WO2020091472A1 - PRODUCTION OF TRANSGENIC DOG OVER-EXPRESSING PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR δ (PPARδ) IN MUSCLE-SPECIFIC MANNER - Google Patents

Abstract

The present invention relates to a transgenic dog over-expressing peroxisome proliferator-activated receptor δ (PPARδ) in a muscle-specific manner, a production method therefor, and a use thereof, and, specifically, to using, as an animal model having increased motor ability due to slow-twitch muscle generation and an increase in muscular endurance, a a dog transformed in order to over-express PPARδ proteins specifically in muscle tissues.

Description

Production of transgenic dogs overexpressing muscle-specific peroxysome proliferator-activated receptor delta (PPARδ)

The present invention relates to a transformed dog for producing a dog with improved athletic ability, and a method for manufacturing the same, and more specifically, to a transformed dog overexpressing the peroxisome proliferator-activated receptor delta (PPARδ) and a method for manufacturing the same It is about.

Blocking foreign influx infectious sources due to increasing frequency of outbreaks of foot-and-mouth disease (FMD), highly pathogenic avian influenza (AI), and so on through various channels following the trend of increasing trade in goods at home and abroad due to increased economic and social and cultural exchanges There are areas in which humans and machines cannot do this, such as quarantine, drug detection, and lifesaving. In order to solve this, there is a need for an animal that is capable of replacing a person or a machine with excellent athletic ability.

To date, animal production studies that have improved energy metabolism and motor performance have developed a model that overexpresses various genes involved in energy metabolism and growth in rats and mice, and in transgenic rodents, muscle mass, muscle strength, muscle fiber, and mitochondrial biosynthesis compared to controls. Changes in energy metabolism are only being comparatively analyzed.

Therefore, there is a need for medium to large animals with improved energy metabolism and motor performance. Considering the genetic, morphological, and behavioral aspects of medium-to-large animals, transgenic dogs with improved energy metabolism and motor performance appear to be useful.

In addition, in the United States, Canada, the United Kingdom, and Australia, the National Detection and Dog Center has been operated for a long time to secure and nurture excellent dogs, and an efficient training program has been systematized with the help of many experts to secure and nurture a variety of dogs. However, even if an excellent dog is found, it has a problem that the ability is not transmitted as it is, so there is a problem that requires constant high cost and training to cultivate a good dog. Therefore, there is a need for a transgenic dog that is easily maintained with the same trait and has a long life.

Peroxisome is one of the organelles in the cell that is related to metabolic dysfunction. 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 peroxysomes have a wide influence on the regulation of cell proliferation and differentiation and the regulation of salt mediators.

Therefore, it was conceived that a nuclear hormone receptor called peroxisome proliferator-activated receptor (PPAR) would be a good target for pharmacological approaches to diseases caused by metabolic abnormalities. PPAR is one of 48 nuclear receptors, and it binds with ligands to regulate the expression of related genes downstream, and the PPARs identified so far 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).

Among them, the peroxisome proliferation-activated receptor delta (PPARδ) is overexpressed, or through selective removal studies of this gene, it has been found that this gene is a very important regulator of the root muscle kinase. PPARδ is a ligand-dependent transcription factor that is highly expressed in the root muscle. In the case of muscle-specific PPARδ transgenic mice, the production of myofibrillar fibers was significantly increased, and as a result, it was reported that the running time and distance increased by 67% and 92%, respectively (Wang, YX. Et al). ., Plos Biol., 2004, 2. e294). On the other hand, in the case where the PPARδ gene was selectively removed, it was reported that the running time was reduced by 62% compared to the normal mouse, and the running distance was also reduced by 66% (Wang, YX. Et al., PlosBiol., 2004, 2. e294). Therefore, it can be seen that PPARδ is an important factor in the formation of the root muscles and the regulation of motor capacity.

Thus, the present inventors applied the technique of transplanting the nucleus of the somatic cells transformed to overexpress PPARδ, which is considered to be an important factor in the formation of muscle roots and regulation of motor ability, in order to produce a dog with improved motor performance, thereby specifically overexpressing PPARδ in muscle. It was confirmed that the dog can be produced and the present invention was completed.

In areas where humans or machines are difficult to perform, there is a need for medium-to-large animals that can perform tasks on their behalf. The present invention aims to provide a medium-large animal with improved energy metabolism and athletic performance and a long life.

In addition, it is an object of the present invention to provide a dog and a method of manufacturing PPARδ overexpressed specifically in muscle tissue or muscle cells.

Another object of the present invention is to provide a method for producing a dog specifically overexpressed in PPARδ in muscle tissue or muscle cells and a muscle specific expression system required for the dog.

Another object of the present invention is to provide cells and nuclear transfer embryos transformed with the above muscle specific expression system.

Another object of the present invention is to provide various uses of the muscle-specific PPARδ overexpressing dog.

Thus, the present inventors for the study of production of animals that have improved energy metabolism and athletic performance, through the nucleus of somatic cells transformed to overexpress PPARδ recognized as a factor that plays an important role in metabolism of oxygen, glucose, lipids, and hormones. By applying the technique of transplanting, it was confirmed that a dog specifically overexpressed with PPARδ can be produced and completed the present invention.

In order to solve the above problems, the present invention provides a muscle-specific PPARδ overexpressing dog and a method for manufacturing the same.

