KR102624831B1 - Production of transgenic dogs overexpressing muscle-specific PCK1 - Google Patents

Abstract

The present invention relates to a transgenic dog that specifically overexpresses PCK1 in muscle, a method of producing the same, and its use. In particular, the present invention relates to a transgenic dog that overexpresses the PCK1 protein specifically in muscle tissue, which improves muscle mass, strength, muscle fiber, and mitochondria. It concerns use as an animal model for research on biosynthesis, changes in energy metabolism, etc.

Description

Production of transgenic dogs overexpressing muscle-specific PCK1

The present invention relates to transgenic dogs, their production methods, and their use for research into the production of animals with improved energy metabolism and exercise capacity, and more specifically, to transgenic dogs overexpressing PCK1 and their production methods.

Following the trend of increasing domestic and international trade in goods due to increased economic, social and cultural exchanges, the frequency of outbreaks of national disaster-type overseas malignant infectious diseases such as foot-and-mouth disease (FMD) and highly pathogenic avian influenza (AI) is increasing through various channels, blocking infectious agents from abroad. This is necessary, and there are fields that cannot be performed by humans or machines, such as drug detection and lifesaving. To solve this problem, animals with excellent athletic ability that can replace people or machines are needed.

In general, the ability to synthesize glucose from metabolites is very important for most organic life forms. Our body must produce more new glucose than is supplied each day. The process for this activity is gluconeogenesis, which means the production of new glucose.

In animals, almost all gluconeogenesis occurs in the liver and kidneys. The main places where gluconeogenesis occurs are the liver and kidneys, which are responsible for about 90% and 10% of glycogen synthesis activity, respectively. Glucose produced by gluconeogenesis in the liver and kidneys is released into the blood and continues to meet metabolic needs. to be absorbed by the brain, heart, muscles and red blood cells. Pyruvate and lactic acid produced in these tissues return to the liver and kidneys to be used as substrates for gluconeogenesis.

To date, research on animal production that has improved energy metabolism and exercise capacity has mainly developed models in rats and mice that overexpress various genes involved in energy metabolism and growth, and has improved muscle mass, strength, muscle fiber, mitochondrial biosynthesis, and muscle mass in transgenic rodents compared to the control group. Changes in energy metabolism are only being compared and analyzed.

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

In addition, in overseas countries such as the United States, Canada, the United Kingdom, and Australia, the National Detection Dog Center has been operating for a long time to secure and train excellent dogs, and with the help of many experts, efficient training programs have been systematized to secure and foster a large number of dogs of various breeds. However, even if a dog with excellent abilities is discovered, there is a problem in that the ability cannot be passed on to future generations, so there is a problem that continuous high costs and training are required to foster excellent dogs. Therefore, there is a need for transgenic dogs that can easily maintain the same traits and have a long lifespan.

Meanwhile, PCK1 (=PEPCK-C, cytosolic form of phosphoenolpyruvate carboxykinase) is a precursor for the gluconeogenesis process in mammalian mitochondria and plays a role in improving bioenergy metabolism and vitality. PCK1 catalyzes an irreversible step in gluconeogenesis. PCK1, which is an important factor in gluconeogenesis and is involved in bioenergy metabolism and vitality improvement, can be an important target gene for research on animal production with improved energy metabolism and exercise capacity.

In particular, overexpression of PCK1 in muscle, which consumes most glucose and is important for energy metabolism and exercise performance, is thought to be useful for research on animal production with improved energy metabolism and exercise performance.

There is a need for medium to large-sized animals that can perform tasks on behalf of people or machines in areas that are difficult to perform. The primary purpose of the present invention is to provide medium to large-sized animals with improved energy metabolism and locomotion and a long lifespan.

Additionally, the purpose of the present invention is to provide a dog in which PCK1 is specifically overexpressed in muscle tissue or muscle cells and a method for producing the same.

Another object of the present invention is to provide a method for producing a dog in which PCK1 is specifically overexpressed in the muscle tissue or muscle cell, and a muscle-specific expression system required therefor.

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

Another object of the present invention is to provide various uses of the muscle-specific PCK1 overexpressing dog.

Therefore, in order to study the production of animals with improved energy metabolism and exercise capacity, the present inventors have developed a technology to transplant the nucleus of somatic cells transformed to overexpress PCK1, which is considered an important factor in gluconeogenesis, bioenergy metabolism, and improvement of vitality. By applying this method, it was confirmed that a muscle-specific dog with PCK1 overexpression could be produced, and the present invention was completed.

In order to solve the above problems, the present invention provides a muscle-specific PCK1-overexpressing dog and a method for producing the same.

As an embodiment of the present invention,

By knocking in (KI) the base sequence encoding PCK1 and the α-skeletal muscle actin promoter (ACTA promoter), which is specifically expressed in muscle tissue or muscle cells, are operably linked. Transgenic dogs overexpressing PCK1 are provided.

Knock In (Knock In) is an operably linked nucleotide sequence encoding human-derived PCK1 with the α-skeletal muscle actin promoter (ACTA promoter), which is specifically expressed in muscle tissue or muscle cells. KI) to provide transgenic dogs overexpressing PCK1.

As an embodiment, the present invention provides a recombinant vector for producing muscle-specific PCK1 over-expressing animals, which includes an ACTA promoter specifically expressed in muscle tissue or muscle cells and a base sequence encoding PCK1.

As an embodiment, the present invention provides a recombinant vector for producing muscle-specific PCK1 overexpressing animals, which includes an ACTA promoter specifically expressed in muscle tissue or muscle cells and a base sequence encoding human-derived PCK1.

At this time, the vector may be a vector containing one or more transposable elements.

The transposon may be a piggyBac transposon or a Sleeping Beauty transposon.

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

The transposable element may include an enzyme that mediates translocation, such as piggyBac transposase or Sleeping Beauty transposase.

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

At this time, 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 a retroviral vector, an adenovirus vector, an adeno-related virus vector, a herpes virus vector, an avipox virus vector, and a lentivirus vector. It can be a vector.

The above vector can be used to provide muscle-specific PCK1-overexpressing transformed cells containing the ACTA promoter and PCK1 gene specifically expressed in muscle tissue or muscle cells.

After producing embryos using the transgenic cells, the transgenic dogs can be provided by somatic cell nuclear transfer (SCNT).

Dogs overexpressing muscle-specific PCK1 are characterized by improved gluconeogenesis, bioenergy metabolism, vitality, and exercise capacity.

Therefore, the present invention provides a method for producing a muscle-specific PCK1 overexpressing dog, which is characterized in producing offspring by transplanting the nucleus of a nuclear donor cell into which the above-described recombinant vector has been introduced into an enucleated egg.

At this time, the nuclear donor cell may be a dog embryonic cell, somatic cell, or stem cell. In one embodiment of the present invention, canine adult fibroblasts or adipose stem cells can be used.

Therefore, the method for producing muscle-specific PCK1 overexpressing dogs of the present invention may include the following steps as a more specific example.

(a) Preparation of nuclear donor cells, including culturing somatic cells or stem cells isolated from dogs;

(b) introducing a recombinant vector containing the ACTA promoter and a base sequence encoding PCK1 into the nuclear donor cell;

(c) producing enucleated eggs by removing the nucleus from a dog egg;

(d) microinjecting nuclear donor cells into the enucleated oocyte and fusing them;

(e) activating the fused egg; and

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

The present invention includes all the transformation vectors, cells, nuclear transfer embryos, etc. required for producing muscle-specific PCK1-overexpressing dogs that can be obtained in the process of producing muscle-specific PCK1-overexpressing dogs.

That is, the present invention is another embodiment,

It is possible to provide transgenic cells for producing muscle-specific PCK1 overexpressing animals into which a recombinant vector containing the ACTA promoter and a base sequence encoding PCK1 has been introduced,

Dog nuclear transfer embryos formed by transplanting or fusing the nucleus of a dog-derived somatic cell or stem cell transformed with a recombinant vector containing the base sequence encoding the ACTA promoter and PCK1 to an enucleated egg can also be provided.

The muscle-specific PCK1 overexpressing dog of the present invention is a dog in which muscle mass, strength, muscular endurance, muscle fiber, mitochondrial biosynthesis and activity, energy metabolism and gluconeogenesis are increased by muscle-specific overexpressed PCK1, and is used for energy metabolism-related research. , It can be used in a variety of ways, such as research on exercise performance/muscle strength improvement.

Additionally, the present invention provides various uses of the muscle-specific PCK1 overexpressing dog.

As an example, a method for screening drugs for improving energy metabolism is provided, including:

As another example, a method of screening drugs for improving athletic performance, including the following, is provided.

As such, the present invention provides a muscle-specific PCK1 overexpression animal model with improved energy metabolism, vitality, and exercise capacity, and various uses thereof.

