KR102221865B1 - 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 the peroxisome proliferative activating receptor alpha (PPARα) specifically in muscle, and a method for producing the same, and to the use thereof, in particular, a transformation to specifically overexpress the PPARα protein in muscle tissue. It relates to the use of the dog as an animal model of obesity resistance with increased fatty acid oxidation and energy expenditure.

Description

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

The present invention relates to a transgenic dog for metabolic disease research, a method for producing the same, and its use, and more specifically, to a transgenic dog overexpressing a peroxisome proliferative activating receptor alpha (PPARα) for metabolic disease research. It is used as a model.

In living organisms, due to internal and external factors, the function of the living body is degraded, resulting in loss of homeostasis in cells, and the metabolic efficiency of sugars and fats is also reduced. In addition, overnutrition due to changes in eating habits increases metabolic dysfunction such as obesity, hyperlipidemia, diabetes, and cardiovascular diseases such as hypertension and atherosclerosis, and these diseases are a complex cause including genetic and nutritional factors. Have.

Peroxisome (Peroxisome) is one of the organelles in the cell that is related to this metabolic dysfunction, and through many studies, it has been found that it plays an important role in the metabolism of oxygen, glucose, lipids, and hormones. In addition, it has been reported that peroxisomes have a wide range of effects on the regulation of cell proliferation and differentiation and the regulation of inflammatory mediators.

Nuclear hormone receptors, called peroxisome proliferator-activated receptors (PPARs), have been shown to be a good target for pharmacological approaches to diseases caused by metabolic dysfunction. PPAR is one of 48 nuclear receptors, which binds to ligands and regulates the expression of related genes in the downstream, and PPARs that have been identified to date have three isoforms: PPARα, PPARδ, and PPARγ (J Steroid Biochem. Molec. Biol., 51, 157, 1994; Gene Expression, 4, 281, 1995; Biochem. Biophys. Res. Commun., 224, 431, 1996).

PPARα is mainly found in blood vessel walls, liver, heart, muscle, kidney, and brown adipose tissue, and fibrates such as fenofibrate, bezafibrate, ciprofibrate, and gemfibrozil. (fibrate) is an agonist of PPARα, which prevents or delays the onset of arteriosclerosis in rats and humans, and is known to have anti-obesity action through promotion of fatty acidation. In addition, PPARα plays an important function in promoting keratinocyte differentiation and proliferation in the epidermis of the skin, promoting skin barrier formation through lipid metabolism, suppressing inflammation, and healing wounds. The inhibitory effect of erythema production has also been reported.

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

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

Since PPAR plays an important role in fat metabolism, many studies are being conducted to develop therapeutic agents for metabolic diseases targeting PPAR. In particular, it has been reported that PPARα regulates lipid metabolism, inflammation and arteriosclerosis, and in recent years, many studies on PPARα as a kind of target protein for improving and treating inflammation, arteriosclerosis, obesity, and cancer have been conducted.

PPARα increases the expression of several catabolic enzymes related to β-oxidation in mitochondria and peroxisomes and ω-oxidation in mitochondria, and thus in liver or muscle. It is known to enhance the oxidation of fatty acids. In recent studies, it has been reported that when PPARα activity is artificially activated in adipocytes and muscle cells, the expression of genes involved in fatty acid oxidation and energy consumption is increased. In particular, it has been reported that lipid accumulation is decreased as a result of treatment with a ligand or agonist for PPARα in experimental obese mice (Wang Y. X. et al., Cell, 113: 159, 2003).

In addition, Wy14,643, the most well-known PPARα ligand, reduces triglyceride (TG) and blood cholesterol levels in human metabolism, while high density lipoprotein (HDL) in the intestine and liver. It is known to actively regulate cholesterol remodeling and cholesterol outflow (Peter Zahradka, Cardiovascular Drug Reviews, 25: 99, 2007; Caroline Duval et al., Biochemica et Biophtsica Acta, 1771: 961, 2007). In addition, the ligand of PPARα is known to stimulate the ABCA1 pathway to promote cholesterol removal from blood vessels while reducing the formation of dysfunction (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 hyperinsulinic patients with metabolic syndrome. In addition, Apo AI and A-II related to PPARα lipoprotein metabolism, as major proteins of HDL, have the effect of preventing vascular disease by moving excess cholesterol in tissues to the liver, increasing the expression of PPARα Apo AI and A-II genes. It is known to decrease the incidence of arteriosclerosis by increasing the HDL concentration (Stael B. et al., Circulation, 98: 2088-2093, 1998).

In addition, it is reported that PPARα KO mice exhibit obesity and hypertriglyceridemia even if the daily caloric intake is not increased. This effect is mainly explained by the reduction of fatty acid absorption and inhibition of fatty acid oxidation by the liver (J. Biol. Chem., 1998, 273, 29577-29585). These research reports indicate that PPARα is widely involved in fat breakdown and is an important target protein for metabolic diseases caused by obesity.

Accordingly, the present inventors applied a technique for transplanting the nucleus of somatic cells transformed to overexpress PPARα, which is considered to be an important target protein for metabolic diseases, to produce an animal model usable for the study of metabolic diseases. It was confirmed that an overexpressed dog model could be produced, and the present invention was completed.

US published patent US 2004-0038249 A1 US published patent US 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 PPARα overexpression model and a method for producing the same.

Another object of the present invention is to provide a method for overexpressing muscle-specific PPARα using a muscle-specific expression system, which is necessary for the method of manufacturing a dog for the muscle-specific PPARα overexpression model.

Another object of the present invention is to provide a muscle-specific expression system required for the method of manufacturing a dog for the muscle-specific PPARα overexpression model, cells transformed with the muscle-specific expression system, and nuclear transfer embryos.

Another object of the present invention is to provide various uses of dogs for the muscle-specific PPARα overexpression model.

