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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
  • C12N15/877—Techniques for producing new mammalian cloned embryos
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0603—Embryonic cells ; Embryoid bodies
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/10—Cells modified by introduction of foreign genetic material
  • C12N5/12—Fused cells, e.g. hybridomas
  • C12N5/16—Animal cells

Definitions

• the present invention relates to a method for producing cloned canines, and more particularly, to a method of producing a nuclear transfer embryo by enucleating an optimal mature oocyte collected from the living body of a canine to prepare an enucleated recipient oocyte and transferring a canine somatic cell as a nuclear donor cell into the enucleated recipient oocyte in optimized conditions, and a method of producing a cloned canine by transferring the produced nuclear transfer embryo into the oviduct of a surrogate mother. Also, the present invention relates to a cloned canine produced by the method.
• Somatic cell nuclear transfer technology refers to a technique of creating a new individual from a single adult cell without performing processes which occur after the fertilization of an oocyte by a sperm in a generative process. A cloned embryo produced using the technique is transferred into an estrus-synchronized surrogate mother to create a new individual.
• Somatic cell nuclear transfer process when an immature oocyte is cultured and grown in a medium supplemented with various hormones and growth factors for 24-72 hours, it is matured to metaphase II of meiosis and is referred to as an in vitro-matured oocyte. An oocyte collected by superovulation with hormones is referred to as an in vivo-matured oocyte.
• the haploid of the mature oocyte produced using this method is removed by a micromanipulator, and the somatic cell of an animal to be cloned is injected into the perivitelline space or cytoplasm of the enucleated oocyte. Then, the somatic cell injected into the perivitelline space or cytoplasm is physically fused with the enucleated oocyte by electrical stimulation. The fused oocyte is activated either by electrical stimulation or a chemical substance. The cloned embryo thus produced is transferred into the oviduct or uterus of a surrogate mother by a surgical or non-surgical procedure to allow living offspring to be born. Since Dr.
• the reproductive physiology of canines differs from that of other mammals, because ovulation in canines does not occur in a matured state and a maturation process occurs in the oviduct after ovulation. For this reason, in canines, there is a disadvantage in that an oocyte produced by in vitro maturation cannot be used. Accordingly, an oocyte ovulated and matured in vivo can be used in somatic cell nuclear transfer.
• the present inventors have conducted studies on canine cloning and, as a result, have found that the optimal time of ovulation can be determined through the pattern of an increase or decrease in serum progesterone concentration, and based on this finding, have found that the optimal time for collection of good quality oocytes differs between individuals.
• the present inventors have found that, when electrical fusion is performed at a voltage level lower than a voltage level known as an optimal condition prior to the present invention, a higher fusion rate is shown. Accordingly, the present inventors have found conditions for activation of nuclear transfer embryos and a more effective method for producing cloned canines, and based on these findings, have produced healthy cloned dogs, thereby completing the present invention.
• the present inventors have found that the optimal time of ovulation can be determined through the pattern of an increase or decrease in serum progesterone concentration, and based on this finding, have found that the optimal time for collection of good quality oocytes differs between individuals.
• the present inventors have found that the present inventors have found conditions for activation of nuclear transfer embryos and a more effective method for producing cloned canines, and based on these findings, have produced healthy cloned dogs, thereby completing the present invention.
• the present invention provides a method of producing a canine nuclear transfer embryo using somatic cell nuclear transfer technology in optimal conditions and a canine nuclear transfer embryo produced thereby.
• the present invention also provides a method for producing a cloned canine, which comprises a step of transferring a nuclear transfer embryo, produced by said method, into a surrogate method to allow living offspring to be born, and a cloned canine produced thereby.
• nuclear transfer refers to a gene manipulation technique that yields identical characteristics and qualities by artificially combining an enucleated cell with the nuclear DNA of another cell.
• nuclear transfer embryo refers to an embryo injected or fused with a nuclear donor cell.
• cloned refers to a gene manipulation technique for preparing a new individual unit having a gene set identical to that of another individual unit.
• the term “cloned” is used herein to mean that a cell, an embryonic cell, a fetal cell, and/or an animal cell has a nuclear DNA sequence substantially similar or identical to the nuclear DNA sequence of another cell.
• nuclear donor cell refers to a cell or a nucleus from a cell which transfers the nucleus into a recipient oocyte functioning as a nuclear acceptor.