Figure 1 is a schematic diagram of one specific example of a recombinant vector for the production of muscle-specific PCK1 overexpressing dogs.
Figure 2 shows the results of RT-PCR analysis of adult fibroblasts and adipose stem cells into which the pPB-ACTAp-PCK1-Red/Puro vector was introduced.
Figure 3 shows the results of confirming the expression pattern of red fluorescent protein, a marker gene, in adult fibroblasts and adipose stem cells into which the pPB-ACTAp-PCK1-Red/Puro vector was introduced.
Figure 4 shows the results of RT-PCR analysis of adipose stem cells into which pPB-ACTAp-PCK1-Red/Puro vector was introduced.
Figure 5 shows the results of confirming the expression pattern of red fluorescent protein, a marker gene, in adipose stem cells into which the pPB-ACTAp-PCK1-Red/Puro vector was introduced.
Figure 6 shows the results of confirming the expression of the mRuby gene in somatic cell nuclear transfer cloned eggs produced using muscle-specific PCK1-expressing transformed cells.
Figure 7 shows the genomic DNA PCR results of a transgenic cloned dog expressing muscle-specific PCK1, where N represents a negative control and S represents a transgenic cloned dog expressing muscle-specific PCK1.
Figure 8 shows the Southern blot results of a transgenic cloned beagle expressing muscle-specific PCK1, where N represents a negative control and S represents a transgenic cloned dog expressing muscle-specific PCK1.
Figure 9 shows the results of serum chemistry analysis at 1, 2, 4, and 6 months of age in muscle-specific PCK1-expressing transgenic cloned dogs (PCK1) and control dogs (ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase ; BUN, blood urea nitrogen; CREA, creatinine; TC, total cholesterol; GLU, glucose; ALB, albumin; TP, total protein; TG, triglyceride).
Figure 10 is data showing the spectrum of MRS imaging results for control group 1.
Figure 11 is data showing the spectrum of MRS imaging results of control group 2.
Figure 12 is data showing the spectrum of MRS imaging results of control group 3.
Figure 13 is data showing the spectrum of MRS imaging results of control group 4.
Figure 14 is data showing the spectrum of MRS imaging results of control group 5.
Figure 15 is data showing the spectrum of MRS imaging results of a cloned dog overexpressing muscle-specific PCK1.
Figure 16 shows chest radiography results of a cloned dog overexpressing muscle-specific PCK1.
Figure 17 shows abdominal radiography results of a cloned dog overexpressing muscle-specific PCK1.
Figure 18 shows the results of skull radiography of a cloned dog overexpressing muscle-specific PCK1.
Figure 19 shows lumbar radiography results of a cloned dog overexpressing muscle-specific PCK1.
Figure 20 is a pelvic radiograph of a cloned dog overexpressing muscle-specific PCK1.
Figure 21 is a CT image of a cloned dog overexpressing muscle-specific PCK1, showing the normality of the occipital bone and cervical spine visualized through VR.

Best mode for carrying out the invention

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

“Muscle-specific expression system” is a general term for a system that allows any gene or protein to be expressed in muscle tissue or cells forming muscle tissue. The muscle-specific expression system includes regulatory elements, such as promoters and enhancers, that can specifically control the expression of any gene or protein in muscle tissue or cells forming muscle tissue. The expression system may include a vector system, a virus system, etc., which include a nucleic acid sequence encoding an arbitrary gene, but also includes any system capable of expressing an arbitrary gene or protein within a cell. .

“Vector” or “expression vector” refers to a plasmid, a vector containing a transposable element, or a viral vector known in the art into which a nucleic acid encoding a structural gene can be inserted and can express the nucleic acid in a host cell. or any other medium. Preferably, it may be a transposon-containing vector or a viral vector.

“Recombinant vector” refers to a vector capable of expressing a target protein or RNA of interest in a suitable host cell, and refers to a genetic construct containing essential regulatory elements operably linked to express the gene insert.

“Expression control sequence” or “regulatory element” means a DNA sequence that regulates the expression of an operably linked nucleic acid sequence in a specific host cell. Such regulatory sequences include optional operator sequences to regulate transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences that regulate termination of transcription and translation. In the present invention, the regulatory factor may be a promoter, enhancer, etc.

“Promoter” means a DNA sequence that, when linked to a specific sequence, can regulate the transcription of a specific nucleotide sequence into mRNA. Typically, a promoter, although not applicable in all cases, is present 5' (i.e. upstream) of the nucleotide sequence of interest to be transcribed into mRNA and represents a site where RNA polymerase and other transcription factors for transcription initiation specifically bind. to provide. The promoter of the present invention is a constitutive promoter. The term “constitutive,” as used in connection with a promoter, means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence without stimulation (e.g., 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 control muscle-specific transcription of a specific sequence linked to the promoter. The ACTA promoter may be of dog origin.

“Operably linked” refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or RNA to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to affect expression of the encoding nucleic acid sequence. Operable linkages with recombinant vectors can be prepared using genetic recombination techniques well known in the art.

“Transformation” means changing the genetic properties of an organism through DNA given from outside. Transformation methods include various conventionally known methods, such as microinjection, electroporation, particle bombardment, sperm-mediated gene transfer, and viral infection. It can be appropriately selected and applied among techniques using viral infection, direct muscle injection, insulator, and transposon. Preferably, in the present invention, canine fetal fibroblasts can be transformed with an expression vector containing human PCK1 through viral infection.

""Animal" or "laboratory animal" means any mammalian animal other than a human. Such animals include animals of all ages, including embryos, fetuses, neonates, and adults. Animals for use in the present invention include , for example, from commercial sources, such as laboratory animals or other animals, rabbits, rodents (e.g., mice, rats, hamsters, gerbils and guinea pigs), cattle, sheep, pigs, goats, Animals include, but are not limited to, horses, dogs, cats, birds (e.g., chickens, turkeys, ducks, geese), and primates (e.g., chimpanzees, monkeys, rhesus monkeys).The most preferred animal is a dog.

“Overexpression” means that any gene or protein is expressed above normal levels. In the present invention, the expression of PCK1 protein means that it is expressed above the expression level of normal cells. In particular, it may be expressed above the normal level specifically in muscle tissue or cells forming muscle tissue. The overexpressing transformant may be a transformed cell, transgenic tissue, or transgenic animal in which the PCK1 protein is expressed above normal levels. For example, it may be an overexpression transformant created by exposing the cell, tissue, or animal to a material containing a muscle-specific expression system, or introducing the material.

“Nuclear transplantation” refers to a genetic manipulation technology that artificially combines another cell or nucleus with an enucleated egg to have the same characteristics. “Nuclear transfer egg” refers to an egg into which a nuclear donor cell has been introduced or fused.

“Cloning” is a genetic manipulation technique that creates a new individual with the same set of genes as that of one individual. In particular, in the present invention, somatic cells, embryonic cells, fetal-derived cells, and/or adult-derived cells are substantially identical to the nuclear DNA sequence of another cell. refers to having a nuclear DNA sequence. The present invention uses technology to clone dogs using nuclear transfer technology. In particular, the somatic cell nuclear transfer technology is a technology that allows the birth of offspring without going through meiosis and haplochromosome-bearing gametes, which are common during the reproductive process. It is a technology that transfers polyploid-bearing somatic cells from an adult into an egg whose nucleus has been removed to create a fertilized egg. This is a method of generating a new entity by producing and transplanting the fertilized egg into a living body.

“Nuclear donor cell” refers to the cell or nucleus of a cell that transfers the nucleus to the recipient egg, which is the nuclear recipient. “Oocyte” preferably refers to a mature egg that has reached the second meiotic metaphase. In the present invention, dog somatic cells or stem cells can be used as the nuclear donor cells.

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

And/or it means that the time course of progress is slowed down or lengthened.

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

Throughout this specification, unless the context otherwise requires, the words "comprise" and "comprise" 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 to imply that these groups are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to a transgenic dog with improved energy metabolism and exercise capacity due to knock-in (KI) of the base sequence encoding the ACTA promoter and PCK1, a method of producing the same, and its use.

More specifically, it relates to a transgenic dog with improved energy metabolism and exercise capacity due to KI in the base sequence encoding the dog-derived ACTA promoter and PCK1, a method of producing the same, and its use.

Models that overexpress various genes involved in energy metabolism and growth are being developed, but to date, research on animal production with improved energy metabolism and exercise capacity has mainly been conducted in rats and mice.

Additionally, the need for medium to large-sized animals with improved motor skills that can take on tasks that humans or machines cannot do is increasing. Accordingly, in light of genetic, morphofunctional, and behavioral aspects, dogs appear to be useful as transgenic animals with improved energy metabolism and locomotion. Therefore, more specifically, there is a need for transgenic dogs with improved energy metabolism and exercise capacity.

In order to improve the exercise capacity of medium to large-sized animals, various genes involved in energy metabolism or growth can be overexpressed. More specifically, the gene may be PCK.

PCK (Phosphoenolpyruvate carboxykinase) is also called PEPCK and exists in two isoforms depending on the location. There is a cytosolic isoform (PCK1) that exists in the cytoplasm and a mitochondrial isoform (PCK2) that exists in the mitochondria. Among these, PCK1 corresponds to an enzyme that converts oxaloacetate (OAA) into phosphoenolpyruvate (PEP) in the cytoplasm during gluconeogenesis and glyceroneogenesis.

One aspect of the invention may be a transgenic dog in which PCK1 is specifically overexpressed in muscle tissue or muscle cells. Any cell of the transgenic dog may contain a foreign base sequence encoding PCK1 in addition to the inherent base sequence encoding PCK1. More may be included.

The muscle tissue or muscle cells of the transgenic dog may further include an exogenous base sequence encoding PCK1 in addition to the inherent base sequence encoding PCK1.

The muscle tissue or muscle cells of the transgenic dog may contain two or more base sequences encoding PCK1.

The PCK1 expression level of any cell of the transgenic dog may be higher than the PCK1 expression level of any cell of the wild-type dog.

The PCK1 expression level in the muscle tissue or muscle cells of the transgenic dog may be higher than the PCK1 expression level in the muscle tissue or muscle cells of the wild-type dog.

The PCK1 expression level in the muscle tissue or muscle cells of the transgenic dog may be significantly higher than the PCK1 expression level in the muscle tissue or muscle cells of the wild-type dog.

The blood lipase concentration value of the transgenic dog has a value of about 0.5 times, about 0.4 times, about 0.3 times, about 0.2 times, or about 0.1 times or lower than the blood lipase concentration value of the wild type dog. You can.

In another embodiment, in order to produce the above-mentioned transgenic dog, transgenic cells in which the base sequence encoding the ACTA promoter and PCK1 are KI can be provided.

In order to produce the above-mentioned transgenic dog, a transgenic cell in which the dog-derived ACTA promoter and base sequence encoding PCK1 are KI and a method for producing the same cell can be provided.