The present invention is an embodiment,

The alpha-skeletal muscle actin promoter (ACTA promoter) specifically expressed in muscle tissue or muscle cells and the nucleotide sequence encoding PPARδ are operably linked (Knock In, KI). Provided are transgenic dogs overexpressed with PPARδ.

The present invention provides a recombinant vector for producing a muscle-specific PPARδ overexpressing animal comprising an ACTA promoter specifically expressed in muscle tissue or muscle cells and a base sequence encoding PPARδ.

In this case, the vector may be a vector including one or more transfer factors.

The transfer factor may be a piggyBac transfer factor or Sleeping Beauty transfer factor.

The metastasis factor may include an inverted terminal repeat sequence (ITR) and / or a direct repeat (DR) sequence at both ends.

The enzyme that mediates the translocation factor may include piggyBac transferase (transposase) or Sleeping Beauty transferase (transposase).

At this time, the vector may be a piggyBac vector or a Sleeping Beauty vector.

In this case, the vector may be a plasmid or a viral vector, and the viral vector may be one or more selected from the group consisting of retrovirus vector, adenovirus vector, adeno-associated virus vector, herpes virus vector, abipox virus vector, and lentivirus vector. It can be a vector.

Using the above vector, it is possible to provide muscle-specific PPARδ overexpressing transformed cells containing the ACTA promoter and PPARδ gene specifically expressed in muscle tissue or muscle cells.

After the transfer embryo is prepared using the transformed cells, the transformed dog may be provided by somatic cell nuclear transfer (SCNT).

The muscle-specific PPARδ overexpressing dog is characterized by an individual with an increased muscular dystrophy, increased muscular dystrophy, increased muscular endurance, increased running ability (time and / or distance), improved bioenergy metabolism, vitality, and athletic performance.

Therefore, the present invention provides a method for producing a muscle-specific PPARδ overexpressing dog, characterized in that the nucleus of the nuclear donor cell introduced with the above-described recombinant vector is transplanted into a denuclearized egg to produce an acid.

In this case, the nuclear donor cell may be a dog embryo cell, a somatic cell or a stem cell. In one embodiment of the present invention, adipose-derived stem cells (ASC) can be used.

Therefore, the method for producing a muscle-specific PPARδ overexpressing dog of the present invention may include the following steps as a more specific example.

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

(b) introducing a recombinant vector comprising an ACTA promoter and a base sequence encoding PPARδ into the nuclear donor cell;

(c) removing nuclei from dog eggs to produce denuclearized eggs;

(d) microinjecting and fusing nuclear donor cells into the denuclearized egg;

(e) activating the fused egg; And

(f) The activated egg is transplanted into the fallopian canal.

The present invention includes all of the transformation vectors, cells, and nuclear transfer eggs required for the production of muscle-specific PPARδ overexpressing dogs, which can be obtained in the process of the method for preparing the muscle-specific PPARδ overexpressing dogs.

That is, the present invention is another specific example,

It is possible to provide a transformed cell for producing a muscle-specific PPARδ overexpressing animal into which a recombinant vector comprising a base sequence encoding the ACTA promoter and PPARδ is introduced,

A nuclear transfer embryo of a dog formed by transplantation or fusion of a nucleus of a dog-derived somatic cell or stem cell transformed with a recombinant vector containing a base sequence encoding the ACTA promoter and PPARδ into a denuclearized egg can also be provided.

As an example, the muscle-specific PPARδ overexpressing dog is provided as a model dog for analysis of a special purpose dog or a motor mechanism.

In this case, the special purpose dog may be a dog with improved athletic performance compared to a normal dog.

In this case, the dog with improved athletic performance may be a dog having one or more effects of increased muscle atrophy, increased muscular dysfunction, increased muscle endurance, and increased running ability (time and / or distance) compared to a normal dog.

In this case, the general dog may be a dog without transformation for muscle-specific PPARδ overexpression.

The muscle-specific PPARδ overexpressing dog of the present invention has an increased muscle kinesis, increased muscle kinetic activity, increased muscle endurance, increased ability to run (time and / or distance), and increased metabolic disease resistance by muscle-specific overexpressed PPARδ. Eggplant, as a dog, can be used in a variety of metabolic disease-related studies, such as obesity (obesity resistance studies, etc.), exercise / strength improvement studies (including muscle atrophy treatment studies, etc.), and special purpose dogs with improved exercise performance.

1 is a schematic diagram as one embodiment of a recombinant vector for the production of muscle-specific PPARδ overexpressing dogs.

2 is a result of RT-PCR of adipose stem cells into which a recombinant vector has been introduced.

3 is a result of observing green fluorescent protein expression in adipose stem cells into which PB-ACTAp-caPPARδ recombinant vector is introduced.

4 is a result of observing the expression of the GFP gene in somatic cell nuclear transfer replication.

Figure 5 is a genomic DNA PCR results of the muscle-specific PPARδ-expressing transgenic cloned dog, N is a negative control, TG is a muscle-specific PPARδ expressing transgenic cloned dog.

FIG. 6 shows the results of southern bolts of a clone dog expressing transgenic muscle-specific PPARδ, N is a negative control, and TG shows a clone transgenic dog expressing muscle-specific PPARδ.

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

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

"Vector" or "expression vector" is a plasmid known in the art capable of inserting a nucleic acid encoding a structural gene and expressing the nucleic acid in a host cell, a vector containing a transposable element, a viral vector Or other medium. Preferably, it may be a vector containing a transfer factor or a viral vector.