• recipient oocyte refers to an oocyte which receives a nucleus from a nuclear donor cell after an original nucleus has been removed through enucleation.
• mature oocyte refers to an oocyte which has reached the metaphase II of meiosis.
• nucleated oocyte refers to an oocyte which its nucleus has been removed.
• fusion refers to a combination between a nuclear donor cell and the lipid membrane of a recipient oocyte.
• the lipid membrane may be the plasma membrane or nuclear membrane of a cell. Fusion may occur upon application of an electrical stimulus between a nuclear donor and a recipient oocyte when they are placed adjacent to each other or when a nuclear donor is placed in the perivitelline space of a recipient oocyte .
• activation refers to stimulating a cell to divide, before, during or after the nuclear transfer step.
• it means stimulating a cell to divide after the nuclear transfer step.
• living offspring refers to an animal which can survive ex utero. Preferably, it means an animal which can survive for one second, one minute, one day, one week, one month, six months, or more than one year. The animal may not require an in utero environment for survival.
• canines as used herein is meant to include dogs, wolves, foxes, jackals, coyotes, Korean wolves and raccoon dogs. Preferably, they include dogs or wolves.
• the dogs are known to result from the domestication of wild wolves, and thus, they have the same chromosome number and show similarity in gestation period and sex hormone changes
• the present invention provides a method for producing a canine nuclear transfer embryo for use in producing a cloned canine by somatic cell nuclear transfer technology using an oocyte collected at the optimal time, wherein the canine nuclear transfer embryo is produced in the optimized conditions for electrical fusion and activation. Also, the present invention provides a nuclear transfer embryo produced by said method, and a method for producing a cloned canine, which comprises a step of transferring said nuclear transfer embryo into a surrogate mother to allow living offspring to be born.
• a method for producing a canine nuclear transfer embryo comprising the steps of: (a) enucleating the mature oocyte of a canine to prepare an enucleated recipient oocyte; (b) isolating a somatic cell from the tissue of a donor canine to prepare a nuclear donor cell; and (c) microinjecting the nuclear donor cell into the enucleated oocyte and electrically fusing the nuclear donor cell with the enucleated oocyte at a voltage of 1.5-2.5 kV/cm.
• the method of the present invention may further comprise a step of activating the oocyte fused in step (c) .
• the mature oocyte of the canine in step (a) is preferably collected from the living body of the canine, and the collection of the mature oocyte from the canine is preferably carried out at 70-90 hours after ovulation in the canine.
• the somatic cell in step of the inventive method may be any one selected from the group consisting of canine cumulus cells, epithelial cells, fibroblasts, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, erythrocytes, macropharges, monocytes, muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, fetal cells, placental cells and embryonic cells.
• the somatic cell may be a fibroblast or a cumulus cell.
• the electrical fusion in step (c) may be carried out 1-3 times at a direct current voltage of 1.5-2.5 kV/cm for 30-60 ⁇ s .
• the activation of the fused oocyte may be carried out by an electrical method. Specifically, the activation of the fused oocyte may be carried out 1-3 times at a direct current voltage of 1.5-2.5 kV/cm for 30-60 ⁇ s, which are the same conditions as those of the electrical fusion of the oocyte.
• the activation of the fused oocyte may also be carried out using a chemical method by treating the fused oocyte with 6-dimethyl aminopurine (DMAP) for 2-4 hours.
• DMAP 6-dimethyl aminopurine
• the fused oocyte is treated with 6-dimethyl aminopurine (DMAP) for 2 hours.
• the canine is preferably selected from the group consisting of dogs, wolves, foxes, jackals, coyotes, Korean wolves and raccoon dogs.
• the canine may be a dog or a wolf.
• Step 1 Enucleation of recipient oocyte
• immature oocytes become mature oocytes while staying in the oviduct for several times after ovulation, unlike the case of other mammals.
• the oocytes of mammals are ovulated in mature oocytes, i.e., metaphase II stage of meiosis, whereas canine oocytes are ovulated at prophase I stage of meiosis and matured while staying in the oviduct for 48-120 hours.
• canine oocytes matured in vivo are preferably collected for use as recipient oocytes.