In another embodiment, a recombinant vector containing a dog-derived ACTA promoter and a nucleotide sequence encoding PCK1 may be provided to produce the transformed cells.

[Muscle-specific PCK1 expression system]

PCK1

Looking at the mechanisms related to PCK1 in the body, they are as follows.

When energy is needed, such as during exercise, glucose and fatty acid in the blood move into muscle tissue. Glucose produces pyruvate through the glycolysis process. After pyruvate moves into the mitochondria and becomes acetyl CoA, ATP is produced in the TCA cycle.

Fatty acid produces acetyl CoA through bate oxidation, and then this acetyl CoA enters the TCA cycle to produce ATP.

When blood glucose is depleted below a certain level, malate produced during the TCA cycle comes out of the mitochondria, becomes OAA, and PEP is produced by PCK1. Additionally, pyruvate is synthesized from lactate and alanine in the cytoplasm, and OAA is produced, and then PEP is produced by PCK1 (glyceroneogenesis).

Triglyceride is produced from PEP, and fatty acid and glycerol are produced from triglyceride, which are used for energy production through the TCA cycle.

Therefore, when PCK1 is specifically overexpressed in muscle tissue or muscle cells, animals with improved energy metabolism and exercise capacity can be produced.

Therefore, the purpose of the present invention is to produce transgenic animals with increased expression of PCK1 by artificially inserting the base sequence encoding PCK1 into the genome of a specific cell.

The present invention can use a base sequence encoding dog-derived PCK1 or a base sequence encoding a material functionally equivalent to the dog-derived PCK1 gene.

The present invention can use a base sequence encoding human-derived PCK1 or a base sequence encoding a material functionally equivalent to the human-derived PCK1 gene.

Muscle-specific PCK1 expression recombinant vector

The present invention provides a recombinant vector containing a promoter and a base sequence encoding an arbitrary substance.

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

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

In one example, a recombinant vector containing a base sequence encoding the ACTA promoter and PCK1 is provided.

In one example, a recombinant vector containing a base sequence encoding the ACTA promoter and PCK1 derived from any animal is provided.

In one example, a recombinant vector containing a dog-derived ACTA promoter and a nucleotide sequence encoding PCK1 is provided.

In one embodiment, the ACTA promoter and PCK1 gene may be operably linked. The ACTA promoter and PCK1 gene may not be operably linked. In certain embodiments, the vector may be a vector further containing one or more transposable elements.

The transposon may be a piggyBac transposon or a Sleeping Beauty transposon. The transposable enzyme may include piggyBac transposase or Sleeping Beauty transposase.

In certain embodiments, the transposon may further include an inverted terminal repeat sequence (ITR) and/or direct repeat (DR) at both ends.

In certain embodiments, the vector may be a piggyBac vector or a Sleeping Beauty vector.

As an example, a recombinant vector containing an ACTA promoter, a base sequence encoding PCK1, a transposon, a repeat sequence (ITR and/or DR), and a selection marker may be provided.

In one embodiment, a recombinant vector containing an ACTA promoter, a base sequence encoding PCK1, a transposable element, a repeat sequence (ITR and/or DR), and a selection marker arranged in the same order as shown in Figure 1 can be provided. there is.

The recombinant vector can be used to knock in (KI) a base sequence encoding a promoter and an arbitrary substance into the genome of any cell.

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

The recombinant vector can be used to knock in a base sequence encoding a promoter and an arbitrary substance into the genome of muscle tissue or muscle cells.

The base sequence encoding PCK1 can be appropriately used by those skilled in the art from sequences having non-limiting base sequences encoding PCK1 known in the art.

The base sequence encoding PCK1 can be prepared by genetic recombination methods known in the art. For example, PCR amplification to amplify nucleic acids from the genome, chemical synthesis or cDNA sequence manufacturing technology, Fmoc technology, etc.

Additionally, the base sequence may have a base sequence encoding each functional equivalent of PCK1.

The functional equivalent refers to an amino acid sequence that is about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, and about 76% of the wild type amino acid sequence 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 Homology of 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. It refers to a polypeptide that exhibits substantially the same physiological activity as PCK1 disclosed in the specification, which means a base sequence including a sequence having a polypeptide. The term “equivalent physiological activity” refers to activity that regulates mitochondrial gluconeogenesis, bioenergy metabolism, glucose homeostasis, or exercise capacity.

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

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

The muscle-specific PCK1 expression recombinant vector of the present invention is a plasmid, transposon-containing vector, viral vector, or vector known in the art that can specifically express the nucleic acid encoding the PCK1 gene in muscle tissue or cells constituting muscle tissue. Other vectors are preferably transposon-containing vectors or viral vectors.

The viral vector may be, but is not limited to, a retroviral vector, an adenovirus vector, a herpes virus vector, an avipoxvirus 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, a neomycin resistance gene, or a fluorescent protein such as a green fluorescent protein or a red fluorescent protein, but is not limited thereto.

Additionally, in order to confirm whether the PCK1 protein is overexpressed, the vector of the present invention may further include a tag sequence for protein isolation, purification or confirmation. The tag sequence may be GFP, mRuby, GST (Glutathione S-transferase)-tag, HA, His-tag, Myc-tag, or T7-tag, etc. However, the tag sequence of the present invention is limited by the above examples. It doesn't work. In a specific example, the expression and expression level of the tag sequence can be confirmed using GFP or mRuby.

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

Transformed dogs can be provided using the above transformed cells.

In other words, a transgenic dog with an activated gluconeogenesis reaction in the muscle itself can be provided using the recombinant vector disclosed in the specification.

Using the recombinant vector disclosed in the specification, transgenic dogs with improved energy metabolism and exercise capacity can be provided by increasing glucose production in the muscles themselves.

transformed cells

The present invention provides transformed cells in which a promoter and a base sequence encoding a specific substance have been knocked in.

The present invention provides transformed cells in which the ACTA promoter and the base sequence encoding PCK1 have been knocked in.

The base sequences encoding the ACTA promoter and PCK1 can be knocked in using known methods well known to those skilled in the art. The transformed cells can be produced by introducing the recombinant vector into a nuclear donor cell.

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

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

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

The nuclear donor cells include, but are not limited to, cumulus cells, epithelial cells, fibroblasts, neurons, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, macrophages, monocytes, muscle cells, B lymphocytes, These include T lymphocytes, embryonic stem cells, embryonic germ cells, fetal-derived cells, placental cells, and embryonic cells. Additionally, adult stem cells may be derived from various origin tissues, for example, stem cells derived from tissues such as fat, uterus, bone marrow, muscle, placenta, umbilical cord blood, or skin (epithelium).

Additionally, the nuclear donor cell may be a non-human host embryo, and is generally at the 2-cell stage, 4-cell stage, 8-cell stage, 16-cell stage, 32-cell stage, 64-cell stage, and loss. It may be an embryo, or an embryo containing a blastocyst.

Additionally, the nuclear donor cells may be fetal-derived cells, adult fibroblasts, or adipose stem cells.

Additionally, the nuclear donor cells may be canine adipose stem cells or adult fibroblasts. The characteristics of these cells are that a large number of cells can be obtained upon initial isolation, cell culture is relatively easy, and they are easy to culture and manipulate in vitro.

As an example, the embryonic cells, somatic cells, or stem cells serving as nuclear donor cells can be obtained from a method of preparing surgical specimens or biopsy specimens using conventional methods known in the art.

The recombinant vector according to the present invention can be introduced into cells by 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 producing transgenic animals by a method.

Meanwhile, the recombinant vector can be used to KI a specific gene in the genome of a transformed cell. To specifically overexpress PCK1 in muscle tissue or muscle cells, specific regions within the cell genome can be used.

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 where the insertion of a foreign gene does not cause serious side effects, such as cancer, and the foreign gene inserted into the specific region is permanently and safely expressed at a high level. possible.

In one embodiment, in order to produce the transgenic dog, transformed cells in which the dog-derived ACTA promoter and the nucleotide sequence encoding PCK1 are KI may be provided.

The transformed cell may further include an exogenous base sequence encoding PCK1 in addition to the inherent base sequence encoding PCK1.

The transformed cell may further include an exogenous base sequence encoding PCK1 in addition to the inherent base sequence encoding PCK1.

The transformed cell may contain two or more base sequences encoding PCK1.

The transgenic dog can be produced using the transgenic cell.

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

The transformed cells can be proliferated or cultured in medium. The medium may be any medium that has been developed for the culture of animal cells, mammalian cells, or can be prepared in the laboratory with appropriate components necessary for animal cell growth, such as assimilable carbon, nitrogen and/or trace nutrients. can be used.

The medium may be any basic medium suitable for animal cell growth, including, but not limited to, Minimal Essential Medium (MEM), Dulbecco modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), and 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 expansion medium (Chemicon). In addition to this, any badge used in the industry can be used without restrictions. An assimilable source of carbon, nitrogen and micronutrients, a serum source, growth factors, amino acids, antibiotics, vitamins, reducing agents, and/or sugar sources may be further added to the medium.

A person skilled in the art can select or combine appropriate media and properly culture using known methods. In addition, culture can be performed while adjusting conditions such as appropriate culture environment, time, and temperature based on common knowledge in this field.

Transgenic dogs can be produced using the transgenic cells.

As a method, somatic cell nuclear transfer (SCNT) can be used.

The nucleus of the transformed cell is transplanted into a fertilized egg of an animal, and the fertilized egg into which the nucleus has been transplanted is implanted to produce a transformed individual in which a promoter and a base sequence encoding a specific substance are knocked in. The nucleus of the transformed cell is transferred to an animal. It is possible to produce a transgenic individual in which the ACTA promoter and the nucleotide sequence encoding PCK1 have been knocked in by implanting the fertilized egg into a fertilized egg and implanting the fertilized egg into which the nucleus has been transferred.