In order to solve the above problems, the present invention provides a dog for a muscle-specific PPARα overexpression model and a method of manufacturing the same.

The present invention as an embodiment,

It provides a recombinant vector for constructing a muscle-specific PPARα overexpression animal model containing a human myoglobin promoter and a PPARα gene, which overexpresses peroxisome proliferative activation receptor alpha (PPARα) specifically for muscle.

At this time, the vector may be a viral vector, and the viral vector is at least one vector selected from the group consisting of a retrovirus vector, an adenovirus vector, an adeno-related virus vector, a Herpes virus vector, an abipox virus vector, and a lentiviral vector. I can.

As another embodiment of the present invention,

A dog for a muscle-specific PPARα overexpression model characterized by overexpressing a muscle-specific PPARα protein transformed by a vector containing a human myoglobin promoter and a PPARα gene is provided.

The PPARα protein overexpression is expressed in dog muscle tissue or cells constituting the muscle tissue.

The dog for the muscle-specific PPARα overexpression model may be transformed with the vector described above. In particular, the transformed cell is a human myoglobin (myoglobin) promoter and PPARα gene inserted in the genome, and the muscle-specific PPARα overexpression model dog is a somatic cell nuclear transfer technology (somatic cell nuclear transfer) to the nucleus of the transformed cell. , SCNT).

The muscle-specific PPARα overexpression model dog is characterized in that the individual for the obesity resistance model having obesity resistance.

Therefore, the present invention is another embodiment,

It provides a method for producing a dog for a muscle-specific PPARα overexpression model, characterized in that the nucleus of a nuclear donor cell into which the recombinant vector has been introduced is transplanted into an enucleated egg to produce a litter.

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, an adult fibroblast, or a cumulus cell. In one embodiment of the present invention, fibroblasts isolated from fetuses and adults are used.

Therefore, the method of manufacturing a dog for a muscle-specific PPARα 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 dogs;

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

(c) removing the nucleus from the dog's egg to prepare an enucleated egg;

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

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

(f) implanting the activated egg into the fallopian tube of the surrogate mother dog.

The present invention includes all of the transformation vectors, cells, and nuclear transfer embryos required for the production of a muscle-specific PPARα overexpression model that can be obtained in the course of the method for manufacturing a dog for the muscle-specific PPARα overexpression model.

That is, the present invention is another specific example,

A recombinant vector containing a PPARα gene and a human myoglobin promoter can be introduced, and a transformed cell for producing a muscle-specific PPARα overexpression animal model can be provided,

A canine nuclear transfer embryo formed by transplanting the nucleus of a dog-derived somatic cell or stem cell transformed with a recombinant vector containing a PPARα gene and a human myoglobin promoter into an enucleated egg can also be provided.

In addition, the present invention provides various uses of dogs for the muscle-specific PPARα overexpression model.

As an example, there is provided a method for screening a medicament for maintaining or increasing obesity resistance, comprising:

1) administering a candidate substance capable of maintaining or increasing obesity resistance to the muscle-specific PPARα overexpression model dog of the present invention;

2) After administration of the candidate substance, analyzing the muscle tissue of the dog by comparing the control group to which the candidate substance was not administered and obesity resistance.

As described above, the present invention provides a muscle-specific PPARα overexpression animal model having resistance to metabolic diseases and various uses thereof in metabolic diseases (eg, obesity, etc.).

The dog for the muscle-specific PPARα overexpression model of the present invention is a dog model having improved body function and resistance to obesity as the expression of genes involved in fatty acid oxidation and energy consumption is increased by muscle-specific overexpression of PPARα. It can be used in various studies related to metabolic diseases such as studies related to metabolic diseases (obesity resistance studies, etc.), motor performance/muscle enhancement studies (including muscle atrophy treatment studies, etc.), and anti-aging studies.

1 is a schematic diagram as a specific example of a recombinant vector comprising a PPARα gene and a human myoglobin promoter.
2 is a fluorescence microscopic observation photograph of GFP expression in fibroblasts transformed with a recombinant vector.
3 is a PCR result of Puro R and GFP expression of fibroblasts transformed with a recombinant vector.
4 is a schematic diagram of somatic cell nuclear transplantation technology.
5 shows PCR primers for confirming the expression of PPARα in dogs generated through somatic cell nuclear transplantation technology.
6 is a summary of the number of dogs generated through somatic cell nuclear transplantation technology.
7 is an image of a dog generated through somatic cell nuclear transplantation technology.
8 is a PCR result confirming the expression of PPARα in dogs generated through somatic cell nuclear transplantation technology.
9 is a graph showing changes in weight of a dog generated through somatic cell nuclear transplantation technology.
Figure 10 is a result of confirming long-term gene insertion in a transgenic individual by southern blotting (N, non-cloned dog, P, vector, α 1-3, transgenic cloned dogs).
Figure 11 shows the serum lipase concentration in the transgenic individuals of 2 months old, 6 months old and 2 years old and the control group suitable for each age.
Figure 12 is a graph comparing the amount of weight gain, (A) the total weight gain of three transgenic dogs (AL dogs) and (B) only two transgenic dogs (AL1 and AL2 who ate all feed for 6 weeks) Is the result of analysis.

Definitions of representative terms used in the present invention are as follows.

“Muscle-specific expression system” is a collective term for a system that allows any gene or protein to be expressed in muscle tissue or cells constituting muscle tissue. The muscle-specific expression system is a regulatory element capable of specifically controlling the expression of any gene or protein in muscle tissue or cells constituting the muscle tissue, for example, a promoter, an enhancer. The expression system may include a vector system, a virus system, etc. including a nucleic acid sequence encoding an arbitrary gene, but also includes any system capable of expressing an arbitrary gene or protein in a cell. .

