic cells, somatic cells or stem cells provided as nuclear donor cells can be obtained from a method for preparing surgical specimens or biopsy specimens using conventional methods known in the art.

The recombinant vector according to the present invention can be introduced into cells by a method known in the art.

For example, but not by way of limitation, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran The present invention relates to a method for introducing a nucleic acid into a cell, such as, for example, DEAEDextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, Into a cell for the production of a transgenic animal.

Meanwhile, a nuclear donor cell transformed with a recombinant vector containing a human myoglobin promoter (hMb p ) and a PPARα gene for producing a muscle specific PPARα overexpression model according to the present invention can be produced by a method known in the art ≪ / RTI >

Suitable media are those which have been developed for the cultivation of animal cells and in particular mammalian cells, or for any use which may be produced in the laboratory together with suitable components necessary for animal cell growth, such as assimilable carbon, nitrogen and / or micronutrients Possible media can be used.

The medium may be any basic medium suitable for animal cell growth, including but not limited to MEM (Minimal Essential Medium), DMEM (Dulbecco modified Eagle Medium), RPMI (Roswell Park Memorial Institute Medium) and K-SFM (Keratinocyte Serum Free Medium). In addition, there is no limit to the use of any medium used in the industry. Preferably, the cells are cultured in a culture medium such as? -MEM medium (GIBCO), K-SFM medium, DMEM medium (Welgene), MCDB 131 medium (Welgene), IMEM medium (GIBCO), DMEM / F12 medium, PCM medium, M199 / (GIBCO), and MSC expansion medium (Chemicon).

To this base medium may be added an assimilative source of carbon, nitrogen and micronutrients, as a non-limiting example, a serum source, a growth factor, an amino acid, an antibiotic, a vitamin, a reducing agent, and / or a sugar source.

It will be apparent to those skilled in the art that suitable media may be selected or combined and cultured appropriately in a known manner. In addition, it is apparent that culturing can be carried out while adjusting conditions such as a suitable culture environment, time, temperature and the like on the basis of ordinary knowledge in this field.

[Transgenic Animal]

In one embodiment, the nucleus of a nuclear donor cell into which a recombinant vector containing a PPARα is introduced is transplanted into an enucleated oocyte to produce a PPARα protein specifically in cells constituting muscle tissue or muscle tissue, Specific overexpression of the PPAR? Overexpression model, and a dog for the muscle specific PPAR? Overexpression model produced thereby.

In certain embodiments, the embryo can be conceived under conditions suitable to produce a dog that specifically overexpresses the PPAR alpha protein in cells that make up muscle tissue or muscle tissue.

For example, the somatic cell nuclear transfer method (SCNT) described above can be used using transgenic somatic cells.

In certain embodiments, the present invention produces a clone of the muscle specific PPAR? Overexpression model of the invention by somatic cell nuclear transfer (SCNT) using a transgenic cell line knocked out of the PPARa gene.

In one embodiment, the method for producing a dog for a muscle-specific PPAR? Overexpression model of the present invention comprises:

(a) preparing a nuclear donor cell comprising culturing somatic cells or stem cells isolated from the dog;

(b) introducing into the nuclear donor cell a recombinant vector containing a human myoglobin promoter (hMb p ) and a PPAR alpha gene;

(c) removing the nucleus from the eggs to prepare a enucleated oocyte;

(d) injecting and fusing nuclear donor cells of step (b) into the enucleated oocytes of step (c);

(e) activating the oocyte fused in step (d); And

(f) transplanting the activated oocyte into a surrogate mother's oviduct.

The conventional technology for each step can be understood with reference to a conventional method for producing a cloned animal using a somatic cell nuclear transfer technique known in the art.

In one embodiment, the nucleus of a somatic cell, an embryo cell or a stem cell derived from a canine transformed with a composition containing a recombinant vector containing PPAR alpha is transplanted into an enucleated oocyte, It is possible to provide a transfer line.

In another embodiment, there is provided a method for producing a transgenic dog for a muscle-specific PPAR? Overexpression model knocking a PPAR? Gene comprising transplanting the nuclear transfer embryo into a tubal plug of a surrogate mother to produce a live animal, and a method for producing the PPAR? Specific < / RTI > PPARa overexpression model.