[Transgenic animals]

The present invention relates to a transgenic dog with improved energy metabolism and exercise capacity due to knock-in (KI) of the base sequence encoding the ACTA promoter and PCK1, a method of producing the same, and its use.

More specifically, it relates to a transgenic dog with improved energy metabolism and exercise capacity due to KI in the base sequence encoding the dog-derived ACTA promoter and PCK1, a method of producing the same, and its use.

More specifically, it relates to a transgenic dog with improved energy metabolism and exercise capacity due to KI in the nucleotide sequence encoding the dog-derived ACTA promoter and the human-derived PCK1, a method of producing the same, and its use.

Any cell of the transgenic dog may further contain an exogenous base sequence encoding PCK1 in addition to the inherent base sequence encoding PCK1.

The muscle tissue or muscle cells of the transgenic dog may further include an exogenous base sequence encoding PCK1 in addition to the inherent base sequence encoding PCK1.

The muscle tissue or muscle cells of the transgenic dog may contain two or more base sequences encoding PCK1.

The PCK1 expression level of any cell of the transgenic dog may be higher than the PCK1 expression level of any cell of the wild-type dog.

The PCK1 expression level in the muscle tissue or muscle cells of the transgenic dog may be higher than the PCK1 expression level in the muscle tissue or muscle cells of the wild-type dog.

The PCK1 expression level in the muscle tissue or muscle cells of the transgenic dog is about 1-fold, about 1.1-fold, about 1.2-fold, and about 1.3-fold the PCK1 expression level in the muscle tissue or muscle cells of the wild-type dog. 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 a factor of 2 or any multiple higher.

The expression level of PCK1 in the muscle tissue or muscle cells of the transgenic dog may be higher than the expression level of PCK1 in the muscle tissue or muscle cells of the wild-type dog.

The expression level of PCK1 in the muscle tissue or muscle cells of the transgenic dog may be significantly higher than the expression level of PCK1 in the muscle tissue or muscle cells of the wild-type dog.

The blood lipase concentration value of the transgenic dog has a value of about 0.5 times, about 0.4 times, about 0.3 times, about 0.2 times, or about 0.1 times or lower than the blood lipase concentration value of the wild type dog. You can.

The blood lipase concentration value of the transgenic dog may be about 2 times, about 3 times, about 4 times, about 5 times, or about 6 times or more than the blood lipase concentration value of the wild type dog. there is.

The blood lipase concentration value of the transgenic dog may be 0.25 times or less or 4 times more than the blood lipase concentration value of the wild type dog.

The transformed cells can be used to produce the transformed dog. In one embodiment, pregnancy can be induced by implanting the transformed cells under appropriate conditions.

By that method, for example, transformed cells can be used to induce pregnancy through somatic cell nuclear transfer (SCNT).

In one embodiment, the present invention uses a transgenic cell line in which the base sequence encoding the dog-derived ACTA promoter and PCK1 has been knocked in to produce a transgenic animal of the present invention by somatic cell nuclear transfer (SCNT). can be produced.

The transgenic animal may be a dog overexpressing PCK1.

The transgenic animal may be a dog that overexpresses PCK1 in muscle tissue or muscle cells.

In a specific embodiment, the method for producing transgenic dogs of the present invention,

(a) Preparation of nuclear donor cells, including culturing somatic cells or stem cells isolated from dogs;

(b) introducing a recombinant vector containing the ACTA promoter and a base sequence encoding PCK1 into the nuclear donor cell;

(c) producing enucleated eggs by removing the nucleus from a dog egg;

(d) microinjecting nuclear donor cells into the enucleated oocyte and fusing them;

(e) activating the fused egg; and

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

General technical details regarding each step can be used by referring to methods for producing cloned animals using conventional somatic cell nuclear transfer technology known in the art.

As one specific example, a method for producing a canine nuclear transfer embryo formed by transplanting the nucleus of the transformed cell into an enucleated egg, and a nuclear transfer embryo produced thereby can be provided.

As an example, a method for producing a transgenic dog overexpressing PCK1 in which the base sequence encoding the ACTA promoter and PCK1 has been knocked in, comprising transplanting the nuclear transfer embryo into the oviduct of a surrogate mother to produce offspring, or the ACTA promoter and PCK1 produced thereby. Provided is a dog overexpressing PCK1, characterized in that the base sequence encoding .

As an example, a method for producing the transgenic animal may be provided.

The transformation may use nuclease.

The method exposes the embryo or cell to a recombinant vector (e.g., a recombinant vector containing PCK1),

It may include cloning cells in the surrogate mother or transferring embryos in the surrogate mother.

The nuclease can specifically bind to a target chromosome region within an embryo or cell and cause changes in the base sequence of the cell chromosome.

[Usage]

One embodiment of the present invention provides the use of a dog in which the PCK1 gene is knocked in and the PCK1 protein is overexpressed in a muscle specific manner.

Transgenic dogs overexpressing muscle-specific PCK1 in which the PCK1 gene has been knocked in according to the present invention can be produced through crossbreeding, and the foreign gene can be transmitted to future generations.

Therefore, the dog characterized by the knock-in of the PCK1 gene of the present invention and the knock-in PCK1 gene being specifically expressed as a protein in muscle has the advantage of easy reformability, and the PCK1 protein is specifically expressed in muscle. It can be useful as an animal model with improved bioenergy metabolism and exercise capacity and/or an animal model with resistance to metabolic diseases, such as diabetes.

In other words, the muscle-specific PCK1-overexpressing dog of the present invention can be used as a quarantine dog requiring improved exercise capacity or for studying mechanisms involved in causing or treating metabolic diseases, the role of PCK1, muscle mass, strength, muscle fiber, mitochondrial biogenesis, It can be used in a variety of ways, including as a model to study changes in energy metabolism.

Therefore, in another aspect, the present invention may encompass various uses of muscle-specific PCK1 overexpressing dogs in the above methods.

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

As one specific example, the screening method of the present invention

1) Administering a candidate substance that can maintain or increase muscle endurance in muscle-specific PCK1-overexpressing dogs;

2) After administration of the candidate substance, it may include analyzing the dog's muscle tissue by comparing it with a control group that did not administer the candidate substance.

At this time, energy metabolism may mean mitochondrial biosynthesis, mitochondrial activity, gluconeogenesis, etc.

The candidate material may be any one selected from the group consisting of peptides, proteins, non-peptide 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 may be a well-known compound. These candidates may form salts.

Methods for administering the above candidate substances may include, for example, oral administration, intravenous injection, subcutaneous administration, intradermal administration, or intraperitoneal administration. Appropriate selection can be made based on the symptoms of the target animal, the properties of the candidate substance, etc. Additionally, the dosage of the candidate substance can be appropriately selected depending on the administration method or the properties of the candidate substance.

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

As one specific example, the screening method of the present invention

1) Administering a candidate substance that can maintain or increase obesity resistance in muscle-specific PCK1-overexpressing dogs;

2) After administration of the candidate substance, it may include analyzing the tissue of the dog compared to a control group that did not administer the candidate substance.

At this time, exercise capacity may be muscle mass, strength, muscular endurance, etc. compared to the control group.

The candidate material may be any one or more selected from the group consisting of peptides, proteins, non-peptide 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 new compound or a well-known compound. These candidate substances may form salts. Methods for administering the above candidate substances include, for example, oral administration, intravenous injection, subcutaneous administration, intradermal administration, or intraperitoneal administration, and can be appropriately selected according to the symptoms of the target animal, the properties of the candidate substance, etc. Additionally, the dosage of the candidate substance can be appropriately selected depending on 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 there is excessive adipose tissue in the body, and refers to a phenomenon in which energy consumed from food is not balanced with energy consumed through physical activity, etc., and excess energy is accumulated as body fat. If body fat increases abnormally due to energy imbalance over a long period of time, it can cause various metabolic diseases and adult diseases such as diabetes, hyperlipidemia, heart disease, stroke, arteriosclerosis, and fatty liver.

From a morphological perspective, obesity may be caused by an increase in the size (hypertrophy) or number (hyperplasia) of fat cells in the body.

Obesity is caused by abnormal hypertrophy of subcutaneous fat tissue caused by excess energy being accumulated as fat in the body when an imbalance in the metabolic process occurs due to endocrine factors, genetic factors, social and environmental factors, etc. Hypertrophy of adipose tissue is a phenomenon in which the size of fat cells increases (adipocyte hypertrophy) or their number increases (adipocyte hyperplasia), which also affects the congestion of the local venous-lymphatic system, leading to vascular tissue disease in the dermal-subcutaneous tissue. It can also cause

Excessive neutral fat accumulated in obese patients can be stored not only in adipose tissue but also in the liver and muscles, leading to insulin resistance. Therefore, consumption of excessively stored neutral fat can prevent and treat fundamental obesity and subsequent metabolic diseases.

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

When administered to a specific part of the body using the recombinant vector, obesity and metabolic diseases can be treated or alleviated.

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

The obesity or metabolic disease includes, but is not limited to, metabolic syndrome, hypertriglyceridemia, hyperdensity lipidemia, hypodensity lipidemia, angina pectoris, myocardial infarction, sexual dysfunction, sleep apnea, premenstrual syndrome, and stress urinary incontinence. Urinary incontinence, hyperactivity disorder, chronic fatigue syndrome, osteoarthritis, cancer related to weight gain, orthostatic hypotension, pulmonary hypertension, menstrual disorders, diabetes, hypertension, impaired glucose tolerance, coronary thrombosis, fainting, depression, anxiety, psychosis, Tardive dyskinesia, drug addiction, drug abuse, cognitive impairment, Alzheimer's disease, cerebral ischemia, obsessive-compulsive behavior, panic attacks, social phobia, bulimia, atherosclerosis, gallbladder diseases such as cholelithiasis, anorexia, and reproductive disorders such as polycystic ovarian disease. , one or more types selected from the group consisting of infection, varicose veins, skin diseases such as epidermal hyperplasia and eczema, insulin resistance, chronic arterial occlusion, orthopedic injury, thromboembolism, heart disease, urological disease, lipid syndrome, hyperglycemia, and stress. It may be an obesity-related disease.