"Vector" or "expression vector" is a plasmid known in the art that can insert a nucleic acid encoding a structural gene and express the nucleic acid in a host cell, a vector including a transposable element, and a viral vector. Or other media. Preferably, it may be a vector containing a transfer factor or a viral vector.

The term "recombinant vector" refers to a vector capable of expressing a protein of interest or RNA of interest in a suitable host cell, and refers to a genetic construct comprising essential regulatory elements operably linked so that a gene insert is expressed.

The term "expression control sequence" or "regulatory element" refers to a DNA sequence that controls the expression of a nucleic acid sequence operably linked in 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 regulating the termination of transcription and translation. In the present invention, the regulatory factor may be a promoter or enhancer.

"Promoter" refers to a DNA sequence capable of controlling the transcription of a specific nucleotide sequence into mRNA when linked to a specific sequence. Typically, the promoter is not applied in all cases, but exists 5'(i.e., upstream) of the desired nucleotide sequence to be transcribed into mRNA, and the site to which RNA polymerase and other transcription factors for initiating transcription 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 is capable of directing the transcription of an operably linked nucleic acid sequence without stimulation (eg, 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 specific sequence linked to the promoter is transcribed specifically for muscle.

"Operably linked" refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to affect the expression of the encoding nucleic acid sequence. The operative linkage with a recombinant vector can be made using genetic recombination techniques well known in the art.

"Transformation" means to change the genetic properties of an organism by DNA given from outside. Transformation methods include various methods known in the art, such as microinjection, electroporation, particle bombardment, sperm-mediated gene transfer, and viral infection. It can be appropriately selected and applied from techniques using virtual infection, direct muscle injection, insulator, and transposon. Preferably, in the present invention, the expression vector containing human PPARα can be transformed into canine fetal fibroblasts through viral infection.

“Cell”, “host cell”, “modified host cell” and the like refer to a particular target cell as well as progeny or potential progeny of such cells. Because certain modifications may occur laterally by mutation or environmental influences, such progeny, in fact, will not be identical to the parental cell, but are still included within the term and scope used herein.

The term "model animal for disease research" refers to an animal having a disease very similar to a human disease or resistant to a disease. The significance of disease model animals in human disease research is based on physiological or genetic similarities between humans and animals. In disease research, model animals for biomedical disease research provide materials for research on various causes, pathogenesis, and diagnosis of diseases. Through the study of model animals for disease research, genes related to diseases are identified, and interactions between genes are performed. It is possible to understand and obtain basic data to determine the possibility of practical use through the actual efficacy and toxicity tests of the developed new drug candidate.

"Animal" or "experimental animal" means any mammalian animal other than human. The animal includes animals of all ages, including embryos, fetuses, newborns and adults. Animals for use in the present invention can be used, for example, from commercial sources. These animals include laboratory animals or other animals, rabbits, rodents (e.g. mice, rats, hamsters, gerbils and guinea pigs), cattle, sheep, pigs, goats, horses, dogs, cats, birds (e.g., Chickens, turkeys, ducks, goose), primates (eg, chimpanzees, monkeys, rhesus monkeys). The most preferred animal is a dog.

“Overexpression” means that any gene or protein is expressed above a normal level. In the present invention, it means that the expression of the PPARα protein is expressed above the expression level of normal cells, and in particular, expression may be expressed above the normal level in muscle tissue or cells constituting the muscle tissue. The overexpressing transformant may be a transformed cell, a transformed tissue, or a transformed animal in which the PPARα protein is expressed above a normal level. For example, it may be an overexpression transformant produced by exposing the cells, tissues, or animals to a substance including a muscle-specific expression system, or introducing the substance.

"Nuclear transplantation" refers to a genetic engineering technology that artificially binds another cell or nucleus to an enucleated egg to have the same traits. "Nuclear transplantation" refers to an egg into which a nuclear donor cell has been introduced or fused.

"Replication" is a genetic engineering technology to create a new individual having the same set of genes as one individual. In particular, in the present invention, dog somatic cells, embryonic cells, fetal-derived cells and/or adult-derived cells are substantially identical to the nuclear DNA sequence of other cells. It refers to having a nuclear DNA sequence. The present invention uses a technique for cloning a dog using a nuclear transfer technique. In particular, somatic cell nucleus transplantation technology is a technology that allows progeny to be born without passing through meiosis and haploid-bearing germ cells, which are generally performed in the reproductive process. It is a method of producing and transplanting the fertilized egg into a living body to generate a new individual.

"Nuclear donor cell" refers to a cell or nucleus of a cell that transfers the nucleus to a nuclear receptor, a recipient egg. "Ovum" preferably refers to a mature egg that has reached the second stage of meiosis. In the present invention, canine somatic cells or stem cells may be used as the nuclear donor cells.

A "somatic cell" is a cell other than a germ cell among cells constituting a multicellular organism, and the ability to differentiate into a differentiated cell that specializes for a certain purpose and does not become a cell other than that, and a cell with various other functions. Includes cells with

"Stem cell" means a cell that can develop into any tissue. There are two basic characteristics. First, it has the ability to differentiate itself into cells with specific functions depending on the environment, self-renewal, which creates oneself by repetitive division.

"Cultivation" is the growth of living organisms or parts of living organisms (organs, tissues, cells, etc.) under appropriately artificially controlled environmental conditions. In this case, external conditions include temperature, humidity, light, and gaseous composition (carbon dioxide or oxygen). Partial pressure), etc. are important, and the most important direct influence on the organism to be cultured is the medium (incubator), which is the direct environment of the organism and the supply of various nutrients necessary for survival or proliferation.

"In vitro culture" refers to a series of laboratory processes in which cells are cultured in a laboratory incubator under conditions similar to the internal environment in a manner that is distinct from the state in which cells grow in the body.

"Medium" or "medium composition" refers to a mixture for growth and proliferation of cells, etc. in vitro, including essential elements such as sugar, amino acids, various nutrients, serum, growth factors, minerals, etc. essential for the growth and proliferation of cells.