A "recombinant vector" refers to a gene construct comprising essential regulatory elements operably linked to express a gene insert as a vector capable of expressing a target protein or target RNA in a suitable host cell.

By "expression control sequence" or "regulatory element" is meant a DNA sequence that regulates the expression of nucleic acid sequences operably linked in a particular host cell. Such regulatory sequences include any operator sequence to regulate transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that regulates the termination of transcription and translation. In the present invention, the regulatory factor may be a promoter, an enhancer, or the like.

“Promoter” refers to a DNA sequence that can regulate the transcription of a specific nucleotide sequence into mRNA when linked to a specific sequence. Typically, the promoter is not applicable in all cases, but is located at 5 '(i.e., upstream) of the desired nucleotide sequence to be transcribed into mRNA, and a site to which the 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 a nucleic acid sequence to which it is operatively linked without stimulation (eg heat shock, chemicals, etc.). The promoter of the present invention may preferably be an alpha-skeletal muscle actin promoter (ACTA promoter). The ACTA promoter can be regulated such that specific sequences linked to the promoter are muscle-specific. The ACTA promoter may be from a dog.

"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 are operably linked to influence the expression of the encoding nucleic acid sequence. Operational linkage with recombinant vectors can be made using genetic recombination techniques well known in the art.

"Transformation" means changing the genetic properties of an organism by DNA given from outside. As a method of transformation, various methods known in the art, for example, microinjection, electroporation, particle bombardment, and sperm-mediated gene transfer, virus infection It can be appropriately selected and applied from techniques using a viral infection, direct muscle injection, insulator, and transposon. Preferably, in the present invention, an expression vector containing human PPARδ can be transformed into a dog fetal fibroblast through a virus infection method.

"" Animal "or" experimental animal "means any mammalian animal other than humans. The animal includes animals of all ages, including embryos, fetuses, newborns, and adults. , Eg commercial sources, such as laboratory animals or other animals, rabbits, rodents (eg mice, rats, hamsters, gerbils and guinea pigs), cattle, sheep, pigs, goats, Horses, dogs, cats, birds (eg, chickens, turkeys, ducks, geese), primates (eg, chimpanzees, monkeys, rhesus monkeys), but is not limited to the most preferred animals are dogs.

"Overexpression" means that any gene or protein is expressed above a normal level. In the present invention, it means that the expression of PPARδ protein is expressed above the expression level of normal cells, and specifically, it may be expressed specifically above the normal level in muscle tissue or cells constituting muscle tissue. The overexpressed transformant may be a transformed cell in which the PPARδ protein is expressed above a normal level, a transformed tissue, or a transformed animal. For example, it may be an overexpressed transformant produced by exposing the cell, tissue, or animal to a substance containing a muscle-specific expression system or introducing the substance.

"Nuclear transplant" refers to a genetic manipulation technique that artificially binds other cells or nuclei to a denuclearized egg to have the same trait. "Nuclear transplant" refers to an egg in which nuclear donor cells have been introduced or fused.

"Replication" is a genetic manipulation technique to create a new individual with the same gene set as one individual. In particular, in the present invention, the dog's somatic cells, embryonic cells, embryo-derived cells and / or adult-derived cells are substantially identical to the nuclear DNA sequences of other cells. Refers to having a nuclear DNA sequence. The present invention uses the technique of cloning a dog using nuclear transfer technology. In particular, the somatic cell nuclear transfer technology is a technology capable of producing offspring without passing through meiosis and haploid chromosome-bearing germ cells, which are generally performed in the reproductive process. It is a method of producing and transplanting the fertilized egg in vivo to generate a new individual.

“Nuclear donor cell” refers to a cell or nucleus of a cell that delivers the nucleus to a nuclear receptor, a nuclear recipient. The "ovum" preferably refers to a mature egg that has reached the middle of the second meiosis. In the present invention, dog cells or stem cells may be used as the nuclear donor cells.

“Obesity resistance” refers to a state in which the incidence of obesity is suppressed or reduced by suppressing 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.

And / or the time course of progress is delayed or lengthened.

"About" means 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 for reference amount, level, value, number, frequency, percent, dimension, size, amount, weight or length , Amount, level, value, number, frequency, percent, dimension, size, amount, weight or length, varying by 3, 2 or 1%.

Throughout this specification, unless otherwise required by context, the words “comprises” and “comprises” include a given step or element, or group of steps or elements, but any other step or element, or step or element. It should be understood that it implies that the group of the people is not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to a transformed dog having improved nucleotide sequence encoding an ACTA promoter and a PPARδ (Knock in, KI) and improved energy metabolism and exercise ability, a method for manufacturing the same, and use thereof.

More specifically, the nucleotide sequence encoding the ACTA promoter and PPARδ derived from a dog is KI, and thus relates to a transformed dog having improved energy metabolism and athletic ability, a method of manufacturing the same, and use thereof.

Models that overexpress various genes involved in energy metabolism or growth are being developed, but until now, animal production studies that improved energy metabolism and motor performance were mainly conducted in rats and mice.

In addition, there is an increasing need for medium-to-large animals with improved motor skills that can replace areas that humans or machines cannot. Accordingly, in light of genetic, morphological, and behavioral aspects, dogs are likely to be useful as transgenic animals with improved energy metabolism and motor performance. Therefore, more specifically, there is a need for a transgenic dog with improved energy metabolism and athletic performance.