In the muscle-specific PCK1 overexpression model dog of the present invention, as the PCK1 protein is overexpressed in muscle tissue, the activity of PCK1 is artificially increased, activating the gluconeogenesis reaction in the muscle itself, leading to an increase in glucose production, and also energy Since it induces improvements in metabolism and exercise capacity, it can be provided as an animal model with obesity resistance and improved energy metabolism and exercise capacity.

In addition, in the muscle-specific PCK1 overexpressing dog of the present invention, as the PCK1 protein is overexpressed in muscle tissue, the activity of PCK1 is artificially increased, activating the gluconeogenesis reaction in the muscle itself, leading to an increase in glucose production, and also energy Since it induces improvements in metabolism and exercise capacity, it can be provided as an animal model for research on energy metabolism/exercise performance improvement.

The present invention provides a muscle-specific PCK1-overexpressing dog and various uses thereof, thereby activating the gluconeogenesis reaction by the muscle-specifically overexpressed PCK1, resulting in increased glucose production, improved energy metabolism and exercise capacity, and obesity using dogs. It can be used in a variety of ways, such as metabolic disease-related research (obesity resistance research, etc.), energy metabolism/exercise improvement research, etc.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and 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.

Forms for practicing the invention

Example 1. Construction of vector for production of muscle-specific PCK1 overexpressing dogs

The PCK1 gene is expressed in various tissues, including liver, kidney, adipocytes, small intestine, lung, muscle, and brain. Since PCK1 is expressed in a wide range of tissues, it is important to select a promoter that can increase expression limited to specific tissues to reduce side effects. In this study, to produce dogs that specifically express PCK1 in muscle cells, the muscle-specific promoter, α-skeletal muscle actin promoter (2,372 bp), and the human-derived PCK1 gene (1,896 bp) were synthesized, and then muscle-specific PiggyBac vector cloning was synthesized into which the PCK1 gene was introduced under the control of an appropriate promoter. The mRuby gene was used as a marker gene (Figure 1). The nucleotide sequence of the ACTA promoter is SEQ ID NO: 1, the nucleotide sequence of PCK1 is SEQ ID NO: 2, and the nucleotide sequence of the vector constructed in this experiment is SEQ ID NO: 3, and the 5' PB terminal repeat and 3' PB terminal repeat are SEQ ID NO: 4, respectively. and 5.

Example 2. Confirmation of vector introduction in cells

After introducing the pPB-ACTAp-PCK1-Red/Puro vector into normal beagle adult fibroblasts and adipose-derived stem cells (ASC), molecular biological analysis was performed. RT-PCR showed that the vector It was confirmed that was introduced into the genome, and the expression of mRuby, a marker gene, was confirmed under a fluorescence microscope (Figures 2 and 3).

Among the two cells, the number of cells expressing red fluorescent protein was found to be greater in adipose stem cells. Therefore, it was determined that adipose stem cells were more suitable than adult fibroblasts as a cell line to be used in producing transgenic cloned dogs.

Example 3. Somatic cell nuclear transfer

After constructing a cell line by introducing the pPB-ACTAp-PCK1-Red/Puro vector into the adipose stem cells of a beagle, a detection dog to be cloned, a molecular biological assay was performed, and the cell line in which transfer and expression of the vector were confirmed was used for somatic cell nuclear transfer. (Figures 4 and 5).

After somatic cell nuclear transfer and in vitro culture, the expression of the marker gene mRuby was confirmed in the cloned embryos (Figure 6).

After transplanting somatic cell nuclear transfer cloned eggs into surrogate mothers, a total of one muscle-specific PCK1-expressing transgenic cloned beagle was produced. Genomic DNA PCR and Southern blot were performed using blood samples from the produced individuals to verify that they were stably inserted into the genome (Figures 7 and 8).

Example 4. Serum chemistry and phenotype analysis results of transgenic cloned dogs

As a result of serum chemistry tests conducted on the transgenic cloned dogs and the control group at 1, 2, 4, and 6 months of age, although statistical comparison was not possible because there was only one transgenic cloned dog, the blood triglyceride concentration was higher than the increasing trend in the control group. A rapid increase was confirmed (Figure 9). This proves that as the gluconeogenesis process increases, the concentration of its product, triglyceride, increases.

In addition, blood lipase concentration tended to increase in the control group, whereas in the transgenic individuals, it was about 20 times higher than the control group at 1 month of age and then gradually decreased. This is because in transgenic dogs, when autonomous movement is difficult, the concentration of lipase is high to break down hyperformed triglyceride, but from the time when autonomous movement is possible, energy is released through exercise and fat tissue accumulation decreases and decomposition occurs. It is interpreted that this is because the concentration of triglyceride required has been reduced.

Additionally, PCK1-overexpressing transgenic individuals showed the following phenotype due to increased glyceroneogenesis. It was confirmed that intramuscular PCK1 production, cage activity, exercise capacity, intramuscular mitochondrial number, and feed intake increased. It was confirmed that as the number of mitochondria in the muscle increased, the excessively produced triglyceride could be consumed, and the lactate concentration in the tissue was observed to be lower than that in the control group.

It was confirmed that lifespan was extended and reproductive lifespan was extended.

And as a result of MRI, it was confirmed that the fat tissue volume had decreased. It was confirmed that appetite increased due to a decrease in the concentration of leptin, MCP-1, and insulin in the blood.

Example 5. MRS imaging results of the femoral muscles of transgenic cloned dogs

MRS (magnetic resonance spectrography) imaging of the femoral muscles of the clone detection dog was performed. MRS images were analyzed using TARQUIN (version 4.3.10. Birmingham, UK) software and evaluated according to the MRS criteria.

The results of the muscle characteristic spectrum of the adductor muscle of the cloned detection dog are shown in Table 1 below and Figures 10 to 15. Figures 10 to 14 show the results for Controls 1 to 5, and the spectra of Controls 1 to 3 and Controls 4 and 5 showed similar patterns except for the Lipid13b substance.

Comparing the spectrum of the PCK1-overexpressing cloned dog 'Woojoo' with the spectra of control groups 1 to 5, which are regular cloned dogs, overall, high absolute concentration values of metabolite substances were detected in the PCK1-overexpressed cloned dog.

A comparison of the relative values (metabolite/Cr) obtained by dividing the intramuscular metabolite value by the creatinine (Cr) substance is shown in Table 1 below, and only some metabolite/Cr values were confirmed to be high in PCK1 overexpressing cloned dogs.

MRS imaging results of control and PCK1-expressing cloned dogs Control group 1 Control group 2 Control group 3 Control group 4 Control group 5 PCK1 Ala:Cr 0.590654 2.440945 2.852113 2.53463 1.61 2.28 Asp:Cr 3.439252 2.952756 1.725352 0.765811 1.61 1.87 Cr:Cr One One One One One One GABA:Cr 0 0 0 0 0 0 GPC:Cr 0.842991 0.850394 0.739437 0.583113 0.63 0.24 Glc:Cr 0.623364 0.373228 0.493662 0.551045 0.872 0.264 Gln:Cr 0.86729 0.714173 0.264085 1.000515 0.84 0.141 Glth:Cr 0.324299 0.338583 0.371127 0.251672 0 0.356 Glu:Cr 0.373832 0.565354 0.760563 3.064577 0.731 0.369 Ins:Cr 0 0 0 0 0 0 Lac:Cr 0.7486 1.16535 1.76056 0.93318 0 2.3 Lip09:Cr 2.570093 2.094488 1.598592 17.74347 22.3 13.4 Lip20:Cr 1.33645 1.33858 1.46479 2.58341 2.65 2.72 MM09:Cr 0 0 0 0 0 0 MM17:Cr 0 0 0 0 0 0 MM20:Cr 0.134579 0.262992 0.66831 8.132649 0.43 0.929 NAA:Cr 0.069252 0 0 0 0 0 NAAG:Cr 0.091402 0.024961 0 0 0.0654 0 PCh:Cr 0.12523 0 0.06169 0 0.128 0.273 PCr:Cr 1.130841 0.765354 0.838028 1.075382 0.762 0.404 Scyllo:Cr 0.041402 0 0 0 0.0282 0.00477 Tau:Cr 1.719626 1.291339 0.978873 1.047401 0.641 0.458 TNAA:Cr 0.160748 0.024961 0 0 0.0654 0 TCho:Cr 0.971963 0.850394 0.802817 0.583113 0.757 0.513 TCr:Cr 2.130841 1.771654 1.84507 2.075382 1.76 1.4 Glx:Cr 1.242991 1.275591 1.021127 4.065093 1.57 0.51 TLM09:Cr 2.570093 2.094488 1.598592 3.953779 3.01 2.44 TLM13:Cr 23.5514 18.97638 18.87324 17.74347 33.1 30.5 TLM20:Cr 1.476636 1.598425 2.133803 10.71606 3.08 3.65

Example 6. Evaluation of anatomical normality using X-ray in cloned dogs overexpressing muscle-specific PCK1

As a result of radiography of the thorax, abdomen, skull, lumbar spine, and pelvis, it was confirmed that no abnormalities were observed in control groups 4 and 5 and PCK1-overexpressing cloned dogs (FIGS. 16 to 20).

Referring to the paper Growth and hematologic characteristics of cloned dogs derived from adult somatic cell nuclear transfer (Park JE et al., 2010), the measurement results of the skull, lumbar spine, and pelvis were all judged to be normal results.

It was determined that there was no difference in measured values between PCK1-overexpressing cloned dogs and normal cloned dogs (control groups 1 to 5).

