[go: up one dir, main page]

WO2001000794A1 - Procede de production de tigres clones utilisant une technique de transplantation de noyau inter-especes - Google Patents

Procede de production de tigres clones utilisant une technique de transplantation de noyau inter-especes Download PDF

Info

Publication number
WO2001000794A1
WO2001000794A1 PCT/KR2000/000706 KR0000706W WO0100794A1 WO 2001000794 A1 WO2001000794 A1 WO 2001000794A1 KR 0000706 W KR0000706 W KR 0000706W WO 0100794 A1 WO0100794 A1 WO 0100794A1
Authority
WO
WIPO (PCT)
Prior art keywords
oocytes
embryos
oocyte
tigers
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2000/000706
Other languages
English (en)
Inventor
Byeong-Chun Lee
Tae-Young Shin
Sang-Ho Roh
Jeong-Muk Lim
Jong-Im Park
Jong-Ki Cho
Ki-Yon Kim
Eun-Song Lee
Soo-Jung Shin
Sung-Ki Kim
Hwan-Yul Yong
Gook-Jun Ahn
Sang-Hwan Hyun
Kil-Young Song
Byung-Dong Lee
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020000004382A external-priority patent/KR20010076941A/ko
Priority claimed from KR1020000004381A external-priority patent/KR20010069167A/ko
Application filed by Individual filed Critical Individual
Priority to EP00941006A priority Critical patent/EP1117763A4/fr
Priority to CA002334954A priority patent/CA2334954A1/fr
Priority to NZ508739A priority patent/NZ508739A/xx
Priority to AU55777/00A priority patent/AU753209B2/en
Priority to JP2001506788A priority patent/JP2003503045A/ja
Publication of WO2001000794A1 publication Critical patent/WO2001000794A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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