"Living offspring" refers to an animal that can survive outside the womb. Preferably, it refers to an animal that can survive for one second, one minute, one hour, one day, one week, one month, six months, or one year or more. These animals do not require an intrauterine environment to survive.

"Obesity" refers to a condition in which body fat is excessive, and clinically, the body mass index is 25 in Korea and 30 or more according to the World Health Organization (WHO). At this time, the body mass index (BMI) is calculated as ^{the weight of the square of the height (kg/m 2 ).} In general, it means when the weight is higher than the normal value, but even if the weight is not much, it is diagnosed as obesity when the proportion of body fat among the components of the body is high, and it is a disease that occurs in both adults and children. In addition, obesity is a disease caused by inactivation or abnormality/disorder of mechanisms or factors related to oxidation of fatty acids, energy consumption, and the like.

"Obesity resistance" refers to a state in which the occurrence of obesity is suppressed or reduced by inhibiting or inhibiting factors and environments that may cause obesity by increasing or activating mechanisms or factors that inhibit obesity, such as fatty acid oxidation and energy consumption.

“Treatment” refers to an approach to obtain beneficial or desirable clinical outcomes. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (i.e., not exacerbation), delay or decrease in disease progression, disease state Amelioration or temporal alleviation and alleviation of (partially or entirely), detectable or undetectable. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include the disorder to be prevented as well as the treatment required for a disorder that has already occurred. "Palliating" the disease is a reduced or prolonged time course of progression and/or the extent of the disease state and/or undesirable clinical signs compared to without treatment. Means to lose.

“About” means 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 for a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length. , Means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length varying by 3, 2 or 1%.

Throughout this specification, unless the context requires otherwise, the terms "include" and "comprise" include, but are not limited to, a given step or element, or a group of steps or elements, but any other step or element, or step or element. It should be understood to imply that the group of people is not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to a muscle-specific PPARα expression animal model.

More specifically, it relates to a muscle-specific PPARα overexpressing dog model, a method of making the same, and its use.

The muscle-specific PPARα overexpression dog model of the present invention is characterized in that it is transformed and used to specifically overexpress the PPARα protein in muscle tissue or cells constituting muscle tissue.

In this case, the PPARα gene can be transformed using any known method, but a somatic cell nuclear transfer (SCNT) method is preferably used. That is, a muscle-specific PPARα overexpression model cloned animal of the present invention is produced using a somatic cell nucleus transplantation (SCNT) method using a transformed cell line overexpressing a specific gene PPARα.

Therefore, for example, the method for producing muscle-specific PPARα overexpression model dogs of the present invention through somatic cell nuclear transplantation method,

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

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

(c) removing the nucleus from the dog's egg to prepare an enucleated egg;

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

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

(f) implanting the activated egg into an oviduct of a surrogate mother dog.

The general technical content of each step can be understood by referring to a method for preparing a cloned animal using a conventional somatic cell nuclear transplantation technique known in the art.

[Muscle-specific PPARα expression system]

Muscle specific PPARα Expression recombinant vector

The present invention provides a muscle-specific PPARα expression vector that specifically overexpresses PPARα in muscle tissue or cells constituting muscle tissue.

In particular, the vector is characterized by comprising a human myoglobin (myoglobin) promoter (hMb p). The hMb p and PPARα genes may be operably linked.

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

In the present invention, PPARα is one of the PPARs, and is a protein encoded and expressed in the PPARα (or PPARA) gene, and is a protein composed of 468 amino acids, and is also known as NR1C1. Mainly expressed in blood vessel wall, liver, heart, muscle, kidney, brown adipose tissue, and several catabolic activities related to β-oxidation in mitochondria and peroxisomes and ω-oxidation in mitochondria. It is known to increase the expression of catabolic enzymes, thereby enhancing the oxidation of fatty acids in the liver or muscle. In particular, it has been reported that PPARα regulates lipid metabolism, inflammation and arteriosclerosis, and recently, PPARα is attracting attention as a kind of target protein that improves and treats inflammation, arteriosclerosis, obesity, and cancer.

The muscle-specific PPARα expression recombinant vector of the present invention can be used for transformational mutations that knock-in the PPARα gene into the genome of a cell, wherein the knock-in is a specific foreign gene expression It means that it is introduced into the host's genome so that it can be made. Using somatic cells into which the recombinant vector has been introduced, it is possible to produce a transgenic individual knocked-in with the PPARα gene by performing nuclear transfer into an animal's fertilized egg, and implanting a nuclear-transferred fertilized egg.

The nucleic acid encoding PPARα can be suitably used by those skilled in the art from a sequence known in the art and having a nucleotide sequence encoding non-limiting PPARα.

The nucleic acid encoding PPARα can be prepared by genetic recombination methods known in the art. For example, for example, PCR amplification for amplifying a nucleic acid from a genome, a chemical synthesis method, or a technique for preparing a cDNA sequence.

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

The functional equivalent is one having at least 70%, preferably 80%, more preferably 90% or more sequence homology with a wildtype amino acid sequence as a result of amino acid addition, substitution or deletion, and the PPARα of the present invention. It refers to a polypeptide that exhibits a physiological activity substantially equivalent to that of. The "equal physiological activity" refers to increasing the expression of several catabolic enzymes related to β-oxidation in mitochondria and peroxisomes and ω-oxidation in mitochondria. Therefore, it refers to the activity of inducing the oxidation of fatty acids.

At this time, the amino acid deletion or substitution is preferably located in a region 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 a plasmid, viral vector, or other medium known in the art capable of expressing a nucleic acid encoding a PPARα gene specifically in muscle tissue or cells constituting muscle tissue, and is preferable. It may be a viral vector.

Examples of the viral vector include, but are not limited to, a retroviral vector, an adenovirus vector, a Herpes virus vector, an abipox virus vector, a lentiviral vector, and the like.