• Methods for determining the proper time of ovulation in canines to collect mature oocytes include, for example, but are not limited to, a method of regarding the time point, at which keratinized epithelial cells reach more than 70% as determined by a vaginal smear test, as the optimum time for oocyte collection, a method of examining the growth and development of oocytes in real time by ultrasonographic imaging, and a method of determining the optimal time for oocyte collection by measuring plasma progesterone concentration.
• the degree of maturation of oocytes is preferably determined by measuring the blood progesterone concentration of a donor dog providing recipient oocytes.
• the day on which plasma progesterone concentration reaches 4.0-7.5 ng/mL is regarded as the day of ovulation, and then oocytes are collected.
• a surgical method including anesthetizing an animal to be cloned, followed by laparotomy can be used.
• the collection of oocytes matured in vivo can be performed using salpingectomy by any method known in the art.
• the salpingectomy is a method of collecting the oocytes by surgically excising the oviduct, flushing an oocyte collection medium into the oviduct to obtain a flushing solution and collecting the oocytes from the flushing solution.
• oocytes matured in vivo can be collected by inserting a catheter into the fimbriated end of the oviduct, and injecting a flushing solution into the uterotubal junction using a needle indwelling catheter. This method has an advantage in that it does not cause damage to the oviduct, and thus allows an oocyte donor animal to be used for the next estrus.
• the collection of oocytes matured in vivo is preferably preformed using the method including the use of the catheter that does not damage the oviduct. More preferably, in the method of collecting oocytes using the catheter, oocytes may also be collected using a 16-guage Sonde (needle) developed by the present inventors, which has a rounded front end such that it is easily inserted into the entrance of the oviduct.
• a 16-guage Sonde needle
• the method of collecting oocytes using the needle developed by the present inventors comprises inserting and ligating the oocyte retrieval needle having a rounded front end into the oviduct, followed by flushing an oocyte collection medium into the uterotubal junction so as to allow the flushing solution to flow into the oocyte retrieval needle, and observing the flushing solution with a microscope so as to select mature oocytes .
• the haploid nuclei of the oocytes are removed.
• the enucleation of the oocytes can be performed by any method known in the art (US Patent No. 4994384, US Patent No. 5057420, US Patent No. 5945577, EP Publication No. 0930009 Al, Korean Patent , Kanda et al, J. Vet. Med. Sci., 57 (4) : 641-646, 1995; Willadsen, Nature, 320:63-65, 1986, Nagashima et al., MoI. Reprod. Dev. 48:339-343 1997; Nagashima et al . , J.
• the enucleation of recipient oocytes can be performed by either of the following two methods.
• One method comprises removing the cumulus cells of mature recipient oocytes, incising a portion of the zona pellucida of the recipient oocytes using a microneedle to give a slit, and removing the first polar body and the oocyte nucleus and cytoplasm (the smallest possible amount) through the slit.
• Another method comprises removing the cumulus cells of recipient oocytes, staining the oocytes, and removing the first polar body and nucleus of the oocytes using an aspiration pipette.
• a slit is formed in oocytes, which are then enucleated by a squeezing method.
• the nucleation is performed by holding recipient oocytes with a holding micropipette, and then removing the first polar body and the oocyte nucleus and cytoplasm.
• a more specific method is as follows. For example, excised oocytes are rotated such that the split is vertically located. Then, a holding pipette is located under the oocytes, such that the oocytes are held by the pipette so as not to move downward. Then, the oocytes are pressed down with an incision pipette to remove 10-30% of cytoplasm including the first polar body.
• the slit of oocytes is rotated vertically from the 3 o'clock direction, and then the oocytes are pressed up and down with a helding pipette and an incision pipette to remove 10-30% of cytoplasm including the first polar body.
• the enucleated oocytes are washed three times with TCM-W and incubated in TCM 199 (B-(D) for IVM before nuclear transfer. Because the enucleated oocytes are very weak, a mouse pipette having an inner diameter of more than 300 ⁇ m must be used such that the oocyte cytoplasm does not come off through the slit after an operation.
• Step 2 Preparation of nuclear donor cells
• somatic cells derived from canines can be used.
• somatic cells used in the present invention may be canine embryonic cells, fetal cells, juvenile cells, or adult cells, and preferably, cells originated from the tissue such as cumulus, skin, oral mucosa, blood, bone marrow, liver, lungs, kidneys, muscles and reproductive tract etc. that can be obtained from the adult cells.