Example 7. Normality evaluation using echocardiography in cloned dogs overexpressing muscle-specific PCK1

As a result of Teichholz measurement in M-mode in the right parasternal long axis 5 chamber view, the data values of control group 4, control group 5, and PCK1 overexpressing cloned dogs were all judged to be within the normal range (Table 2).

Teichholz measurement results Teichholz Control group 4 Control group 5 PCK1 IVSd(mm) 10 7 8 LVIDd(mm) 27 26 26 LVPWd(mm) 10 8 11 IVSs (mm) 13 10 13 LVIDs (mm) 17 15 14 LVPWs (mm) 13 14 12 EDV(ml) 26 24 26 ESV(ml) 8 6.2 4.7 EF(%) 75.6 79.6 81.6 FS(%) 37.6 41.2 48.4

As a result of comparing cardiac function between PCK1-overexpressing cloned dogs and regular cloned dogs, it was determined that there was no difference.

As a result of measuring the LA/AO ratio in the right parasternal long axis 5 chamber view, the data values for control group 4, control group 5, and PCK1 overexpressing cloned dogs were all judged to be within the normal range (Table 3).

LA/AO ratio measurement results Control group 4 Control group 5 PCK1 LA/AO 1.13 1.01 1.02

As a result of measuring RVTO flow in the right parasternal long axis 5 chamber view, the data values of control group 4, control group 5, and PCK1 overexpression cloned dogs were all judged to be within the normal range (Table 4).

RVOT flow measurement results Control group 4 Control group 5 PCK1 pV 96.1 147.6 108.2 AT/ET 0.41 0.39 0.46

As a result of mitral flow and TDI measurements in the left apical 4 chamber view, the data values of control group 4, control group 5, and PCK1 overexpressing cloned dogs were all judged to be within the normal range (Table 5).

Mitral flow and TDI measurement results Control group 4 Control group 5 PCK1 Mitral flow E/A 1.61 1.83 1.67 TDI E/E' 8.92 11.87 8.89

It was confirmed that congenital heart disease and structural abnormalities were not observed in the 2D evaluation of the heart, reflux of cardiac blood flow in the color Doppler test, and cardiac systolic/diastolic function tests in control group 4, control group 5, and PCK1-overexpressing cloned dogs. .

In the end, the hearts of control group 4, control group 5, and PCK1-overexpressing cloned dogs were all judged to be normal.

Example 8. Evaluation of anatomical normality using CT in cloned dogs overexpressing muscle-specific PCK1

CT scans were performed to evaluate the anatomical normality of muscle-specific PCK1-overexpressing cloned dogs. It was determined that malformations of the hepatobiliary system and occiput-cervical spine did not occur in control group 4, control group 5, and PCK1 overexpressing cloned dogs (Figure 21)