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

These may be collectively referred to as'a structure comprising a nucleic acid encoding PPARα' or a'DNA structure including a nucleic acid encoding hMb p -PPARα'.

That is, the vector may be composed of a human myoglobin promoter (hMb p ); and a behavior containing the PPARα 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, and a fluorescent protein such as a green fluorescent protein and a red fluorescent protein, but is limited thereto. It doesn't work.

In addition, in order to confirm whether the PPARα protein is overexpressed, a tag sequence for protein isolation or purification may be additionally included in the vector of the present invention. Examples of the tag sequence include GFP, GST (Glutathione S-transferase)-tag, HA, His-tag, Myc-tag, T7-tag, and the like, but the tag sequence of the present invention is not limited by the above examples. In a preferred embodiment of the present invention, the presence or absence of expression and the amount of expression were confirmed using GFP.

Preferably, the vector prepared by the above method may be the vector disclosed in FIG. 1.

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

Introduction to nuclear donor cells

In addition, the present invention relates to a method for introducing the recombinant vector containing PPARα into a nuclear donor cell, and to a cell into which the recombinant vector has been introduced.

A recombinant vector composed of a behavior containing a human myoglobin promoter (hMb p ); and a PPARα gene is introduced into a nuclear donor cell.

In one embodiment, the nuclear donor cells may be canine embryonic cells, somatic cells, or stem cells.

In certain embodiments, for example, but not limited to, cumulus cells, epithelial cells, fibroblasts, nerve cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, macrophages, monocytes, muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, embryo-derived cells, placental cells, and embryonic cells. In addition, adult stem cells derived from various tissues of origin, for example, stem cells derived from tissues such as fat, uterus, bone marrow, muscle, placenta, umbilical cord blood, or skin (epithelial) may be used. The non-human host embryo may generally be an embryo 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, a morula, or a blastocyst. .

More preferably, it may be fetal-derived cells, adult fibroblasts, and cumulus cells. Most preferably, fibroblasts isolated from fetuses and adults of dogs are used. The characteristics of these cells are that a large number of cells can be obtained during initial separation, cell culture is relatively easy, and culture and manipulation in vitro are easy.

The embryonic cells, somatic cells or stem cells provided as nuclear donor cells can be obtained from a method of preparing a surgical specimen or a biopsy specimen using a conventional method 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 limited thereto, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran -Mediated transfection (DEAEDextran-mediated transfection), polybrene-mediated transfection, electroporation, gene gun and other known methods for introducing nucleic acids into cells It can be introduced into cells for production of transgenic animals by the method.

On the other hand, for the production of a muscle-specific PPARα overexpressing dog model according to the present invention, a human myoglobin promoter (hMb p ); and a nuclear donor cell transformed with a recombinant vector containing a PPARα gene is a method known in the art. It can be proliferated and cultured according to.

Appropriate media are any use that can be developed for the cultivation of animal cells and, in particular, mammalian cells, or that can be prepared in vitro with the appropriate components necessary for animal cell growth, such as anabolic carbon, nitrogen and/or micronutrients Any possible medium can be used.

The medium is any basic medium suitable for animal cell growth, as a non-limiting example, and as a basic medium generally used for culture, MEM (Minimal Essential Medium), DMEM (Dulbecco modified Eagle Medium), RPMI (Roswell Park Memorial Institute) Medium) and K-SFM (Keratinocyte Serum Free Medium), and in addition, any medium used in the industry can be used without limitation. Preferably, α-MEM medium (GIBCO), K-SFM medium, DMEM medium (Welgene), MCDB 131 medium (Welgene), IMEM medium (GIBCO), DMEM/F12 medium, PCM medium, M199/F12 (mixture) (GIBCO), and MSC expansion medium (Chemicon) may be selected from the group consisting of.

To this basal medium, anabolic sources of carbon, nitrogen and micronutrients, such as, but not limited to, serum sources, growth factors, amino acids, antibiotics, vitamins, reducing agents, and/or sugar sources can be added.

It will be apparent that one of ordinary skill in the art can select or combine a suitable medium and appropriately cultivate it by a known method. In addition, it is obvious that the culture can be cultured while adjusting conditions such as suitable culture environment, time, temperature, etc. based on common knowledge in this field.

[Transgenic animals]

As an embodiment of the present invention, by transplanting the nucleus of a nuclear donor cell into which a recombinant vector containing PPARα has been introduced into an enucleated oocyte to produce a litter, the PPARα protein is specifically produced in muscle tissue or cells constituting the muscle tissue. It relates to a method for preparing a transgenic dog for an overexpressing muscle-specific PPARα overexpression model, and to a dog for a muscle-specific PPARα overexpression model prepared thereby.

In certain embodiments, embryos can be conceived under suitable conditions to produce dogs that specifically overexpress the PPARα protein in muscle tissue or cells constituting the muscle tissue.

For example, the somatic cell nucleus transplantation method (SCNT) described above can be used using somatic cells of a transformed dog.

In certain embodiments, the present invention produces a muscle-specific PPARα overexpression model cloned animal of the present invention by somatic cell nuclear transfer (SCNT) using a transformed cell line knocked in with a PPARα gene.

In one embodiment, the method for producing a dog for a muscle-specific PPARα overexpression model of the present invention,

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

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

(c) removing the nucleus from the dog's egg to prepare an enucleated egg;

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

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

(f) implanting the activated egg into the oviduct of the surrogate mother dog.

The general technical content of each step can be understood by referring to a method for preparing a cloned animal using a conventional somatic cell nuclear transplantation technique known in the art.

In one embodiment, a method for preparing a nuclear transfer egg for a dog formed by transplanting the nucleus of a dog-derived somatic cell, embryonic cell, or stem cell transformed with a composition containing a recombinant vector containing PPARα into an enucleated egg, and a nucleus produced thereby Transplanted eggs can be provided.