• somatic cells which can be used in the present invention include, but are not limited to, cumulus cells, epithelial cells, fibroblasts, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, erythrocytes, macropharge, monocytes, muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, fetal cells, placental cells and embryonic cells.
• somatic cells which can be used in the present invention may include fetal and adult fibroblasts, and cumulus cells.
• the nuclear donor cells used in the present invention may be those obtained by transforming wild-type somatic cells with certain genes by a gene transfer method or a gene targeting method.
• the gene transfer or gene targeting method can be easily practiced by any person skilled in the art, because it is known in the art.
• the somatic cells which are provided as the nuclear donor cells can be obtained by a method of preparing surgical samples or biopsy samples, and from the samples, single cells can be obtained by any method known in the art. For example, some of tissue from an animal to be cloned is aseptically incised to obtain a surgical sample or a biopsy sample, and the sample is minced, treated with trypsin, and then cultured in a tissue culture medium. After culturing for 3-4 days in the tissue culture medium, the growth of the cells on a culture dish is confirmed. When the cells completely grow, some of the tissue is frozen and stored in liquid nitrogen for later use, and the remnants are subcultured for use in nuclear transfer.
• the cells to be continuously cultured for use in nuclear transfer are subcultured up to 10 times so as to prevent the cells from growing excessively.
• the tissue culture medium used as described above may be one known in the art, and examples thereof include TCM-199, and DMEM (Dulbecco's modified Eagle's medium).
• Step 3 Microinjection and fusion/activation of nuclear donor cells
• the microinjection of nuclear donor cells into enucleated oocytes can be performed by microinjecting the nuclear donor cells between the cytoplasm and zona pellucida of the enucleated oocytes using a transfer pipette.
• the enucleated oocytes microinjected with nuclear donor cells are electrically fused with nuclear donor cells using a cell manipulator.
• the electrical fusion can be performed with direct or alternating current. Preferably it can be performed at a voltage of 1.5-4.0 kV/cm.
• the electrical fusion can be performed 1-3 times at a direct current voltage of 1.5-2.5 kV/cm for 30-60 ⁇ s. Most preferably, it can be performed once for 30 ⁇ s . If the voltage in the electrical fusion is less than 2.5 kV/cm or more than 2.5 kV/cm, the fusion rate between the oocytes and the nuclear donor cells will be very low. In a prior art method for producing cloned canines, it is known that it is most preferable to perform electrical fusion twice with direct current for 15 ⁇ s, but in the present invention, it was found that, when electrical fusion was performed once with direct current for 30 ⁇ s, a high fusion rate was shown.
• the fusion of nuclear donor cells with oocytes by electrical stimulation can be performed in a fusion medium.
• the fusion medium used in the present invention may be a medium containing mannitol, MgSO, Hepes and BSA.
• the fusion can be performed in a medium containing 0.3-0.5 M mannitol, 0.1-0.3 mM MgSO 4 , 0.1-0.3 mM Hepes and 0.05-0.1 % (w/v) BSA.
• Step 4 Activation of fused nuclear transfer embryos
• Activation of the fused nuclear transfer embryos is a step of reactivating the cell cycle temporarily arrested in metaphase II of meiosis in in vitro maturation.
• the activity of materials which arrest the cell cycle such as MPF, CSF etc., has to be reduced, and the activation of APC which promotes the metaphase II-to-anaphase II transition has to be increased.
• intracellular Ca 2+ ion concentration must be increased to induce chromosomal condensation and embryonic development.
• a physical method there is a method that uses a mechanical stimulus, heat, and direct current.
• the chemical method there is a method of treating unclear transfer embryos with a material such as ethanol, inositol trisphosphate, Ca 2+ or Sr 2+ , cytochalasin B, calcium ionophore, 6-dimethylaminopurine, cycloheximide, or phorbol 12-myristate 13-acetate.
• a method of treating the fused nuclear transfer embryos with 6-dimethylaminopurine (DMAP) for 2-4 hours can be used in the present invention. More preferably, the nuclear transfer embryos are treated with 5- 10 ⁇ M calcium ionophore at 37-39 °C for 3-6 minutes and then with 1.5 mM-2.5 mM 6-dimethylaminopurine at 37-39 °C for 4-5 hours .
• DMAP 6-dimethylaminopurine
• the nuclear transfer embryos were observed for the change in chromatin in a medium containing 6-dimethylamino ⁇ urine.