20191031_OPP19-079-PCT_Sequence List.app

<110> Seoul National University Industry Foundation <120> Production of transgenic dogs that overexpress PCK1 in a muscle specific manner <130> OPP19-079-PCT <150> KR 10-2018-0131498 <151> 2018-10-31 <160 > 5 <170> KoPatentIn 3.0 <210> 1 <211> 2372 <212> DNA <213> Artificial Sequence <220> <223> ACTA (alpha-skeletal muscle actin) promoter <400> 1 aagctttctg taaggaaagg taagagttga actgagcaag agttttgaaa aatagtgaca 60 atcccattct cctttggaat gcgcacaaat attgaggtat ccagtgaacg gcagcaaatt 120 tcctaccttc aaggcccaaa tgtaagctag tccccttacg ttacatgcag ctcatttgct 180 aagtggtttt tttctagtat ctccactact cgctgacaca ggaggacaca ggatgttaaa 2 40 aaggaaatac agttctgtca attattcact tactctccaa aatacttgga agaactaaat 300 atggaaccat aggagacttt atcctcaccg catagtccct atactagtca aactccttat 360 tttttaattg atcattttta ggaaggtagc attttattca ctagaacatt tttgttaata 420 cttgtttat tt tgggatga actgccatga tgtgggctac agaggagggt cgcatatgct 480 tccatccccc ttttagagaa tccacacctg tcccagttgc tgggttccac taccaaaagt 540 gaattgcaac tattttagga gcacttaagc acatccgaaa aatgagtgat tctgttctgg 600 cccacaccac atcactgatg taccccctta aagcatgtcc ctgagttcat cacagaagac 660 tgctcctcct gtg ccctcca caaggttaga actgtccttg tcttagggaa aaaggagaga 720 gagagagaga gagagagaga gagagagaga gagagagaga gagagggaca ggcaccaact 780 gggtaacctc tgctgacccc cactctactt taccataagt agctccaaat ccttctagaa 840 aatctgaaag gcatagcccc atatatcagt gatataaata gaacctg cag caggctctgg 900 taaaatgatga ctacaaggtg gactgggagg cagcccggcc ttggcaggca tcatcctcta 960 aatataaaga tgagtttgtt cagcctttgc agaaggaaaa actgccaccc atcctagagt 1020 gccgcgtcct tgtcccccca ccccctccaa tttattggga ggaaggacca gctaagcctc 1080 atctaggaag agcccct cac ccatctccac ctccactcca ggtctagcca gtcctgggtt 1140 gtgacccttg tctttcagcc ccaggagagg gacacacata gtgccaccaa agaggctggg 1200 ggagggcctc agcccaccaa aacctggggc cagtgcgtcc tacaggaggg gaaccctcac 1260 cccttcaatc cctttaggag acccaagggc gctgcgcgtc cctgaggcgg acagctccgt 1320 gtgctcaggc tttgcgcctg acaggcctat ccccgggagc ccccgcgcct cctccccggc 1380 gctccgccct cgcctccccc cgccagttgt ctatcctgcg acagctgcgc gccctccggc 1440 cgccggtggc cctctgtgcg gtgggggaag gggtcgacgt ggctcagctt tttggattca 1500 gggag ctcgg gggtgggaag agagaaatgg agttccaggg gcgtaaagga gagggagttc 1560 gccttccttc ccttcctgag actcaggagt gactgcttct ccaatcctcc caagcccacc 1620 actccacacg actccctctt cccggtagtc gcaagtggga gtttggggat ctgagcaaag 1680 aac ccgaaga ggagttgaaa tattggaagt cagcagtcag gcaccttccc gagcgcccag 1740 ggcgctcaga gtggacatgg ttggggaggc ctttgggaca ggtgcggttc ccggagcgca 1800 ggcgcacaca tgcacccacc ggcgaacgcg gtgaccctcg ccccacccca tcccctccgg 1860 cgggcaactg ggtcgggtca ggaggggcaa acccgctagg gagacactcc atatacggcc 1920 cggcccgcgt tacctgggac cggg ccaacc cgctccttct ttggtcaacg caggggaccc 1980 gggcggggggc ccaggccgcg aaccggccga gggagggggc tctagtgccc aacacccaaa 2040 tatggctcga gaagggcagc gacattcctg cggggtggcg cggaggggaat gcccgcgggc 2100 tatataaaac ctgag cagag ggacaagcgg ccaccgcagc ggacagcgcc aagtgaagcc 2160 tcgcttcccc tccgcggcga ccaggggcccg agccgagagt agcagttgta gctacccgcc 2220 caggtagggc aggagttggg aggggacagg gggacagggc actaccgagg ggaacctgaa 2280 ggactccggg gcagaaccca gtcggttcac ctggtcagcc ccaggcctgc gccctgagcg 2340 ctgtgcctcg tctccggagc cacacgcgct tt 23 72 <210> 2 <211> 1869 <212> DNA <213> Artificial Sequence <220> <223> PCK1 <400> 2 atgcctcctc agctgcaaaa cggcctgaac ctctcggcca aagttgtcca gggaagcctg 60 gacagcctac cccaggcagt gagggagttt ctcgagaata acgctgagct gtgtcagcct 120 gatcacatcc acatctgtga cggctctgag gaggagaatg ggcggcttct gggccagatg 180 gaggaaga gg gcatcctcag gcggctgaag aagtatgaca actgctggtt ggctctcact 240 gaccccaggg atgtggccag gatcgaaagc aagacggtta tcgtcaccca agagcaaaga 300 gacacagtgc ccatccccaa aacaggcctc agccagctcg gtcgctggat gtcagaggag 360 gattttgaga aagcgttcaa tgccaggttc ccagggtgca tgaaaggtcg caccatgtac 420 gtcatcccat tcagcatggg gccgctgggc tcgcctctgt caaagatcgg catcgagctg 480 acggattcac cctacgtggt ggccagcatg cggatcatga cgcggatggg cacgcccgtc 540 ctggaagcag tgggcgatgg ggagtttgtc aaatgcctcc attctgtggg gtgccctctg 600 cct ttacaaa agcctttggt caacaactgg ccctgcaacc cggagctgac gctcatcgcc 660 cacctgcctg accgcagaga gatcatctcc tttggcagtg ggtacggcgg gaactcgctg 720 ctcgggaaga agtgctttgc tctcaggatg gccagccggc tggccaagga ggaagggtgg 78 0 ctggcagagc acatgctgat tctgggtata accaaccctg agggtgagaa gaagtacctg 840 gcggccgcat ttcccagcgc ctgcgggaag accaacctgg ccatgatgaa ccccagcctc 900 cccgggtgga aggttgagtg cgtcggggat gacattgcct ggatgaagtt tgacgcacaa 960 ggtcatttaa gggccatcaa cccagaaaat ggctttttcg gtgtcgctcc tgggacttca 1020 gtgaagacca accccaatg c catcaagacc atccagaaga acacaatctt taccaatgtg 1080 gccgagacca gcgacggggg cgtttactgg gaaggcattg atgagccgct agcttcaggt 1140 gtcaccatca cgtcctggaa gaataaggag tggagctcag aggatgggga accttgtgcc 1200 caccccaact cgaggttctg cacccctgcc agccagtgcc ccatcattga tgctgcctgg 1260 gagtctccgg aaggtgttcc cattgaaggc attatctttg gaggccgtag acctgctggt 1320 gtccctctag tctatgaagc tctcagctgg caacatggag tctttgtggg ggcggccatg 1380 agatcagagg ccacagcggc tgcagaacat aaaggcaaaa tcatcatgca tgaccccttt 1440 gccatgcggc ccttctttgg ctacaacttc gg caaatacc tggcccactg gcttagcatg 1500 gcccagcacc cagcagccaa actgcccaag atcttccatg tcaactggtt ccggaaggac 1560 aaggaaggca aattcctctg gccaggcttt ggagagaact ccagggtgct ggagtggatg 1620 ttcaaccgga tcgatggaaa agccagcacc aag ctcacgc ccataggcta catccccaag 1680 gaggatgccc tgaacctgaa aggcctgggg cacatcaaca tgatggagct tttcagcatc 1740 tccaaggaat tctggggagaa ggaggtggaa gacatcgaga agtatctgga ggatcaagtc 1800 aatgccgacc tcccctgtga aatcgagaga gagatccttg ccttgaagca aagaataagc 1860 cagatgtaa 1869 <210> 3 <211> 1141 0 <212> DNA <213> Artificial Sequence <220> <223> Vector <400> 3 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 60 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 120 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 180 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttata a atcaaaagaa 240 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 300 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 360 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcact aaa tcggaaccct 420 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 480 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 540 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc 600 aggctgcgca actgttggga agggcgatcg gtgc gggcct cttcgctatt acgccagctg 660 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 720 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg 780 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg 840 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg 900 tacgttaaag ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag 960 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata 1020 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa caaaaactca 1080 aaatttct tc tataaagtaa caaaactttt atgagggaca gccccccccc aaagccccca 1140 gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct 1200 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg 1260 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag 1320 acacctgggg ggatacgggg aaaaggcctc cacggccact agtggcgcgc cataccggta 1380 agctttctgt aaggaaaggt aagagttgaa ctgagcaaga gttttgaaaa atagtgacaa 1440 tcccattctc ctttggaatg cgcacaaata ttgaggtatc cagtgaacgg cag caaattt 1500 cctaccttca aggcccaaat gtaagctagt ccccttacgt tacatgcagc tcatttgcta 1560 agtggttttt ttctagtatc tccactactc gctgacacag gaggacacag gatgttaaaa 1620 aggaaataca gttctgtcaa ttattcactt actctccaaa atacttggaa gaactaaata 1680 tggaaccata ggagacttta tcctcaccgc atagtcccta tactagtcaa actccttatt 1740 ttttaattga tcatttttag gaaggtagca ttttattcac tagaacattt ttgttaatac 1800 ttgttattt ttgggatgaa ctgccatgat gtgggctaca gaggagggtc gcatatgctt 1860 ccatccccct tttagagaat ccacacctgt cccagttgct gggttccact accaaaagtg 1920 aatt gcaact attttaggag cacttaagca catccgaaaa atgagtgatt ctgttctggc 1980 ccacaccaca tcactgatgt acccccttaa agcatgtccc tgagttcatc acagaagact 2040 gctcctcctg tgccctccac aaggttagaa ctgtccttgt cttagggaaa aaggagagag 2100 aga gagagag agagagagag agagagagag agagagagag agagggacag gcaccaactg 2160 ggtaacctct gctgaccccc actctacttt accataagta gctccaaatc cttctagaaa 2220 atctgaaagg catagcccca tatatcagtg atataaatag aacctgcagc aggctctggt 2280 aaatgatgac tacaaggtgg actgggaggc agcccggcct tggcaggcat catcctctaa 2340 atataaagat gagtttgttc agcctt tgca gaaggaaaaa ctgccaccca tcctagagtg 2400 ccgcgtcctt gtccccccac cccctccaat ttattgggag gaaggaccag ctaagcctca 2460 tctaggaaga gcccctcacc catctccacc tccactccag gtctagccag tcctgggttg 2520 tgacccttgt ctttcagcc c caggagaggg acacacatag tgccaccaaa gaggctgggg 2580 gagggcctca gcccaccaaa acctggggcc agtgcgtcct acaggagggg aaccctcacc 2640 ccttcaatcc ctttaggaga cccaagggcg ctgcgcgtcc ctgaggcgga cagctccgtg 2700 tgctcaggct ttgcgcctga caggcctatc cccgggagcc cccgcgcctc ctccccggcg 2760 ctccgccct c gcctcccccc gccagttgtc tatcctgcga cagctgcgcg ccctccggcc 2820 gccggtggcc ctctgtgcgg tgggggaagg ggtcgacgtg gctcagcttt ttggattcag 2880 ggagctcggg ggtgggaaga gagaaatgga gttccagggg cgtaaaggag agggagtt cg 2940 ccttccttcc cttcctgaga ctcaggagtg actgcttctc caatcctccc aagcccacca 3000 ctccacacga ctccctcttc ccggtagtcg caagtgggag tttggggatc tgagcaaaga 3060 acccgaagag gagttgaaat attggaagtc agcagtcagg caccttcccg agcgcccagg 3120 gcgctcagag tggacatggt tggggaggcc tttgggacag gtgcggttcc cggagcgcag 3180 gcgcacacat gcacccaccg gcga acgcgg tgaccctcgc cccaccccat cccctccggc 3240 gggcaactgg gtcgggtcag gaggggcaaa cccgctaggg agacactcca tatacggccc 3300 ggcccgcgtt acctgggacc gggccaaccc gctccttctt tggtcaacgc aggggacccg 3360 ggcgggggcc caggccg cga accggccgag ggagggggct ctagtgccca acacccaaat 3420 atggctcgag aagggcagcg acattcctgc ggggtggcgc ggagggaatg cccgcgggct 3480 atataaaacc tgagcagagg gacaagcggc caccgcagcg gacagcgcca agtgaagcct 3540 cgcttcccct ccgcggcgac cagggcccga gccgagagta gcagttgtag ctacccgccc 3600 aggtagggca ggagttggga ggggacaggg ggacag ggca ctaccgaggg gaacctgaag 3660 gactccgggg cagaacccag tcggttcacc tggtcagccc caggcctgcg ccctgagcgc 3720 tgtgcctcgt ctccggagcc acacgcgctt tggatccagt gtacagccac catgcctcct 3780 cagctgcaaa acggcctgaa cctct cggcc aaagttgtcc agggaagcct ggacagccta 3840 ccccaggcag tgagggagtt tctcgagaat aacgctgagc tgtgtcagcc tgatcacatc 3900 cacatctgtg acggctctga ggaggagaat gggcggcttc tgggccagat ggaggaagag 3960 ggcatcctca ggcggctgaa gaagtatgac aactgctggt tggctctcac tgaccccagg 4020 gatgtggcca ggatcgaaag caagacggtt atcgtcaccc aagagcaaag aga cacagtg 4080 cccatcccca aaacaggcct cagccagctc ggtcgctgga tgtcagagga ggattttgag 4140 aaagcgttca atgccaggtt cccagggtgc atgaaaggtc gcaccatgta cgtcatccca 4200 ttcagcatgg ggccgctggg ctcgcctctg tcaaaga tcg gcatcgagct gacggattca 4260 ccctacgtgg tggccagcat gcggatcatg acgcggatgg gcacgcccgt cctggaagca 4320 gtgggcgatg gggagtttgt caaatgcctc cattctgtgg ggtgccctct gcctttacaa 4380 aagcctttgg tcaacaactg gccctgcaac ccggagctga cgctcatcgc ccacctgcct 4440 gaccgcagag agatcatctc ctttggcagt gggtacggcg ggaactcg ct gctcgggaag 4500 aagtgctttg ctctcaggat ggccagccgg ctggccaagg aggaagggtg gctggcagag 4560 cacatgctga ttctgggtat aaccaaccct gagggtgaga agaagtacct ggcggccgca 4620 tttcccagcg cctgcgggaa gaccaacctg gccatgatga acccc agcct ccccgggtgg 4680 aaggttgagt gcgtcgggga tgacattgcc tggatgaagt ttgacgcaca aggtcattta 4740 agggccatca acccagaaaa tggctttttc ggtgtcgctc ctgggacttc agtgaagacc 4800 aaccccaatg ccatcaagac catccagaag aacacaatct ttaccaatgt ggccgagacc 4860 agcgacgggg gcgtttactg ggaaggcatt gatgagccgc tagcttcagg tgtcaccatc 4920 a cgtcctgga agaataagga gtggagctca gaggatgggg aaccttgtgc ccaccccaac 4980 tcgaggttct gcacccctgc cagccagtgc cccatcattg atgctgcctg ggagtctccg 5040 gaaggtgttc ccattgaagg cattatcttt ggaggccgta gacctgctgg tgtcc ctcta 5100 gtctatgaag ctctcagctg gcaacatgga gtctttgtgg gggcggccat gagatcagag 5160 gccacagcgg ctgcagaaca taaaggcaaa atcatcatgc atgacccctt tgccatgcgg 5220 cccttctttg gctacaactt cggcaaatac ctggcccact ggcttagcat ggcccagcac 5280 ccagcagcca aactgcccaa gatcttccat gtcaactggt tccggaagga caaggaaggc 5340 aaattcctct ggccagg ctt tggagagaac tccagggtgc tggagtggat gttcaaccgg 5400 atcgatggaa aagccagcac caagctcacg cccataggct acatccccaa ggaggatgcc 5460 ctgaacctga aaggcctggg gcacatcaac atgatggagc ttttcagcat ctccaaggaa 5520 ttctgggaga aggaggtgg a agacatcgag aagtatctgg aggatcaagt caatgccgac 5580 ctcccctgtg aaatcgagag agagatcctt gccttgaagc aaagaataag ccagatgtaa 5640 acgcgtcagg taccaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc 5700 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatc 5760 tgtgccttct agttg ccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 5820 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 5880 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 5940 ggaagagaat agcaggcatg ctggggagcg gccgcaagga tctgcgatcg ctccggtgcc 6000 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc 6060 aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac 6120 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg 6180 aacgttcttt ttc gcaacgg gtttgccgcc agaacacagc tgaagcttcg aggggctcgc 6240 atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg gttgagtcgc 6300 gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta ggta agttta 6360 aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc tagactcagc 6420 cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt ttcgttttct 6480 gttctgcgcc gttacagatc caagctgtga ccggcgccta cgatatcgcc accatggcca 6540 gctccgagga tgtcatcaaa gagtttatga gatttaaggt caagatggag ggaagcgtca 6600 acggacacga gttcgagatt gagggagaag gagaaggccg gcctta cgag ggcacacaaa 6660 ccgctaagct caaggtcaca aaaggaggac ccctcccctt ctcctgggat attctgagcc 6720 ctcagttcca gtacggaagc aaagcctatg ttaaacaccc tgccgacatc cctgactatc 6780 tgaagctctc cttccctgaa ggcttcaagt ggg agagatt catgaacttc gaggacggag 6840 gcgtggtgac agtcacacaa gatagcaccc tccaggacgg agagtttat tataaggtga 6900 aactcagagg aaccaacttc ccctccgatg gccctgtcat gcaaaaaaaaa acaatgggat 6960 gggaagcctc caccgagaga atgtatcctg aggatggcgc tctgaaaggc gaaattaaaa 7020 tgagactgaa actcaaagac ggaggacact acgatgccga ggtcaaaaca acctacaagg 7080 ccaagaaaca agtgcagctg cctggcgcct acatgactga tattaaactc gacattatca 7140 gccataatgg ggactacacc atcgtggaac aatatgagag agctgagggc agacatagca 7200 caggcgctgg agagggcaga ggaagtcttc taacatgcgg tgacgtggag gagaatcccg 7260 gccctatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg 7320 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc 7380 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc 7440 tcgacatcgg caaggtgtgg gtcgcggacg acggcgcc gc ggtggcggtc tggaccacgc 7500 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga 7560 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca 7620 aggagcccgc gtggttcc tg gccaccgtcg gcgtctcgcc cgaccacag ggcaagggtc 7680 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct 7740 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca 7800 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg 7860 cctgaaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg 7920 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gcgttaacta 7980 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 8040 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 8100 tatcatgtct ggaattgact caaatgatgt caattagtct atcagaagct atctggtctc 8160 ccttccgggg gacaagacat ccctgtttaa tatttaaaca gcagtgttcc caaactgggt 8220 tcttatatcc cttgctctgg tcaaccaggt tgcagggttt cctgtcctca caggaacgaa 8280 gtccctaaag aaacagtggc agccaggttt agccccggaa ttgactggat tcctttttta 8340 gggcccat tg gtatggcttt ttccccgtat ccccccaggt gtctgcaggc tcaaagagca 8400 gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc 8460 gcacgctgcc ggctcgggga tgcgggggga gcgccggacc ggagcggagc ccc gggcggc 8520 tcgctgctgc cccctagcgg gggagggacg taattacatc cctgggggct ttgggggggg 8580 gctgtccctg atatctataa caagaaaata tatatataat aagttatcac gtaagtagaa 8640 catgaaataa caatataatt atcgtatgag ttaaatctta aaagtcacgt aaaagataat 8700 catgcgtcat tttgactcac gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt 8760 gacaagcacg cctcacggga gctccaagc g gcgactgaga tgtcctaaat gcacagcgac 8820 ggattcgcgc tatttagaaa gagagagcaa tatttcaaga atgcatgcgt caattttacg 8880 cagactatct ttctagggtt aatctagctg catcaggatc atatcgtcgg gtcttttttc 8940 cggctcagtc atcgcccaag ct ggcgctat ctgggcatcg gggaggaaga agcccgtgcc 9000 ttttcccgcg aggttgaagc ggcatggaaa gagtttgccg aggatgactg ctgctgcatt 9060 gacgttgagc gaaaacgcac gtttaccatg atgattcggg aaggtgtggc catgcacgcc 9120 tttaacggtg aactgttcgt tcaggccacc tgggatacca gttcgtcgcg gcttttccgg 9180 acacagttcc ggatggtcag cccgaagcgc atcagcaacc cgaacaatac cggcgacagc 9240 cggaactgcc gtgccggtgt gcagattaat gacagcggtg cggcgctg gg atattacgtc 9300 agcgaggacg ggtatcctgg ctggatgccg cagaaatgga catggatacc ccgtgagtta 9360 cccggcgggc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc 9420 cgctcacaat tccacacaac atacgagccg gaagcataaa gt gtaaagcc tggggtgcct 9480 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 9540 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 9600 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 9660 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggata acg 9720 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 9780 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 9840 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgt ttccc cctggaagct 9900 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 9960 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 10020 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 10080 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 101 40 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 10200 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 10260 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgct g 10320 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 10380 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 10440 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 10500 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 10560 taatcagtga ggcacctatc t cagcgatct gtctatttcg ttcatccata gttgcctgac 10620 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 10680 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 10740 gaagggcc ga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 10800 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 10860 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 10920 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 10980 tcggt cctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 11040 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 11100 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 11160 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 11220 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 11280 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 11340 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 11400 gaatactcat 11410 <210> 4 <21 1> 241 <212> DNA <213> Artificial Sequence <220> <223> 5' PB termunal repeat <400> 4 ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg 60 acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa 120 gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt 180 tatttattta ttaa aaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt 240 t 241 <210> 5 <211> 255 <212> DNA <213> Artificial Sequence <220> <223> 3' PB terminal repeat <400> 5 gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata 60 acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca 120 ttttgactca cgcggtcgtt atagttcaaa atcagtgaca ctta ccgcat tgacaagcac 180 gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg 240ctatttagaa agaga 255