In another embodiment, a method for preparing a transgenic dog for a muscle-specific PPARα overexpression model in which the PPARα gene is knocked in, comprising the step of producing a livestock by transplanting the nuclear transfer egg into the fallopian tube of a surrogate mother, and the PPARα gene prepared by knock-in It provides a dog for a muscle-specific PPARα overexpression model, characterized in that.

In certain embodiments, the present invention includes a method of producing a genetically engineered animal,

The method comprises the steps of exposing the embryo or cell to a recombinant vector (e.g., a recombinant vector containing PPARα),

Cloning cells within the surrogate mother or transplanting embryos within the surrogate mother, wherein the targeting nuclease specifically binds to an embryo or an intracellular target chromosome site to cause a change in the cellular chromosome, and the surrogate mother is at the target chromosome site. Genetically engineered animals can be conceived.

In one embodiment of the present invention, it was confirmed that the insertion of the nucleic acid sequence encoding the PPARα gene occurred through the analysis of the gene and protein expression of the transgenic dog knocked-in with the PPARα gene in the present invention.

A dog for a muscle-specific PPARα overexpression model in which the PPARα gene is knocked in is produced through re-cloning using the cells of the transformed dog whose PPARα gene is knock-in confirmed.

The method of re-cloning is not limited, but, for example, the above somatic cell nucleus transplantation method (SCNT) can be used using somatic cells of a transformed dog.

[Usage]

One embodiment of the present invention provides a use of a dog knock-in with a PPARα gene and overexpressing a PPARα protein specifically for muscle.

The transgenic dog for a muscle-specific PPARα overexpression model knocked in the PPARα gene according to the present invention can be produced through mating, and the foreign gene can be transferred later.

Therefore, dogs characterized in that the PPARα gene of the present invention is knocked-in and that the knocked-in PPARα gene is specifically expressed as a protein in muscle has the advantage of easy remodeling, so that the PPARα protein is specifically used for muscle. By expressing, it is useful as an animal model having resistance to metabolic diseases such as obesity and the like.

That is, using the muscle-specific PPARα overexpression model dog of the present invention, a variety of applications such as a study on the mechanism involved in inducing or treating a metabolic disease of PPARα, a role of PPARα, and a model study having obesity resistance will be possible.

Therefore, in another aspect, the present invention encompasses various uses of dogs for muscle-specific PPARα overexpression models with the above method.

For example, the animal model according to the present invention can be used in studies related to obesity resistance, for example, as a method of screening for drugs capable of maintaining or increasing obesity resistance.

In one embodiment, the screening method of the present invention

1) administering a candidate substance capable of maintaining or increasing obesity resistance to a muscle-specific PPARα overexpression model dog;

2) After the administration of the candidate substance, it may include the step of comparing the tissues of the dog with a control group to which the candidate substance was not administered and analyzing it.

The candidate material is preferably 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, but is not limited thereto. The compound may be a novel compound or a widely known compound. Such candidate substances may form salts.

As a method of administering such a candidate substance, for example, oral administration, intravenous injection, subcutaneous administration, intradermal administration, or intraperitoneal administration can be appropriately selected according to the symptoms of the target animal and the properties of the candidate substance. In addition, the dosage of the candidate substance can be appropriately selected according to the administration method or the properties of the candidate substance.

Obesity and metabolic diseases

In the present invention, obesity generally refers to a state in which adipose tissue is excessive in the body, and is a phenomenon in which excess energy is accumulated as body fat because energy consumed by food cannot be balanced with energy consumed by physical activities. Abnormal increase in body fat due to energy imbalance over a long period of time can lead to various metabolic diseases such as diabetes, hyperlipidemia, heart disease, stroke, arteriosclerosis, fatty liver, etc. and adult diseases.

The obesity is morphologically caused by an increase in the size of fat cells (hypertrophy) or an increase in the number (hyperplasia) of fat cells in the body.

Obesity is caused by abnormal hypertrophy of subcutaneous fat tissue caused by the accumulation of excess energy in the body when an imbalance in metabolic processes occurs due to endocrine factors, genetic factors, social and environmental factors, etc. Adipose tissue hypertrophy is a phenomenon in which the size of adipocytes increases (adipocyte hypertrophy) or the number of adipocytes increases (adipocyte hyperplasia), which also affects the stagnation of the local venous-lymphatic system, resulting in vascular tissue disease of the dermal-subcutaneous tissue It can also cause.

In obese patients, excessively accumulated triglycerides are stored not only in adipose tissue, but also in the liver and muscles to induce insulin resistance. Therefore, the consumption of excessively stored triglycerides can be the prevention and treatment of obesity and metabolic diseases resulting therefrom.

The obesity or metabolic disease includes, but is not limited to, metabolic syndrome, hypertriglyceridemia, hyperdensity lipidemia, low density lipidemia, angina, myocardial infarction, hypogonadism, sleep apnea, premenstrual syndrome, and stress urinary incontinence. Urinary incontinence, hyperactivity disorder, chronic fatigue syndrome, osteoarthritis, weight gain-related cancer, orthostatic hypotension, pulmonary hypertension, menstrual disorder, diabetes, hypertension, impaired glucose tolerance, coronary artery thrombosis, drowsiness, depression, anxiety, psychosis , Delayed dyskinesia, drug addiction, substance abuse, cognitive impairment, Alzheimer's disease, cerebral ischemia, compulsive behavior, panic attacks, social phobia, gallbladder diseases such as atherosclerosis, cholelithiasis, loss of appetite, polycystic ovary disease. Disorders, infections, varicose veins, skin diseases such as epidermal proliferation and eczema, insulin resistance, chronic arterial obstruction, orthopedic injury, thromboembolism, heart disease, urinary disease, lipid syndrome, hyperglycemia and stress. These include obesity-related diseases.