Various genes involved in energy metabolism or growth may be over-expressed to improve the motor performance of the medium-large animal. More specifically, the gene may be PPARδ.

PPARδ, also called PPARβ, is distributed in many tissues and is especially found in skin, brain, and adipose tissues. PPARδ is involved in reverse cholesterol transport, myelination, and wound recovery, and acts as a very important regulator of fatty acid metabolism and energy homeostasis. In addition, PPARδ is known as one of the factors involved in fast-to-slow muscle fiber transformation.

One aspect of the invention may be a transgenic dog overexpressing PPARδ specifically for muscle tissue or muscle cells. Any cell of the transgenic dog may further include a foreign nucleotide sequence encoding PPARδ in addition to an intrinsic base sequence encoding PPARδ.

The muscle tissue or muscle cells of the transformed dog may further include a foreign nucleotide sequence encoding PPARδ in addition to an intrinsic base sequence encoding PPARδ.

The transgenic dog muscle tissue or muscle cell may include two or more base sequences encoding PPARδ.

The PPARδ expression level of any cell of the transgenic dog may be higher than that of any cell of the wild-type dog.

The expression level of PPARδ in muscle tissue or muscle cells of the transformed dog may be higher than that of wild-type dog muscle tissue or muscle cells.

The expression level of PPARδ in the muscle tissue or muscle cells of the transformed dog may be significantly higher than the expression level of PPARδ in the muscle tissue or muscle cells of the wild-type dog.

In another aspect, in order to produce the transformed dog, it is possible to provide a transformed cell with a base sequence encoding the ACTA promoter and PPARδ.

In order to produce the transformed dog, it is possible to provide a transformed cell having a KI sequence encoding a dog-derived ACTA promoter and PPARδ and a method for manufacturing the transformed cell.

In another aspect, in order to prepare the transformed cell, a recombinant vector including an ACTA promoter derived from a dog and a base sequence encoding PPARδ may be provided.

[Muscle specific PPARδ expression system]

Muscle specific PPARδ expression recombinant vector

The present invention provides a recombinant vector comprising a promoter and a base sequence encoding any material.

The present invention provides a recombinant vector that includes a promoter and a base sequence encoding any substance and is capable of modifying the base sequence of any cell.

In one embodiment, the promoter and the base sequence may be operably linked to each other. The promoter and the nucleotide sequence may not be operably linked to each other.

In one embodiment, a recombinant vector comprising an ACTA promoter and a base sequence encoding PPARδ is provided.

In one embodiment, a recombinant vector comprising an ACTA promoter from any animal and a base sequence encoding PPARδ is provided.

In one embodiment, a recombinant vector comprising an ACTA promoter derived from a dog and a base sequence encoding PPARδ is provided.

In one embodiment, the ACTA promoter and the PPARδ gene may be operably linked. The ACTA promoter and the PPARδ gene may not be operably linked. In certain embodiments, the vector may be a vector further comprising one or more transfer factors.

The transfer factor may be a piggyBac transfer factor or Sleeping Beauty transfer factor. The enzyme that mediates the translocation factor may include piggyBac translocation enzyme (transposase) or Sleeping Beauty translocation enzyme (transposase).

In certain embodiments, inverted terminal repeat sequences (ITR) and / or direct repeat (DR) may be further included at both ends of the metastasis factor.

In a specific embodiment, the vector may be a piggyBac vector or Sleeping Beauty vector.

In one embodiment, it is possible to provide a recombinant vector comprising an ACTA promoter, a base sequence encoding PPARδ, a transfer factor, a repeat sequence (ITR and / or DR), and a selectable marker.

In one embodiment, it is possible to provide a recombinant vector comprising an ACTA promoter, a nucleotide sequence encoding PPARδ, a transfer factor, an iterative sequence (ITR and / or DR), and a selectable marker arranged in the same order as shown in FIG. 1. have.

The recombinant vector may be used for knocking in a nucleotide sequence encoding a promoter and any substance into the genome of any cell (Knock in, KI).

At this time, the knock-in (KI) means that it is introduced into the genome of the host so that a specific foreign gene can be expressed.

The recombinant vector may be used for knocking in a nucleotide sequence encoding a promoter and any substance in the genome of muscle tissue or muscle cells.

The base sequence encoding the PPARδ can be appropriately used by those skilled in the art from sequences having a base sequence encoding a non-limiting PPARδ, which is known in the art.

The base sequence encoding the PPARδ can be prepared by genetic recombination methods known in the art. Examples include PCR amplification to amplify nucleic acids from genomes, chemical synthesis or cDNA sequence production techniques, Fmoc techniques, and the like.

In addition, the base sequence may have a base sequence encoding each functional equivalent of PPARδ.

The functional equivalent is about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76% of the amino acid sequence of the wildtype as a result of amino acid addition, substitution or deletion. , About 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% or more homology Refers to a nucleotide sequence comprising a sequence having a polypeptide having a physiological activity substantially equivalent to PPARδ disclosed in the specification. The "equal physiological activity" refers to the activity of regulating mitochondrial biosynthesis, cholesterol reverse transport, myelination, fatty acid metabolism, energy homeostasis, and fast-to-slow muscle fiber transformation.

At this time, deletion or substitution of amino acids may be preferably located in a region not related to the physiological activity of the polypeptide of the present invention.