In certain embodiments, the invention encompasses a method of producing a GM animal,

The method comprises exposing an embryo or cell to a recombinant vector (e. G., A recombinant vector containing PPARa)

Cloning a cell in a surrogate mother, or transplanting embryos in a surrogate mother, wherein the targeting nuclease specifically binds to an embryonic or intracellular target chromosomal region to cause a change in a cell chromosome, Genetically engineered animals can be conceived.

In one embodiment of the present invention, it was confirmed that insertion of a nucleic acid sequence coding for a PPARα gene was confirmed through analysis of gene expression and protein expression of the PPARα gene knocked in the present invention.

The PPARα gene is transfected using transgenic dog cells that have been confirmed to be knocked out to produce a dog specific for the PPARα overexpression model in which the PPARα gene is knocked down.

There is no limitation on the method of replication, but, for example, somatic cell nuclear transfer (SCNT) can be used using transgenic somatic cells as described above.

[Usage]

One embodiment of the present invention provides for the use of dogs overexpressing PPARa protein with PPARa gene knocked down and muscle specificity.

The transgenic dog for the PPARα overexpression model specific for the PPARα gene knocked in accordance with the present invention can be produced through crossing, and the external gene can be transferred later.

Therefore, the present invention is characterized in that the PPARa gene of the present invention is knocked out and that the knocked PPAR alpha gene is expressed as a protein specifically in muscle, so that the dog has an advantage of easy reconstitution, And is useful as an animal model that is resistant to diseases such as metabolic diseases such as obesity.

That is, it is possible to utilize the muscle specific PPARα overexpression model of the present invention to study mechanisms involved in the induction or treatment of metabolic diseases, the role of PPARα, and model studies with obesity resistance.

The invention therefore, in a different aspect, comprises a variety of uses for the muscle specific PPARa over-expression model in this way.

For example, an animal model according to the present invention can be used as a method for screening for agents that can maintain or increase obesity resistance in related studies, for example, obesity resistance.

In one embodiment, the screening method of the present invention comprises

1) administering a candidate substance capable of maintaining or increasing obesity resistance in a muscle specific PPAR? Overexpression model;

2) After the administration of the candidate substance, the dog tissue may be analyzed in comparison with the control group to which the candidate substance is not administered.

Preferably, the candidate substance is any one selected from the group consisting of peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and plasma. The compound may be a novel compound or a well-known compound. These candidate substances may form salts.

The method of administering the candidate substance may be suitably selected in accordance with the symptoms of the subject animal, the nature of the candidate substance, etc., for example, by oral administration, intravenous injection, subcutaneous administration, intradermal administration or intraperitoneal administration. The dose of the candidate substance can be appropriately selected in accordance with the method of administration, the nature of the candidate substance, and the like.

Obesity and metabolic diseases

In the present invention, obesity generally means that the fat tissue is excessively present in the body, and the energy consumed by the food is not balanced with the energy consumed by the physical activity, and the surplus energy accumulates in the body fat. Unusually high body fat due to energy imbalance over a long period of time can lead to various metabolic diseases and adult diseases such as diabetes, hyperlipidemia, heart disease, stroke, arteriosclerosis and fatty liver.

Obesity is caused by hypertrophy or hyperplasia of body fat cells morphologically.

Obesity is caused by abnormal enlargement of subcutaneous fat tissue caused by accumulation of excess energy in the body when the metabolic process imbalance occurs due to endocrine factors, genetic factors, and socio - environmental factors. The hypertrophy of adipose tissue is a phenomenon that the size of adipocyte increases (adipocyte hypertrophy) and the number of adipocyte hyperplasia increases (adipocyte hyperplasia), which also affects the localization of the venous-lymphatic system, .

The excessively accumulated triglyceride in obese patients is stored not only in adipose tissue but also in the liver or muscle, leading to insulin resistance. Therefore, the consumption of excessively stored triglycerides can be a preventive and therapeutic treatment of essential obesity and thus metabolic diseases.