In the dog for the muscle-specific PPARα overexpression model of the present invention, as the PPARα protein is overexpressed in muscle tissue, the activity of PPARα is artificially increased, thereby increasing the expression of genes involved in fatty acid oxidation and energy consumption, thereby reducing fat accumulation. It can be provided as an animal model with obesity resistance.

In addition, in the dog for the muscle-specific PPARα overexpression model of the present invention, as the PPARα protein is overexpressed in muscle tissue, the activity of PPARα is artificially increased, thereby enhancing the function of the muscle by increasing the expression of genes involved in fatty acid oxidation and energy consumption. Therefore, accordingly, it can be provided as an animal model for motor performance/muscle enhancement studies (including muscle atrophy treatment studies, etc.).

In addition, in the dog for the muscle-specific PPARα overexpression model of the present invention, as the PPARα protein is overexpressed in muscle tissue, the activity of PPARα is artificially increased to promote fatty acid metabolism, thereby inducing effects such as promoting skin barrier formation. , Accordingly, it can be provided as an animal model for anti-aging research.

As such, the present invention provides a muscle-specific PPARα overexpression animal model and various uses thereof, to identify the role of PPARα in metabolic diseases and to study resistance to metabolic diseases (obesity, etc.), and to improve exercise performance/muscle strength (muscular atrophy treatment studies, etc.) Including) and anti-aging research, it will be very useful in various studies.

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art 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 the standard procedures established by the Seoul National University's Guide to the Care and Use of Laboratory Animals and the Committee for Approval of Laboratory Animal Care.

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

In order to produce canine peroxisome proliferator activated receptor alpha (PPARα) overexpressing cells in a muscle-specific manner, a human PPARα (hPPARα) gene was synthesized. A vector 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, as shown in FIG. 1. After introducing the constructed vector into canine fetal fibroblasts, the expression of the marker gene GFP was visually confirmed, and molecular biological analysis was performed by polymerase chain reaction (PCR), confirming that each vector was introduced into the genome.

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

Forward primer (5'-GCAGCAACAGATGGAAGG-3') and reverse primer (5'-GCTCGTAGAAGGGGAGGTT-3') to detect puromycin resistance gene, forward primer (5'-CTACGGCTTCTACCACTTCG-3') and reverse primer to detect copGFP gene A primer (5'-GGATGATCTTGTCGGTGAAG-3') was prepared. PCR was performed according to the manufacturer's protocol using a PCR premix kit, and it was confirmed whether bands were generated at 203 bp and 230 bp regions for the Puromycin resistance gene and the copGFP gene, respectively.

3. Production of PPARα-overexpressing Dogs Using Somatic Cell Nuclear Transplantation

In order to recover mature oocytes in the body, blood collection was performed daily or every other day from pre-estrus female females, analyzed the P4 trend, determined the time to reach 6-10 ng/ml as the time of ovulation, and performed surgery to recover mature oocytes after about 72 hours. Implemented. After respiratory anesthesia, the surgical site was prepared according to general laparotomy, the midline was incised, the uterine ovary was towed, and the ovarian suspension ligament was fixed to expose the ovary. After inserting a flushing needle into the end of the fallopian tube in the direction of the ovary, temporarily fixing it with sutures, and then inserting a vascular catheter in the opposite direction from the junction of the uterine fallopian tube. After connecting the syringe to the catheter and injecting the TCM based HEPES buffered media, the ovulated egg in the oviduct was collected in Petri dish along with the medium and transferred to the laboratory.

After removing the cumulus cells from hyaluronidase, oocytes with the first polar body and 15-25 um wide of the upper ovary were selected and used for somatic cell nuclear transplantation. Muscle-specific PCK1-expressing cells were cultured to have more than 80% confluency on the day of somatic cell nucleus transplantation, and then trypsinized immediately before the experiment to separate them into single cells. Mature oocytes in the body from which the cumulus cells were removed were denuclearized with a micromanipulator by staining the first pole and nucleus with Hoechst, and single cells were injected into the gastric cavity within each enucleated oocyte. Using an Electro-Cell Fusion Apparatus (NEPA GENE, Chiba, Japan), 2 pulses of direct current (72 V for 15 ms) electrical stimulation was used to induce fusion between the ovarian cytoplasm and cells. The fused egg-cell fusion was selected, activated with calcium ionophore, and then a cloned embryo was loaded at the end of the Tomcat catheter and injected into the fallopian tube of the surrogate mother in which the egg donor dog and the ovulation cycle were synchronized.

The pregnancy was diagnosed by screening the abdomen of the surrogate mother by ultrasound on the 26th day after the transplantation date, and the number of fetuses was confirmed by X-ray on the 45th day after the pregnancy was confirmed. From about the 52nd day, blood was collected 1-2 times a day, and then the P4 decrease trend was analyzed and the fetal heart rate was monitored by ultrasound to determine natural delivery or cesarean section. When the P4 fell below 1 or the fetal heart rate decreased below 180 to 200 beats per minute around 60 days, cesarean resection was performed.

4. Identification of transgene in dogs overexpressing PPARα using PCR

As a positive control, donor cells were cultured in a 100 mm culture dish until confluency was reached, and then separated into single cells through trypsin treatment. The cells were collected in a 15ml cornical tube, washed with Dulbecco Modified Eagle Medium, and centrifuged. The supernatant was removed, and DNA was extracted using a pellet according to the manufacturer's protocol using a genomic DNA extraction kit. As a negative control, blood was collected from the lumbar cortical vein of one non-transgenic clone and three transgenic individuals produced, and then DNA was extracted according to the manufacturer's protocol using a genomic DNA extraction kit.