At this time, deletion or substitution of amino acids may be preferably located in a region related to the physiological activity of the polypeptide of the present invention.

The muscle specific PPARδ expressing recombinant vector of the present invention is a plasmid, a vector containing a transfer factor, a viral vector or other known in the art capable of expressing a nucleic acid specifically encoding PPARδ in muscle tissue or cells constituting muscle tissue. As a medium, it may be preferably a vector containing a transfer factor or a viral vector.

The viral vector is not limited thereto, and may be a retrovirus vector, an adenovirus vector, a herpes virus vector, an abipoxvirus vector, or a lentivirus vector.

Meanwhile, the recombinant vector may further include a selection marker. The selection marker may be an antibiotic resistance gene such as a kanamycin resistance gene or a neomycin resistance gene, or a fluorescent protein such as a green fluorescent protein or a red fluorescent protein, but is not limited thereto.

In addition, in order to confirm whether the PPARδ protein is overexpressed, the vector of the present invention may further include a tag sequence for purification or identification of protein separation. The tag sequence may be GFP, mRuby, GST (Glutathione S-transferase) -tag, HA, His-tag, Myc-tag, or T7-tag, but the tag sequence of the present invention is limited by the examples It does not work. In a specific embodiment, the tag sequence can be used to confirm the presence or absence of expression and the amount of expression using GFP or mRuby.

Transformed cells can be provided using the recombinant vector disclosed in the specification,

A transformed dog can be provided using the transformed cells.

That is, by using the recombinant vector disclosed in the specification, muscle tissue or muscle cell-specific PPARδ protein can be overexpressed to induce muscle formation and muscle endurance, thereby inducing an improvement in athletic performance.

Transformed cells

The present invention provides a transformed cell with a knock-in sequence encoding a promoter and a specific substance.

The present invention provides a transformed cell in which the base sequence encoding the ACTA promoter and PPARδ is knocked out.

The base sequence encoding the ACTA promoter and PPARδ can be knocked through a known method obvious to those skilled in the art. The transformed cells can be produced by introducing the recombinant vector into nuclear donor cells.

The present invention provides a method for producing the transformed cell and the cell into which the recombinant vector has been introduced.

The recombinant vector can be introduced into nuclear donor cells by any method apparent to those skilled in the art.

In one embodiment, the nuclear donor cell may be a dog embryonic cell, a somatic cell or a stem cell.

The nuclear donor cells include, but are not limited to, blastocytes, epithelial cells, fibroblasts, neurons, 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 tissues of various origins, for example, fat, uterus, bone marrow, muscle, placenta, cord blood, or skin (epithelial) may be stem cells.

Further, the nuclear donor cell may be a non-human host embryo, and is generally a 2-cell stage, 4-cell stage, 8-cell stage, 16-cell stage, 32-cell stage, 64-cell stage, loss Embryos, or embryos comprising blastocysts.

In addition, the nuclear donor cells may be embryo-derived cells, adult fibroblasts, and adipose stem cells.

In addition, the nuclear donor cells may be adipose stem cells or adult fibroblasts of dogs. The characteristics of the cells have the advantage of being able to obtain a large number of cells at the time of initial separation, cell culture is relatively easy, and it is easy to culture and manipulate in vitro.

In one embodiment, the embryonic cells, somatic cells or stem cells provided as nuclear donor cells can be obtained from a method for preparing a surgical specimen or biopsy specimen using conventional methods known in the art.

Recombinant vectors according to the invention can be introduced into cells by methods known in the art.

For example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran -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 the production of transgenic animals by methods.

Meanwhile, a specific gene can be KI in the genome of a transformed cell using the recombinant vector. Specific regions in the cell genome can be used to overexpress PPARδ specifically in muscle tissue or muscle cells.

As an example, the transgene can be inserted into a safe harbor site in the genome of liver cells.

The safe harbor site is a specific region in the genome that does not cause serious side effects, such as cancer, even when a foreign gene is inserted, and the foreign gene inserted in the specific region has a high level of permanent and safe expression. It is possible.

In one embodiment, in order to produce the transformed dog, it is possible to provide a transformed cell in which the nucleotide sequence encoding the ACTA promoter and PPARδ derived from the dog is KI.

The transformed cell may further include a foreign nucleotide sequence encoding PPARδ in addition to an intrinsic base sequence encoding PPARδ.

The transformed cell may further include a foreign nucleotide sequence encoding PPARδ in addition to an intrinsic base sequence encoding PPARδ.

The transformed cell may include two or more base sequences encoding PPARδ.

The transformed dog can be used to produce the transformed dog.

Meanwhile, the transformed cells may be proliferated or cultured according to methods known in the art.

The transformed cells can be grown or cultured in the medium. The medium can be any medium that can be prepared for incubation of animal cells, mammalian cells, or in vitro with appropriate components necessary for animal cell growth, such as anabolic carbon, nitrogen and / or micronutrients. Can be used.

The medium is any basic medium suitable for animal cell growth, as a non-limiting example, Minimal Essential Medium (MEM), Dulbecco modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Keratinocyte Serum Free (K-SFM) Medium), 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 extended media (Chemicon). In addition, any medium used in the industry can be used without limitation. To the medium, an anabolic source of carbon, nitrogen and micronutrients, serum source, growth factor, amino acid, antibiotic, vitamin, reducing agent, and / or sugar source may be further added.