The obesity or metabolic diseases include but are not limited to metabolic syndrome, hypertriglyceridemia, hyperlipidemia, hypolipidemia, angina pectoris, myocardial infarction, sexual dysfunction, sleep apnea syndrome, premenstrual syndrome, stress urinary incontinence Diabetes, hypertension, impaired glucose tolerance, coronary artery thrombosis, diaphoresis, depression, anxiety, psychosis, hyperlipidemia, hyperlipidemia, hyperlipidemia, hyperlipidemia, , Obesity, polycystic ovarian disease such as dysthymic disorder, drug addiction, drug abuse, cognitive disorders, Alzheimer's disease, cerebral ischemia, obsessive-compulsive behavior, panic attacks, social phobia, bulimia such as atherosclerosis, atherosclerosis, gallstone disease, Disorders, infections, skin diseases such as varicose veins, epidermal hyperplasia and eczema, insulin resistance, chronic arterial occlusion, orthopedic injuries, blood Related diseases selected from the group consisting of pre-eclampsia, heart disease, urinary disease, lipid syndrome, hyperglycemia, and stress.

As the PPARα overexpression model of the present invention overexpresses PPARα protein in the muscle tissue, the activity of PPARα is artificially increased to increase the expression of genes involved in fatty acid oxidation and energy consumption, thereby decreasing fat accumulation RTI ID = 0.0 > animal models with < / RTI >

In addition, the PPARα overexpression model of the present invention overexpresses PPARα protein in muscle tissue, resulting in an increase in the activity of PPARα, thereby increasing the expression of genes involved in fatty acid oxidation and energy consumption And thus can be provided as an animal model for the study of athletic performance / muscle strength (including muscle atrophy treatment studies).

In addition, since the PPAR alpha overexpression model of the present invention overexpresses PPAR alpha protein in muscle tissue, the activity of PPAR alpha increases artificially and promotes fatty acid metabolism, thereby promoting the effect of promoting skin barrier formation , Thus providing an animal model for anti-aging studies.

Thus, the present invention provides an animal model of overexpressing PPARα overexpressing muscle and its various uses, thereby making it possible to identify the role of PPARa in metabolic diseases, study resistance to metabolic diseases (such as obesity), exercise / strength improvement studies ) And anti-aging research.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Materials and methods

1. Animals

Animal experiments were conducted according to standard procedures established by the Seoul National University Committee on Laboratory Animal Care and Use Guide and Committee for Approval of Laboratory Animal Care.

2. Production of PPARα-overexpressing cells in a muscle-specific manner

To produce canine peroxisome proliferator activated receptor alpha (PPARα) overexpressing cells in a muscle-specific manner, human PPARα (hPPARα) gene was synthesized. A vector was constructed as shown in FIG. 1, in which the PPARα gene was introduced under the control of the human myoglobin (hMb) promoter, which is a muscle specific promoter, and the copGFP gene was introduced under the control of the EF1 promoter as a marker gene. After the vector was introduced into fetal fibroblasts, the expression of the marker gene, GFP, was visually confirmed and molecular biologic analysis was performed by polymerase chain reaction (PCR). As a result, it was confirmed that each vector was introduced into the genome.

The cells were cultured until reaching confluency in a 100 mm culture dish, and then separated into single cells by trypsin treatment. Cells were collected in 15 ml cornical tubes, washed with Dulbecco Modified Eagle Medium and centrifuged. The supernatant was removed and DNA was extracted according to the manufacturer's protocol using a genomic DNA extraction kit using a pellet.

Forward primer (5'-GCAGCAACAGATGGAAGG-3 ') and reverse primer (5'-GCTCGTAGAAGGGGAGGTT-3') to detect the Puromycin resistance gene, forward primer (5'-CTACGGCTTCTACCACTTCG-3 ' primer (5'-GGATGATCTTGTCGGTGAAG-3 '). PCR was performed using the PCR premix kit according to the manufacturer 's protocol. The presence of the bands at the 203 bp and 230 bp sites was confirmed for the Puromycin resistance gene and the copGFP gene, respectively.

3. Production of overexpressed PPARα using somatic cell nuclear transfer

In order to recover matured oocytes from the ovules, blood samples were collected every day or every day from the estrus biopsy to analyze the P4 trend. When the oocyte reached 6-10ng / ml, the oocyte stage was determined and 72 hours later, Respectively. After the anesthesia with respiration, the operation site was prepared according to general laparotomy and the midline was incised, and the uterine ovary was trained and the ovarian ligament was fixed to expose the ovary. A flushing needle was inserted into the ovarian end of the fallopian tube, temporarily fixed with a suture, and the vessel catheter inserted in the opposite direction from the junction of the fallopian tubes. The catheter was connected to a syringe and TCM based HEPES buffered media was injected, and the ovulated oocytes were recovered into a Petri dish along with the medium and transferred to the laboratory.