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

Example 1: Confirmation of the insertion of foreign genes into the genome

Southern blot was performed to verify the insertion of a foreign gene, that is, an externally introduced PPARα gene into the genome. As a result, it was confirmed that foreign genes were inserted into all three transgenic dogs (AL1, 2, 3) as shown in the figure below, and more than 5 copies of AL1 and AL2, and 3 copies of AL3 were inserted (FIG. 10).

Example 2: Evaluation of internal function of foreign genes

In order to evaluate the function of the foreign gene in the body, blood lipase concentration test and weight gain analysis according to the increase in feed amount were performed. First, the results of the blood lipase concentration test at the age of 2 months, 6 months and 2 years are shown in the table below. In the transgenic subjects, blood lipase concentration tended to be high from childhood, but there was no significant difference until 6 months of age. However, two-year-old individuals showed significantly higher levels of lipase than the control group. It is believed that overexpression of the PPARα gene in the muscle increased the rate of decomposition of fatty acids by β oxidation, and the lipase concentration for fatty acid production increased as a compensation for this (FIG. 11).

Example 3: Analysis of weight gain according to increase in feed amount of transgenic individuals

In order to analyze the weight gain according to the increase in feed amount, the feed amount was doubled for 8 weeks, and the weight gain was analyzed every week compared to the initial weight. AL3 started to leave feed from the 3rd week, and AL1 and AL2 started to leave feed from the 7th and 8th weeks, respectively. The control group of the same age consumed all of the feed for 8 weeks. As a result of analyzing the weight gain of three dogs for 8 weeks (A) and the weight gain of two dogs (AL1, AL2) for 6 weeks (B), it was confirmed that in both cases, the weight gain tends to be low in the transgenic cloned dogs. This is thought to be due to the high basal energy metabolism in transgenic cloned dogs due to an increase in the rate of decomposition of fatty acids by β oxidation due to overexpression of the PPARα gene in muscle (Fig. 12).

Claims (22)

delete delete delete delete It has a genome into which a human myoglobin promoter and a human peroxisome proliferator-activated receptor alpha (PPARα) gene are inserted,
Transgenic dogs in which the PPARα protein is overexpressed in muscle specificity compared to the naturally occurring form of dogs.
The method of claim 5,
The transgenic dog is a transgenic dog, characterized in that PPARα is overexpressed in muscle tissue or cells constituting the muscle tissue.
The method of claim 5,
Transgenic dog, characterized in that the transgenic dog has obesity resistance.
A method for producing a transgenic dog in which a muscle-specific PPARα protein is overexpressed using the nucleus of a cell into which a recombinant vector containing a human myoglobin promoter and a human PPARα gene has been introduced.
The method of claim 8,
The method for producing the transformed dog is a method for producing a transformed dog, characterized in that using somatic cell nuclear transfer (SCNT) technology.
The method of claim 9,
The somatic cell nuclear transfer (SCNT) technology is the production of a transformed dog, characterized in that the nucleus of a cell into which a recombinant vector containing a human myoglobin promoter and a human PPARα gene is introduced is transplanted into an enucleated egg of a dog. Way.
The method of claim 8,
The method for preparing the transgenic dog comprises the following steps: A method for producing a transgenic dog in which the PPARα protein is overexpressed specifically for muscle:
(a) preparing a nuclear donor cell comprising culturing somatic cells or stem cells isolated from dogs;
(b) introducing a recombinant vector comprising a human myoglobin promoter and a human PPARα gene into the nuclear donor cell of step (a);
(c) removing the nucleus from the dog's egg to prepare an enucleated egg;
(d) microinjecting and fusing the nuclei of the nuclear donor cells of the (b) step into the enucleated oocyte of the step (c);
(e) activating the egg fused in step (d); And
(f) implanting the activated egg into the fallopian tube of the surrogate mother dog.
Transformed cells for the production of transgenic dogs in which PPARα is overexpressed specifically for muscle containing a recombinant vector containing a human myoglobin promoter and a human PPARα gene.
The method of claim 12,
The transformed cell for producing a transformed dog is a nuclear donor cell into which a recombinant vector including a human myoglobin promoter and a human PPARα gene has been introduced.
The method of claim 13,
The nuclear donor cell is an embryonic cell, a somatic cell, or a stem cell.
The method of claim 13,
The nuclear donor cell is a transformed cell for producing a transformed dog, characterized in that the fetal-derived cells, adult fibroblasts or cumulus cells.
The method of claim 12,
The transformed cell for the production of the transformed dog is a nuclear transfer egg in which the nucleus of a nuclear donor cell into which a recombinant vector containing a human myoglobin promoter and a human PPARα gene is introduced is transplanted cell.
The method of claim 12,
The transformed cell for producing a transformed dog is a transformed cell for producing a transformed dog, characterized in that a human myoglobin promoter and a human PPARα gene are inserted into the genome.
The method of claim 12,
The transformed cell for producing a transformed dog is a transformed cell for producing a transformed dog, characterized in that overexpressing human PPARα compared to a cell that has not been transformed.
As a drug screening method for maintaining or increasing obesity resistance using the transgenic dog of claim 5, a drug screening method comprising the following:
(i) administering a candidate substance capable of maintaining or increasing obesity resistance to a dog muscle-specifically overexpressing human PPARα;
(ii) After the step (i), analyzing the muscle tissue of a dog in which human PPARα is overexpressed specifically for the muscle is compared with a control group not administered a candidate substance for obesity resistance.
A recombinant vector comprising a human myoglobin promoter and a human peroxisome proliferator-activated receptor alpha (PPARα) gene for the production of dogs for the muscle-specific PPARα overexpression model of claim 5.
The method of claim 20,
The vector is a recombinant vector, characterized in that the viral vector.
The method of claim 21,
The viral vector is a recombinant vector, characterized in that at least one vector selected from the group consisting of a retrovirus vector, an adenovirus vector, an adeno-related virus vector, a Herpes virus vector, an abipox virus vector, and a lentiviral vector.

Images