• condensed chromatin which is an index of good-quality nuclear transfer embryos was higher in the group treated for 2 hours than in the group treated for 4 hours.
• the electrical activation can be performed 1-3 times at a direct current voltage of 1.5-2.5kV/cm for 30- 60 ⁇ s . Preferably, it is performed once for 30 ⁇ s . Therefore, according to a second aspect of the present invention, there is provided a nuclear transfer embryo produced by the above-described method.
• the present inventors deposited a canine nuclear transfer embryo, produced in one example of the present invention, under the accession number KCTC 11331BP on May 6, 2008 with Korean Collection for Type Cultures (KCTC) , Korean Research Institute of Bioscience and Biotechnology (52, Oun-dong, Yusong-gu, Daej eon, Korea) .
• the nuclear transfer embryos are freeze-stored and can be used after dissolution, if needed.
• a method for producing a cloned canine the method comprising a step of transferring the nuclear transfer embryo into the oviduct of a surrogate mother to allow living offspring to be born.
• the canine may be selected from the group consisting of dogs, wolves, foxes, jackals, coyotes, Korean wolves and raccoon dogs.
• the inventive method for producing a cloned canine comprises the following steps.
• a cloned canine produced by the above- described method.
• the present invention provides a method of producing cloned dogs based on determination of the optimal time of ovulation in oocyte donor dogs and on optimization of the conditions for oocyte fusion and activation and provides a method of efficiently producing canines as companion animals using the same.
• the present invention provides a method of producing cloned dogs based on determination of the optimal time of ovulation in oocyte donor dogs and on optimization of the conditions for oocyte fusion and activation and provides a method of efficiently producing canines as companion animals using the same.
• the present invention is highly applicable for the production of drugs or organs using cloned canines in the medical, pharmacological and veterinary fields.
• the technology for producing cloned canines according to the present invention is not limited only to the production of canines, and it can be modified by a broad range of attempts in the future and can also be applied to other species of animals .
• FIG. 1 shows the change in chromatin according to culture time after nuclear fusion of cloned embryos produced according to the present invention.
• A-C chromatin observed when cultured for 2 hours after nuclear fusion
• A-B condensed chromatin
• C metaphase-type chromatin
• D-F chromatin observed when cultured for 4 hours after nuclear fusion.
• FIG. 2 is a set of photographs showing the 25-week-old cloned dog "Soodog” (a) , the 22-week-old cloned dog “Ari” (b) and the 19-week-old cloned dog "Joomong", which were produced according to the method of the present invention.
• Example 1 Collection of recipient oocytes from dogs Dogs used for the collection of recipient oocytes were
• the maturation time of ovulated oocytes is known to be 48-72 hours after ovulation.
• the present inventors retrieved oocytes at 70-90 hours after ovulation in the following manner.
• female dogs which had reached the retrieval time of oocytes matured in vivo were administered with Enrofloxacin, Cefazolin and 0.05 mg/kg of atropin sulfate and systemically anesthetized by administering 0.025 mg/kg of acepromazine maleate and 5 mg/kg of ketamine.
• the anesthesia was maintained by administering isoflurane during a surgical operation.
• the anesthetized female dogs were subjected to an aseptic surgical operation to incise the middle abdominal region by 5-10 cm, thus exposing the oviduct. Then, a specifically manufactured Sonde (16-guage needle) was inserted into the abdominal cavity of the oviduct and held in place with suture. Then, a 24-gauge IV catheter was inserted into the uterotubal junction, and an oocyte collection medium
• Example 2 Enucleation of recipient oocytes 0.1% (v/v) hyaluronidase (Sigma, USA) was added to a TCM-199 medium (Table 2) containing Hepes-buffer . Then, the oocytes obtained in Example 1 were added to the medium and pipetted repeatedly to remove cumulus cells from the oocytes. Then, the oocytes were stained with 5 ⁇ g/mL of bisbenzimide (Hoechst 33342) for 15 minutes and observed under an inverted fluorescence microscope at 20Ox magnification so as to select only oocytes with the first polar body.