Claims (11)

A transgenic dog overexpressing muscle cell-specific PCK1,
-At this time, the transgenic dog contains muscle tissue or muscle cells,
At this time, the genome of the muscle tissue or muscle cell contains an inherent base sequence encoding PCK1; and
Contains at least one sequence in which a human-derived ACTA promoter and a foreign base sequence encoding human-derived PCK1 are operably linked;
At this time, the ACTA promoter works specifically in muscle cells -;
Characterized by
Transgenic dogs with muscle cell-specific PCK1 overexpression. ◈Claim 2 was abandoned upon payment of the setup registration fee.◈ According to claim 1,
The blood lipase concentration value of the transgenic dog is characterized in that it has a value greater than 0 and 0.25 times or less than the blood lipase concentration value of the wild-type dog.
Transgenic dogs with muscle cell-specific PCK1 overexpression. According to claim 1,
The ACTA promoter corresponds to SEQ ID NO: 1,
The foreign base sequence encoding PCK1 is characterized in that it corresponds to SEQ ID NO: 2,
Transgenic dogs with muscle cell-specific PCK1 overexpression. One or more transposons;
One or more inverted terminal repeat sequences or direct repeats; and
Comprising a base sequence in which one or more human-derived ACTA promoters and a foreign base sequence encoding human-derived PCK1 are operably linked
Recombinant vector for producing muscle cell-specific PCK1 overexpressing dogs. ◈Claim 5 was abandoned upon payment of the setup registration fee.◈ According to clause 4,
The transposable factor further includes a transposase,
- At this time, the transposase is piggyBac transposase or Sleeping Beauty transposase -;
Characterized in that an inverted terminal repeat sequence or a trend repeat sequence is located at both ends of the transposable element.
Recombinant vector for producing muscle cell-specific PCK1 overexpressing dogs. ◈Claim 6 was abandoned upon payment of the setup registration fee.◈ The method of claim 5, wherein the recombinant vector is a plasmid or viral vector.
Recombinant vector for producing muscle cell-specific PCK1 overexpressing dogs. ◈Claim 7 was abandoned upon payment of the setup registration fee.◈ According to clause 4,
The recombinant vector is characterized in that it corresponds to SEQ ID NO: 3,
Recombinant vector for producing muscle cell-specific PCK1 overexpressing dogs. As a transformed cell,
- At this time, in the genome of the transformed cell, one or more nucleotide sequences encoding the human-derived ACTA promoter and human-derived PCK1 are knocked in,
At this time, the ACTA promoter works specifically in muscle cells,
At this time, the offspring born through the transformed cells are characterized by specifically overexpressing PCK1 in muscle tissue or muscle cells;
Transformed cells for producing transgenic dogs, characterized in that: ◈Claim 9 was abandoned upon payment of the setup registration fee.◈ According to clause 8,
Characterized in that the transformed cells are adult fibroblasts or adipose stem cells.
Transformed cells for making transgenic dogs. ◈Claim 10 was abandoned upon payment of the setup registration fee.◈ According to clause 8,
The ACTA promoter corresponds to SEQ ID NO: 1,
The foreign base sequence encoding PCK1 is characterized in that it corresponds to SEQ ID NO: 2,
Transformed cells for making transgenic dogs. ◈Claim 11 was abandoned upon payment of the setup registration fee.◈ Preparing the recombinant vector and nuclear donor cells according to any one of paragraphs 4 to 7,
-At this time, the nuclear donor cells are somatic cells or stem cells derived from dogs-;
Inducing a first cell by introducing the recombinant vector into the nuclear donor cell;
Characterized in that it includes introducing the nucleus of the first cell into an enucleated egg to induce a second cell,
Method for producing transgenic cells for producing muscle cell-specific PCK1-overexpressing transgenic dogs.

Images