A person having ordinary skill in the art can select or combine a suitable medium and appropriately culture it by a known method. In addition, it can be cultivated while adjusting conditions such as a suitable culture environment, time, and temperature based on common knowledge in this field.

A transformed dog can be produced using the transformed cells.

Somatic cell nuclear transfer (SCNT) can be used as the method.

The nucleus of the transformed cell is transplanted into the fertilized egg of an animal, and the fertilized egg implanted with the nucleus is conceived to produce a transformed individual encoding a promoter and a specific sequence encoding a specific substance. Implanted in the fertilized egg of the nucleus, and implanted with the nuclear implanted embryo, it is possible to produce a transformed individual encoding the ACTA promoter and PPARδ.

[Transgenic animals]

The present invention relates to a transformed dog having improved nucleotide sequence encoding an ACTA promoter and a PPARδ (Knock in, KI) and improved energy metabolism and exercise ability, a method for manufacturing the same, and use thereof.

More specifically, the nucleotide sequence encoding the ACTA promoter and PPARδ derived from a dog is KI, and thus relates to a transformed dog having improved energy metabolism and athletic ability, a method of manufacturing the same, and use thereof.

Any cell of the transgenic dog may further include a foreign nucleotide sequence encoding PPARδ in addition to an intrinsic base sequence encoding PPARδ.

The muscle tissue or muscle cells of the transformed dog may further include a foreign nucleotide sequence encoding PPARδ in addition to an intrinsic base sequence encoding PPARδ.

The transgenic dog muscle tissue or muscle cell may include two or more base sequences encoding PPARδ.

The PPARδ expression level of any cell of the transgenic dog may be higher than that of any cell of the wild-type dog.

The expression level of PPARδ in muscle tissue or muscle cells of the transformed dog may be higher than that of wild-type dog muscle tissue or muscle cells.

The amount of PPARδ expression in muscle tissue or muscle cells of the transgenic dog is about 1, about 1.1, about 1.2, and about 1.3 times that of wild-type dog muscle tissue or muscle cells. About 1.4 times. About 1.5 times. About 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 It can be as high as any multiple of a fold, or more.

The expression level of PPARδ in muscle tissue or muscle cells of the transformed dog may be significantly higher than that of wild-type dog muscle tissue or muscle cells.

The transformed cells can be used to produce the transformed dog. In one embodiment, the transformed cells may be implanted under suitable conditions to induce pregnancy.

In that way, for example, transformed cells can be induced to conceive through somatic cell nuclear transfer (SCNT).

In one embodiment, the present invention is transformed by the somatic cell nuclear transfer method (somatic cell nuclear transfer, SCNT) using a transformed cell line knocked down the base sequence encoding the ACTA promoter "I PPARδ from a dog" Animals can be produced.

The transformed animal may be a dog overexpressing PPARδ.

The transformed animal may be a dog overexpressing PPARδ in muscle tissue or muscle cells.

In a specific embodiment, the method for producing a transformed dog of the present invention,

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

(b) introducing a recombinant vector comprising an ACTA promoter and a base sequence encoding PPARδ into the nuclear donor cell;

(c) removing nuclei from dog eggs to produce denuclearized eggs;

(d) microinjecting and fusing nuclear donor cells into the denuclearized egg;

(e) activating the fused egg; And

(f) transplanting the activated egg into the fallopian canal.

The general technical content of each step can be used with reference to a conventional method for producing cloned animals using somatic cell nuclear transfer technology known in the art.

As an embodiment, a method for manufacturing a nuclear transfer embryo of a dog formed by transplanting the nucleus of the transformed cell into a denuclearized egg and a nuclear transfer embryo prepared thereby may be provided.

In one embodiment, the nuclear transfer embryo is transplanted into the fallopian tubes of the surrogate mother to produce an acid-containing ACTA promoter and PPARδ encoding a base sequence encoding PPARδ overexpressing transgenic dogs or a method of producing the ACTA promoter and PPARδ produced thereby It provides a dog over-expressing PPARδ, characterized in that the base sequence encoding.

As an embodiment, a method for producing the transgenic animal can be provided.

Nuclease may be used for the transformation.

The method exposes an embryo or cell to a recombinant vector (eg, a recombinant vector containing PPARδ),

Cloning cells in surrogate mothers, or transplanting embryos in surrogate mothers.

The nuclease may specifically bind to a target chromosome site in an embryo or cell, thereby causing a nucleotide sequence change in the cell chromosome.

[purpose]

One embodiment of the present invention provides the use of a dog in which the PPARδ gene is overexpressed, a muscle-specific PPARδ protein is overexpressed.

The PPARδ gene knocked-in muscle-specific PPARδ overexpressing transgenic dog according to the present invention can be produced through crossing, and the transfer of the external gene is possible later.

Therefore, the dog characterized in that the PPARδ gene of the present invention is knocked in and that the knocked-in PPARδ gene is specifically expressed as a protein in muscle. Animal models and / or metabolic diseases that have been expressed and improved bioenergy metabolism and motor performance, such as obesity, hypertriglyceridemia, hyperlipidemia, hypoalphalipoproteinemia, hypercholesterolemia, dyslipidemia or diabetes.

That is, the muscle-specific PPARδ overexpressing dog of the present invention is used as a special-purpose dog requiring improved motor performance or is a mechanism study involved in inducing or treating metabolic diseases, the role of PPARδ, muscle mass, muscle strength, muscle fibers, and mitochondrial biosynthesis In addition, various applications such as models for studying changes in energy metabolism will be possible.