• TCM-199 medium 10% (v/v) FBS and 5 ⁇ g /ml cytochalasin B were added to a TCM-199 medium (Table 2), and the selected oocytes were enucleated in the medium using a micromanipulator (TE2000-E; Nikon Corporation, Tokyo, Japan) . Namely, the oocytes were held with a holding micropipette (150 ⁇ g inner diameter), and then the first polar body, adjacent cytoplasm (less than 5%) and oocyte nuclei were removed using an aspiration pipette. The enucleated oocytes were stored in a TCM-199 medium (Table 2) supplemented with 10% (v/v) FBS. [Table 2] Composition of TCM-199 medium
• Example 3 Preparation of nuclear donor cells
• nuclear donor cells adult fibroblasts collected from dogs were used. For this purpose, an abdominal skin tissue from 5-year-old Golden Retrievers was first isolated. The abdominal skin tissue was washed three times with DPBS (Dulbecco's Phosphate Buffered Saline) and minced with a surgical blade. The minced tissue was added to a medium obtained by mixing a 20% (v/v) FBS-containing Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Rockville, MD) and 4 mg/ml of filtered collagenase (Type II, Life Technologies) at a ratio of 6:1 and was treated in a 5% CO 2 saturated humidity incubator at 38 °C for 24 hours.
• DMEM Dulbecco's modified Eagle's medium
• the cells degraded by collagenase were washed twice with Ca 2+ - and Mg 2+ - free DPBS and once with 10% (v/v) FBS-containing DMEM by centrifugation at 300 x g for 3 minutes for each washing, and then seeded into 100-mm plastic culture dishes.
• the seeded cells were subsequently cultured for 6-8 days in DMEM supplemented with 10% (v/v) FBS, 1 mM mercaptoethanol, 1 ⁇ iM glutamine, 25 mM NaHCO 3 and 1% (v/v) minimal essential medium
• Example 4 Microinjection and fusion of nuclear donor cells into enucleated oocytes
• the nuclear donor cells prepared in Example 3 were microinjected into the enucleated oocytes prepared in Example 2. After an incision pipette on the micromanipulator of Example 2 was replaced with a transfer pipette, the fixed oocytes were treated with a TCM-199 medium (Table 2) containing Hepes-buffer . The slits of the enucleated oocytes were held with a holding pipette, and then inserted with a transfer pipette. Then, the fibroblasts isolated as single cells in Example 3 were injected between the cytoplasm and zona pellucida of the enucleated oocytes by the transfer pipette .
• the oocytes injected with the nuclear donor cells as described above were placed in a fusion medium (containing 0.3 M mannitol, 0.1 mM MgSO 4 , 0.5 mM Hepes and 0.05% BSA), and transferred into a cell fusion chamber equipped with a stainless steel wire electrode (BTX 453, 3.2 mm gap; BTX, San Diego, CA) . After equilibration for 3 minutes, the couplets were applied with direct current in a voltage of 1.5-2.5 kV/cM for 30-60 ⁇ s using a BTX Electro-cell Manipulator, thus fusing the donor cells to the oocytes. The fused nuclear transfer embryos were observed with a stereoscopic microscope to select 1095 fused nuclear transfer embryos. The selected embryos were cultured for 2-4 hours in the modified synthetic oviductal fluid (mSOF) shown in Table 3.
• mSOF modified synthetic oviductal fluid
• the nuclear transfer embryos obtained in Example 4 were activated by an electrical or chemical method, and the degree of growth of the nuclear transfer embryos was observed.
• the nuclear transfer embryos of Example 4 was added to 100 nM calcium (CaCl 2 ) -containing mannitol medium (0.26 M mannitol, 0.1 mM MgSO 4 , 0.5 mM Hepes and 0.05% BSA) and transferred into a cell fusion chamber equipped with a stainless steel electrode (BTX 453, 3.2mm gap; BTX, San Diego, CA) . After equilibration for 3 minutes, the couplets were applied with direct current in a voltage of 1.5-2.5 kV/cM for 30-60 ⁇ s using a BTX Electro-cell
• the nuclear transfer embryos obtained in Example 4 were cultured in mSOF containing 10 ⁇ M ionophore for 4 minutes at 39 " C. The embryos were then washed and additionally incubated for 4 hours in mSOF (Table 3) supplemented with 1.9 mM of 6- dimethylaminopurine. After completion of the culture, the nuclear transfer embryos were transferred into a TCM199 medium (Table 2), and the degree of growth thereof was observed with a stereoscopic microscope at 10Ox magnification.
• the activated nuclear transfer embryos were cultured in mSOF medium (Table 3) in a 5% CO 2 and 5% O 2 incubator at 38- 39 °C for 2-4 hours and observed for the change in chromatin using a confocal microscope (Eclipse Clsi, Nikon Corporation, Tokyo, Japan) .