After removal of cumulus cells from hyaluronidase, oocytes with a first polar body and a width of 15-25 μm were selected for somatic cell nuclear transfer. The muscle-specific PCK1-expressing cells were cultured at 80% confluency on the day of somatic cell nuclear transfer, and were trypsinized immediately before the experiment and isolated into single cells. The mature oocytes were removed from the cumulus cells and stained with Hoechst. The cells were enucleated using a micro - manipulator and single cells were injected into the proliferated eggs of each enucleated oocyte. Cell fusion and cell fusion were induced by electrical stimulation of 2 pulses of direct current (72 V for 15 ms) using an Electro-Cell Fusion apparatus (NEPA GENE, Chiba, Japan). Fused oocyte - cell fusions were selected and activated with Calcium ionophore. The cloned embryos were loaded at the end of the Tomcat catheter and injected into the fallopian tubes of synchronized oocyte donation nests and ovulation cycles.

On the 26th day after the transplantation, the pregnancy diagnosis was made by surrogate mother's abdominal screening with ultrasound and the fetus number confirmed by X-ray after the 45th day after the pregnancy was confirmed. After about 1-25 days of blood sampling from about 52 days, P4 decline was analyzed, and fetal heart rate was measured by ultrasound to determine natural delivery or cesarean section. A cesarean section was performed if P4 fell below 1 or after 60 days of labor, or if the fetal heart rate decreased from 180 to 200 cycles per minute or less.

4. Identification of transgene in PPARα overexpressing dogs by PCR

As a positive control, donor cells were cultured in a 100 mm culture dish until confluence was reached, and then separated into single cells by trypsin treatment. Cells were collected in 15 ml cornical tubes, washed with Dulbecco Modified Eagle Medium and centrifuged. The supernatant was removed and DNA was extracted according to the manufacturer's protocol using a genomic DNA extraction kit using a pellet. Blood was collected from 1 non-transgenic cloned dog and 3 transgenic transgenic canines as negative control, and DNA was extracted according to the manufacturer's protocol using genomic DNA extraction kit.

Forward primer (5'-CTACGGCTTCTACCACTTCG-3 ') and reverse primer (5'-GGATGATCTTGTCGGTGAAG-3') to detect the copGFP gene and forward primer (5'-TCGGTGACTTATCCTGTGGT-3 ') to detect hPPARα gene and reverse primer (5'-AAGGCACTTGTGAAATCGAC-3 '). PCR was performed using the PCR premix kit according to the protocol of the manufacturer and it was confirmed whether the bands were generated at 230 bp and 249 bp regions for the copGFP gene and the hPPARα gene, respectively.

Example 1: Genomic insertion of a foreign gene

Southern blotting was performed to verify the insertion of the exogenous gene, that is, the exogenous PPARα gene into the genome. As a result, it was confirmed that foreign genes were inserted into all three transgenic canines (AL1, 2, 3) as shown in the figure below. More than 5 copies of AL1 and AL2 and 3 copies of AL3 were inserted (FIG.

Example 2: Evaluation of function of foreign genes

To evaluate the function of exogenous genes, we performed blood lipid level test and weight gain analysis according to the increase of feed quantity. First, blood lipid levels were measured at 2 months, 6 months, and 2 years of age. Serum lipase levels were higher in the transgenic animals at the age of 6 months, but not significantly at 6 months of age. However, blood lipid levels were significantly higher in the 2 - year - old group than in the control group. The overexpression of the PPARα gene in the muscle increased the rate of fatty acid degradation by β-oxidation, and the lipase concentration was increased for fatty acid production as a compensating action (FIG. 11).

Example 3: Amount of body weight gain with increasing feed amount of transgenic plants

For the analysis of body weight gain with increasing feed quantity, feeding amount was doubled for 8 weeks and weight gain per week compared to initial body weight was analyzed. From the third week, AL3 began to feed, and AL1 and AL2 started to feed after 7 and 8 weeks, respectively. The same control group fed all feeds for 8 weeks. The body weight gain of two (AL1, AL2) for six weeks (A) and three (8) weeks of body weight gain were analyzed. This is probably because the overexpression of the intramuscular PPAR alpha gene in transgenic cloned dogs results in an increase in the rate of fatty acid degradation due to? Oxidation, resulting in a higher basic energy metabolism (Fig. 12).