Therefore, the present invention may include various uses of muscle-specific PPARδ overexpressing dogs by the above method in another aspect.

For example, the animal model according to the present invention can be used as a method of screening a medicament for improving energy metabolism.

In one embodiment, the screening method of the present invention

1) administering a candidate substance capable of maintaining or increasing muscle endurance in a muscle-specific PPARδ overexpressing dog;

2) After administration of the candidate substance, the muscle tissue of the dog may be analyzed by comparison with a control group not administered with the candidate substance.

At this time, energy metabolism may mean mitochondrial biosynthesis, mitochondrial activity, and the like.

The candidate substance may be 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. However, it is not limited thereto. The compound may be a novel compound or a well-known compound. These candidate substances are capable of salt formation.

The method for administering the candidate substance may be, for example, oral administration, intravenous injection, subcutaneous administration, intradermal administration or intraperitoneal administration. It 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.

In one embodiment, the muscle-specific PPARδ overexpressing dog may be used as a model dog for analysis of a special purpose dog or motor mechanism.

In this case, the special purpose dog may be a dog with improved athletic performance compared to a normal dog.

In this case, the dog with improved athletic performance may be a dog having one or more effects of increased muscle atrophy, increased muscular dysfunction, increased muscle endurance, and increased running ability (time and / or distance) compared to a normal dog.

In this case, the general dog may be a dog without transformation for muscle-specific PPARδ overexpression.

For example, the animal model according to the present invention can be used as a method of screening a medicament for improving athletic performance.

In one embodiment, the screening method of the present invention

1) administering a candidate that can maintain or increase obesity resistance in muscle-specific PPARδ overexpressing dogs;

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

At this time, the athletic performance may be muscle mass, muscle strength, and endurance compared to the control group.

The candidate substance may be any one or more selected from the group consisting of peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma. However, it is not limited thereto. The compound may be a novel compound, or a well-known compound. These candidate substances may form salts. The method for administering the candidate substance as described above may be, for example, oral administration, intravenous injection, subcutaneous administration, intradermal administration, or intraperitoneal administration, and may 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 means that the fat tissue in the body is excessive, and the energy consumed as food does not balance the energy consumed by physical activity and the like, and thus means a phenomenon in which excess energy is accumulated as body fat. 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, and adult disease.

Obesity may be caused by an increase in the size (hypertrophy) or increase in the number of fat cells in the body (hyperplasia).

Obesity is caused by abnormal hypertrophy of the subcutaneous tissue caused by the accumulation of excess energy into the body when an imbalance of metabolic processes occurs due to endocrine factors, genetic factors, and social and environmental factors. Fat tissue enlargement is a phenomenon in which the size of fat cells increases (fat cell enlargement) or increases in number (fat cell hyperplasia), which also affects the stagnation of the local venous-lymph system, resulting in vascular tissue disease of the dermis-subcutaneous tissue. Can also cause

Triglycerides, which are excessively accumulated in obese patients, can be stored in the liver or muscles as well as in adipose tissue, leading to insulin resistance. Therefore, excessively stored consumption of triglycerides can be the prevention and treatment of underlying obesity and metabolic diseases.

The recombinant vector can be used to treat or alleviate the obesity and metabolic disease.

Obesity and metabolic diseases may be treated or alleviated when administered to a specific region of the living body using the recombinant vector.

When administered to a specific part of the living body using the recombinant vector, the amount of adipose tissue or adipocytes may be reduced.

The obesity or metabolic disorders include, but are not limited to, metabolic syndrome, hypertriglyceridemia, high-density lipidemia, low-density lipidemia, angina pectoris, myocardial infarction, hypogonadism, sleep apnea, premenstrual syndrome, stress urinary incontinence Urinary incontinence, hyperactivity disorder, chronic fatigue syndrome, osteoarthritis, weight-related cancer, orthostatic hypotension, pulmonary hypertension, menstrual disorder, diabetes, hypertension, impaired glucose tolerance, coronary thrombosis, somnolence, depression, anxiety, psychosis, Reproductive disorders such as delayed movement disorder, drug addiction, substance abuse, cognitive impairment, Alzheimer's disease, cerebral ischemia, obsessive-compulsive behavior, panic attack, social phobia, bulimia, atherosclerosis, gallbladder disease such as cholelithiasis, anorexia, polycystic ovary disease Skin infections such as infection, varicose veins, epidermal hyperplasia and eczema, insulin resistance, chronic arterial occlusion, orthopedic injury, thromboembolism It may be one or more obesity-related diseases selected from the group consisting of pre-emergency, heart disease, urinary disease, lipid syndrome, hyperglycemia, and stress. As the dog for the muscle-specific PPARδ overexpression model of the present invention, the PPARδ protein is overexpressed in muscle tissue The activity of PPARδ is artificially increased, leading to an increase in the kinetic muscle of the fast-growing muscles, an increase in the development of the muscle roots, an increase in muscle endurance, an increase in running ability (time and / or distance), and resistance to metabolic diseases, and also energy metabolism and motor activity. Since it induces an improvement, it can be provided as an animal model having improved obesity resistance according to energy metabolism and exercise.