• a confocal microscope Eclipse Clsi, Nikon Corporation, Tokyo, Japan
• condensed chromatin and metaphase-type chromatin were larger in the group cultured for 2 hours than in the group cultured for 4 hours.
• the condition in which condensed chromatin and metaphase-type chromatin are larger is the optimal activation condition.
• treating the nuclear transfer embryos in mSOF (Table 3) containing 6-dimethylaminopurine for 2 hours after nuclear fusion was the optimal condition in which chromatin formation could be inhibited and good-quality nuclear transfer embryos could be obtained.
• the present inventors deposited one of the above- produced nuclear transfer embryos under the accession number KCTC 11331BP on May 6, 2008 with Korean Collection for Type Cultures (KCTC) , Korean Research Institute of Bioscience and Biotechnology (52, Oun-dong, Yusong-gu, Daejeon, Korea) .
• KCTC Korean Collection for Type Cultures
• the nuclear transfer embryos from Example 5 were surgically transferred into the oviduct of surrogate mothers. The transfer was conducted depending on the preparation state of surrogate mothers after the activation of the nuclear transfer embryos. Namely, when the surrogate mothers were immediately prepared, the transfer of the nuclear transfer embryos was immediately conducted. As the surrogate mothers, mixed breed dogs were used. The selected dogs were disease-free, showed the repetition of the normal estrus cycle and had a normal uterine condition. For this purpose, the surrogate mothers were anesthetized by vascular injection with 0.1 mg/kg acepromazine and 6 mg/kg propofol, and maintained at the anesthetized state using 2% isoflurane.
• the operation area of the anesthetized female dogs was aseptically treated and incised on the center of the abdomen by 5-10 cm according to general laparotomy so as to expose the oviduct.
• an ovarian hook was used to draw the ovarium, the oviduct and the uterus to the incision slit.
• the mesovarium of the drawn ovarium was carefully handled to recognize the opening of the oviduct, and a 3.5 F Tom cat catheter (Sherwood, St. Louis, MO) equipped with a 1.0 ml tuberculin syringe (Latex free, Becton Dickinson & CO. Franklin lakes, NJ 07417) was inserted into the oviduct to secure a sufficient space in the front of the catheter. Then, the nuclear transfer embryos were injected into the oviduct through the catheter.
• a cloned dog was spontaneously delivered on April 14, 2007, 60 days after the transfer of the nuclear transfer embryos, and was named "Ari”, and a cloned dog was delivered by caesarean section on May 4, 2007, 60 days after the transfer of the nuclear transfer embryos, and was named "Joomong”.
• Test Example 1 Examination of genetic identity of cloned dogs
• genomic DNA was isolated from the somatic cell of each of the cloned dogs, the donor dogs and the surrogate mothers, and then the genetic information of the isolated somatic cell was compared between the cloned dogs, the donor dogs and the surrogate mothers.
• skin tissue fragments were collected from the cloned dogs and the surrogate mothers.
• the somatic cells of the donor dogs were subcultured and used in analysis. Each of the samples was stored in a freezer at -80 °C , and phenol extraction and ethanol precipitation were conducted to isolate genomic DNA from each of the samples.
• the isolated genomic DNA samples were dissolved in 50 ul TE and subjected to microsatellite analysis using canine- specific markers ⁇ PEZ1, FHC 2054, FHC 2010, PEZ5, PEZ20, PEZ12, PEZ3, PEZ6, PEZ8, and FHC 2079> (Ref: Canine STR analyses in forensic practice: Observation of a possible mutation in a dog hair. Int J Legal Med (2002) 116:286-288) .
• the isolated genomic DNA as a template was amplified by PCR using fluorescence-labeled specific primers prepared based on the sequences of the markers located on the genomic DNA. The amplification products were analyzed with an automated DNA sequence analyzer (ABI 373: Applied Biosystems, Foster City, CA) .
• the PCR reaction was performed in the following conditions: predenaturation at 94 °C for 1 min, and then 30 cycles, each consisting of denaturation at 94 °C for 20 sec, annealing at 58 °C for 20 sec and extension at 74 "C for 20 sec by 30 cycles, followed by post-extension at 74 °C for 5 min.