Claims (19)

A recombinant vector for constructing muscle-specific PPARα-overexpressing animal models comprising the myoglobin promoter and the peroxisome proliferator-activated receptor alpha (PPARα) gene.
The method according to claim 1,
Wherein the myoglobin promoter is a human myoglobin promoter. 2. The recombinant vector according to claim 1, wherein the myoglobin promoter is a human myoglobin promoter.
The method according to claim 1,
Wherein said vector is a viral vector. 2. A recombinant vector for constructing an animal model of overexpressing PPAR?
5. The method of claim 4,
Wherein the viral vector is at least one vector selected from the group consisting of a retrovirus vector, an adenovirus vector, an adeno-associated viral vector, a herpes virus vector, an avipox virus vector, and a lentivirus vector. Recombinant vector.
A dog for the muscle-specific PPARα over-expression model, including the insertion of the human myoglobin promoter and the peroxisome proliferator-activated receptor alpha (PPARα) gene in the genome.
6. The method of claim 5,
The dog for the muscle-specific PPAR [alpha] over-expression model is characterized in that PPAR [alpha] is overexpressed in cells constituting muscle tissue or muscle tissue.
6. The method of claim 5,
Wherein the dog for the muscle-specific PPAR? Overexpression model has obesity resistance.
A method for producing a dog for a muscle specific PPAR? Overexpression model using a nucleus of a cell into which a recombinant vector containing a human myoglobin promoter and a PPAR? Gene is introduced.
9. The method of claim 8,
Wherein the method for producing the muscle specific PPAR? Overexpression model uses somatic cell nuclear transfer (SCNT) technology.
10. The method of claim 9,
The somatic cell nuclear transfer (SCNT) technique involves transplanting the nucleus of a cell into which a recombinant vector containing a human myoglobin promoter and a PPAR alpha gene has been introduced into a perinuclear oocyte of a dog, Method for manufacturing dogs for models.
9. The method of claim 8,
Wherein the method for producing a dog for the muscle specific PPAR alpha overexpression model comprises the steps of:
(a) preparing a nuclear donor cell comprising culturing somatic cells or stem cells isolated from the dog;
(b) introducing a recombinant vector comprising a human myoglobin promoter and a PPAR? gene into the nuclear donor cell of step (a);
(c) removing the nucleus from the eggs to prepare a enucleated oocyte;
(d) injecting and fusing nuclei of nuclear donor cells of step (b) into the enucleated oocytes of step (c);
(e) activating the oocyte fused in step (d); And
(f) transplanting the activated oocyte into a surrogate mother's fallopian tube.
Transgenic cells for the production of muscle specific PPAR? Overexpressing animal models into which a recombinant vector containing the human myoglobin promoter and the PPAR? Gene has been introduced.
13. The method of claim 12,
Wherein the transformant for constructing the muscle-specific PPAR alpha overexpressing animal model is a nuclear donor cell into which a recombinant vector containing a human myoglobin promoter and a PPAR alpha gene has been introduced. cell.
14. The method of claim 13,
Wherein the nuclear donor cell is an embryonic cell, a somatic cell, or a stem cell.
14. The method of claim 13,
Wherein the nuclear donor cell is a fetal-derived cell, an adult fibroblast, or a cumulus cell; and the transformed cell for producing a muscle-specific PPAR? Overexpressing animal model.
13. The method of claim 12,
Wherein the transformed cell for constructing the muscle specific PPARα overexpressing animal model is a nuclear transfer embryo of a nuclear donor cell into which a recombinant vector containing a human myoglobin promoter and a PPARα gene has been transferred. Transgenic cells for overexpressing animal models.
13. The method of claim 12,
The transgenic cells for constructing muscle-specific PPARα overexpressing animal models are characterized in that human myoglobin promoter and PPARα gene are inserted into the genome.
13. The method of claim 12,
Wherein the transformed cell for constructing the muscle-specific PPAR? Overexpressing animal model overexpresses PPAR? Compared to the untransformed cell.
A pharmaceutical screening method for maintaining or increasing obesity resistance using a dog for a muscle specific PPAR? Overexpression model, comprising:
(i) administering a candidate substance capable of maintaining or increasing obesity resistance in a dog for a muscle specific PPAR? overexpression model; And
(ii) comparing the muscle tissue for the muscle-specific PPAR? overexpression model with the control group not administering the candidate substance after the step (i), and comparing the resistance to obesity.

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