In addition, the muscle-specific PPARδ over-expressing dog of the present invention artificially increases the activity of PPARδ as the PPARδ protein is over-expressed in muscle tissue, thereby increasing the muscularization of the fast muscles, increasing the occurrence of late muscles, increasing muscle endurance, and running ability (time and / or Or distance) Various metabolic diseases related studies such as obesity using obese dogs with increased and metabolic disease resistance (obesity resistance studies, etc.), exercise / strength improvement studies (including muscle atrophy treatment studies, etc.) Therefore, it can be used as an animal model for energy metabolism / motor enhancement research.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1. Vector construction for production of muscle-specific PPARδ overexpressing dogs

The PPARδ (peroxisome proliferator activated receptors δ) gene is expressed in various tissues such as skeletal muscle, liver, and adipocytes, and plays a role of lipid homeostasis and glucose homeostasis. Since PPARδ is expressed in various tissues, it is important to select a promoter capable of increasing expression by limiting specific tissues to reduce side effects. In this study, the peroxisome proliferator activated receptor delta (PPARδ) gene was synthesized, and the PiggyBac vector in which the PPARδ gene was introduced was constructed and cloned under the control of the muscle-specific promoter α-skeletal muscle actin promoter (ACTA promoter) (FIG. 1). The base sequence of the ACTA promoter is SEQ ID NO: 1, the base sequence of PPARδ is SEQ ID NO: 2, and the base sequence of the vector constructed in this experiment is shown as SEQ ID NO: 3.

Example 2. Confirmation of vector introduction in adipose stem cells

After introducing the constructed vector into Adipose-derived stem cells (ASC), molecular biological analysis was conducted, and it was confirmed that each vector was introduced into the genome in RT-PCR, and the expression of the marker gene, copGFP, was fluorescent. It was confirmed under a microscope (Fig. 2, Fig. 3).

Example 3. Somatic cell nuclear transfer

After the somatic cell nuclear transfer and in vitro culture, the expression of the marker gene copGFP in the cloned embryo was confirmed (FIG. 4).

After transplanting the somatic cell nuclear transfer cloned egg into a surrogate mother, a total of 1 muscle-specific PPARδ-expressing transgenic cloned beagles were produced. Genomic DNA PCR and Southern blot were performed using the blood samples of the produced individuals to verify that they were stably inserted into the genome (FIGS. 5 and 6).

OPP19-080-PCT_sequence list.app

Claims (10)

1. A transgenic dog overexpressed with muscle cell specific PPARδ,

   -At this time, the transformed dog contains muscle tissue or muscle cells,

   At this time, the genome of the muscle tissue or muscle cell has an intrinsic base sequence encoding PPARδ; And

   ACTA promoter derived from one or more dogs and a foreign nucleotide sequence encoding PPARδ includes a sequence operably linked,

   At this time, the ACTA promoter works specifically in muscle cells (working)-;

   Characterized by,

   Transgenic dog overexpressing muscle cell specific PPARδ.
2. According to claim 1,

   The ACTA promoter corresponds to SEQ ID NO: 1,

   The foreign base sequence encoding the PPARδ is characterized in that it corresponds to SEQ ID NO: 2,

   Transgenic dog overexpressing muscle cell specific PPARδ.
3. One or more metastatic factors;

   One or more inverted terminal repeat sequences (direct repeats) or trend repeat sequences (direct repeats); and

   ACTA promoter derived from one or more dogs and a foreign nucleotide sequence encoding PPARδ comprises a operably linked nucleotide sequence

   Recombinant vector for producing muscle cell-specific PPARδ overexpressing animals.
4. According to claim 3,

   The transfer factor further includes a transposase,

   -At this time, the translocation enzyme is piggyBac transferase or Sleeping Beauty transferase-;

   Characterized in that the reverse terminal repeat sequence or trend repeat sequence is located at both ends of the metastasis factor,

   Recombinant vector for producing muscle cell-specific PPARδ overexpressing animals.
5. According to claim 4, The recombinant vector is characterized in that the plasmid or viral vector,

   Recombinant vector for producing muscle cell-specific PPARδ overexpressing animals.
6. According to claim 3,

   The recombinant vector is characterized in that it corresponds to SEQ ID NO: 3,

   Recombinant vector for producing muscle cell-specific PPARδ overexpressing animals.
7. As a transformed cell,

   -At this time, in the genome of the transformed cell, an intrinsic base sequence encoding PPARδ, and

    An ACTA promoter derived from one or more dogs and a foreign nucleotide sequence encoding PPARδ operably linked to the sequence,

    At this time, the ACTA promoter works specifically in muscle cells (working),

   At this time, the birth born through the transformed cells is characterized in that it overexpresses PPARδ specifically in muscle tissue or muscle cells-;

   Characterized in that, the transformed cells.
8. The method of claim 7,

   Characterized in that the transformed cells are fat stem cells,

   Transformed cells.
9. The method of claim 7,

   The ACTA promoter corresponds to SEQ ID NO: 1,

   The foreign base sequence encoding the PPARδ is characterized in that it corresponds to SEQ ID NO: 2,

   Transformed cells.
10. Preparing the recombinant vector and nuclear donor cells of any one of claims 3 to 6,

    -At this time, the nuclear donor cells are somatic cells or stem cells derived from dogs-;

    Introducing the recombinant vector into the nuclear donor cell to induce a first cell;

    Characterized in that it comprises introducing the nucleus of the first cell into the denuclearization egg to induce the second cell,

    Method for producing transfected cells expressing PPARδ specific to muscle cells.

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