• somatic cells of the cloned dog "Soodog” produced according to the present invention were genetically completely identical to the somatic cells isolated from the donor dog Golden Retrievers.
• the cloned dog "Soodog” was genetically different from the surrogate mothers (mixed breed dogs) (Table 6) .
• Test Example 2 Determination of optimal time for collection of canine oocytes
• the day on which plasma progesterone concentration reached 3.5- 4.5ng/mL was regarded as the day of ovulation, and the number of good-quality oocytes collected with the passage of time was counted.
• Test Example 3 Optimization of conditions for electrical fusion of nuclear donor cells to enucleated oocytes
• the nuclear donor cells were microinjected into the enucleated oocytes in the same manner as in Example 4. Then, the fused nuclear transfer embryos were observed with a stereomicroscope to check whether the fusion occurred.
• the oocytes injected with the nuclear donor cells were placed in a cell fusion chamber (0.3 M mannitol, 0.1 mM MgSO4, 0.5 mM Hepes and 0.05% BSA) and transferred into a cell fusion chamber equipped with a stainless steel electrode.
• the oocytes were applied with direct current at each of voltages of 0.5-1.5 kV/cM, 1.5-2.5 kV/cM and 2.5-3.5 kV/cM for 30-60 ⁇ s using an electro-cell manipulator, thus fusing the oocytes to the enucleated cells.
• the fused nuclear transfer embryos were observed with a stereomicroscope to select 187 nuclear transfer embryos.
• the selected nuclear transfer embryos were cultured in the mSOF
• Test Example 4 Optimization of conditions for activation of canine nuclear transfer embryos
• the nuclear transfer embryos obtained in Example 4 were cultured in mSOF containing 10 ⁇ M ionophore at 39 °C for 4 minutes, and then additionally cultured in mSOF (Table 4) containing 1.9 mM 6-dimethylaminopurine for 2-4 hours. After completion of the culture, the embryos were transferred into a TCM199 medium (Table 3), and the degree of growth thereof was observed with a stereomicroscope at 10Ox magnification.
• the activated nuclear transfer embryos were cultured in mSOF medium (Table 3) in a 5% CO 2 and 5% O 2 incubator at 38- 39 °C for 2-4 hours and observed for the change in chromatin using a confocal microscope (Eclipse Clsi, Nikon Corporation, Tokyo, Japan) .
• a confocal microscope Eclipse Clsi, Nikon Corporation, Tokyo, Japan
• condensed chromatin and metaphase-type chromatin were larger in the group cultured for 2 hours than in the group cultured for 4 hours.
• the condition in which condensed chromatin and metaphase-type chromatin are larger is the optimal activation condition.
• treating the nuclear transfer embryos in mSOF (Table 3) containing 6-dimethylaminopurine for 2 hours after nuclear fusion was the optimal condition in which chromatin formation could be inhibited and good-quality nuclear transfer embryos could be obtained.
• Test Example 5 Optimization of conditions for transfer of canine nuclear transfer embryos into surrogate mothers
• the surrogate mothers were anesthetized by vascular injection with 0.1 mg/kg acepromazine and 6 mg/kg propofol, and maintained at the anesthetized state using 2% isoflurane.
• the operation area of the anesthetized female dogs was aseptically treated and incised on the center of the abdomen by 5-10 cm according to general laparotomy so as to expose the oviduct.
• an ovarian hook was used to draw the ovarium, the oviduct and the uterus to the incision slit.
• the mesovarium of the drawn ovarium was carefully handled to recognize the opening of the oviduct, and a 3.5 F Tom cat catheter (Sherwood, St. Louis, MO) equipped with a 1.0 ml tuberculin syringe (Latex free, Becton Dickinson & CO. Franklin lakes, NJ 07417) was inserted into the oviduct to secure a sufficient space in the front of the catheter. Then, the nuclear transfer embryos were injected into the oviduct through the catheter.
• the present invention provides a method of producing cloned dogs based on determination of the optimal time of ovulation in oocyte donor dogs and on optimization of the conditions for oocyte fusion and activation and provides a method of efficiently producing canines as companion animals using the same.
• the present invention is highly applicable for the production of drugs or organs using cloned canines in the medical, pharmacological and veterinary fields.
• the technology for producing cloned canines according to the present invention is not limited only to the production of canines, and it can be modified by a broad range of attempts in the future and can also be applied to other species of animals .

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