Definitions

  • the present invention relates to a method for producing cloned tigers by employing inter-species nuclear transplantation technique, more specifically, a method for producing cloned tigers by employing inter-species nuclear transplantation technique by which nuclei of somatic cells derived from the tissues of tiger are transferred into oocytes originated from cow or cat. It also relates to cloned tiger embryos and cloned tigers produced by the method described above.
  • the first cloned sheep was produced by introducing nuclei from somatic cells (s_e_e.: Wilmut et al., Nature, 385:810-813, 1997), thus making correction in the prior developmental theory and enabling many successful examples to be reported in production of cloned cows (.s_e_e_: Wells et al, Reprod. Fertil. and Develop., 10:369-378, 1998) and pigs.
  • cloned tiger embryos and cloned tigers developed from the said embryos can be produced by inter-species nuclear transplantation technique involving fusion of the oocytes from cow or cat with somatic cells of tiger .
  • a primary object of the present invention is, therefore, to provide a method for producing cloned tigers by inter-species nuclear transplantation technique.
  • the other object of the invention is to provide cloned tiger embryos produced by the said method. Another object is to provide somatic cell-derived cloned tigers produced by the said method.
  • Figure 1 is a photograph of donor somatic cells.
  • Figure 2 is a photograph showing the process of cutting zona pellucida of a recipient oocyte with a holding pipette and cutting pipette.
  • Figure 3 is a photograph showing the process of enucleation by removing the first polar body and nucleus from a recipient oocyte .
  • Figure 4 is a photograph showing the process of transferring a somatic cell into an enucleated oocyte with a holding pipette and injection pipette.
  • the method for producing cloned tigers of present invention comprises the steps of: preparing donor somatic cell lines collected from tiger; maturing oocytes collected from ovary of cow or cat in vitro; removing cumulus cells surrounding the oocytes, cutting a portion of zona pellucida of the matured oocytes and squeezing out a portion of cytoplasm including the first polar body to give enucleated recipient oocytes; transferring a nucleus to the recipient oocyte by injection of the donor cells to the enucleated oocytes, followed by the subsequent electrofusion and activation of the electrofused cells to give embryos; postactivating and culturing the embryos in vitro; and, transferring the cultured embryos into surrogate mothers to produce cloned tigers.
  • the method for producing cloned tigers of the invention is further illustrated as follows.
  • Step 1 Preparation of donor cells
  • Somatic cell lines collected from tiger are prepared as donor cells: although cells collected from a tiger are not limited for donor cells, preferable cell lines include cells collected from uterine flushing fluid, endometrium, oviduct, ear or muscle, cumulus cells or fetal fibroblasts, which are prepared by employing the conventionally known method (.s_e_e.: Mather & Barnes, Methods in Cell Biology, Vol.57, Animal Cell Culture Methods, Academic Press, 1998) with some modifications.
  • cells are collected by the addition of
  • PBS phosphate buffered saline
  • penicillin- streptomycin Gabco; lOOOOU/ml penicillin, lOmg/ml streptomycin
  • DMEM Dulbecco' s modified Eagle's medium
  • FBS fetal bovine serum
  • penicillin-streptomycin under an environment of 39 ° C, 5% C0 2 .
  • Uterine epithelial cells collected from endometrium or oviduct are washed with the said PBS, trypsinized, and cultured under the same conditions as described above.
  • cumulus-oocyte complexes are treated with hyaluronidase solution to isolate cumulus cells surrounding oocytes.
  • the cumulus cells are trypsinized for 30 to 60 minutes under an environment of
  • ear fibroblasts and fetal fibroblasts are obtained from the inner side of skin lined along with cartilage tissue and from tissue collected from trunk and limbs of fetus, respectively, by washing and mincing the tissues aseptically, followed by treatment of trypsin and collagenase type II under an environment of 39 ° C, 5% C0 2 . These cells are also cultured analogously as in the somatic donor cell lines described above.
  • the somatic cell lines are stored by subculture, serum starvation culture or freezing.
  • the subculture of donor cell lines is carried out at regular intervals by changing the old medium to new one after trypsinization.
  • the serum starvation culture is performed by employing DMEM supplemented with 0.5% FBS and the method of Wilmut et al.( ⁇ _e_: Wilmut et al . , Nature, 385:810-813, 1997).
  • the cell lines thus stored are used for later step as donor cells.
  • Step 2 Preparation of recipient oocytes
  • Immature oocytes collected from ovary of cow or cat are matured in vitro: immature oocytes are selected from ovary in TCM199 washing medium containing 10 mM HEPES (N-
  • TCM199-1 culture medium (see : Table 1) is employed.
  • TCM199-2 culture medium (s_e_e_: Table 2) supplemented with human Chorionic Gonatropin (hCG) .
  • hCG human Chorionic Gonatropin
  • Step 3 Enucleation of recipient oocytes
  • a portion of cytoplasm including the first polar body is removed from the oocytes to give enucleated oocytes: first, cumulus cells surrounding the mature oocytes are removed physically with a denuding pipette in TCM199 washing medium containing hyaluronidase . Then, denuded oocytes are washed with TCM199 washing medium and transferred into cytochalasin B solution.
  • a portion of zona pellucida of the denuded oocytes is penetrated by a cutting pipette to give a slit through which 10 to 15 % of cytoplasm including the first polar body can be squeezed out of the oocytes.
  • the enucleated oocytes are washed and incubated in TCM199 culture medium.
  • the said cytochalasin B solution is prepared by diluting cytochalasin B dissolved in DMSO (dimethylsulfoxide) with the TCM199 culture medium.
  • Step 4 Electrofusion of donor cells with recipient oocytes and activation of the electrofused cells
  • the donor cells are transferred to the recipient oocytes, followed by subsequent electrofusion and activation of the electrofused cells: before the injection of donor cells into recipient oocytes, the enucleated oocytes are washed with TCM199 culture medium and transferred to PHA-P (phytohemagglutinin) solution. Then, the donor cells are transferred to the enucleated oocytes by injecting donor cells to the slit made on zona pellucida of the oocytes in PHA-P solution.
  • PHA-P phytohemagglutinin
  • the electrofusion is carried out by employing an Electro Cell Manipulator (BTX ECM2001).
  • BTX ECM2001 Electro Cell Manipulator
  • the reconstructed embryos in mannitol solution supplemented with TCM199 washing solution are placed in a chamber with two electrodes, one on either side.
  • the chamber was filled with mannitol solution.
  • the embryos are electrofused by applying DC pulse of 0.75 to 2.00 kV/cm twice with one second's interval for 15 ⁇ s each time, the electrofused embryos are washed with mannitol solution and TCM199 washing medium, incubated in cytochalasin B solution, and activated.
  • the electrofusion and activation occur in a simultaneous manner provided that the electrofusion is carried out in a mannitol medium containing Ca 2+ . Otherwise, the activation is performed after electrofusion.
  • the activation step is performed by incubating the embryos in ionomycin solution in the dark. Then, ionomycin is removed from the embryos by washing them with TCM199 washing medium containing FBS or BSA. The said ionomycin solution is prepared by diluting ionomycin dissolved in DMSO with TCM199 washing medium containing BSA.
  • Step 5 Postactivation and in vitro culture of embryos
  • the embryos are postactivated and cultured in vitro: the activated embryos incubated in TCM199 washing medium containing FBS or BSA are postactivated by incubating in cycloheximide solution or DAMP (4-dimethylaminopurine) solution, and cultured in vitro under an environment of 5% C0 2 , or a mixture of 5% C0 2 , 7% 0 2 and 88% N 2 .
  • the said cycloheximide solution or DAMP solution is prepared by adding cycloheximide dissolved in ethanol or DAMP to media for in vitro culture, respectively.
  • the media for in vitro culture include mTALP (.s_ae_: Table 3), mSOF (. ⁇ e_e_: Table 4) and mCR2aa (.s_e_e_: Table 5) medium, all of which comprise NaCl, KC1, NaHC0 3 , NaH 2 P0 4 , CaCl 2 , Na-lactate, glucose, phenol red, BSA, kanamycin, essential amino acids, non-essential amino acids and L-glutamine.
  • the embryos cultured in vitro are stored by freezing for later use, and subjected to thawing when they are intended to be used.
  • Step 6 Production of cloned tigers
  • the embryos cultured in vitro are transferred into surrogate mothers to produce tigers: the embryos in PBS containing FBS are implanted to uterus of surrogate mothers.
  • the present inventors produced an embryo, SNU5 (Korean Tiger NT Embryo), by using ear cells of tiger and oocytes of Korean cow as nucleus donors and recipient oocytes, respectively.
  • the embryo was deposited with an international depositary authority, KCTC (Korean Collection for Type Cultures; KRIBB #52, Oun-dong, Yusong-ku, Taejon, 305-333, Republic of Korea) on Mar. 10, 2000, under an accession number of KCTC 0752BP.
  • Example 1 Preparation of donor cells and recipient oocytes
  • tissue was washed with PBS and minced into 100 mesh size. Then, the tissue was incubated in PBS containing 0.25% trypsin, ImM EDTA and lmg/ml collagenase type II for lhr under an environment of 39 ° C, 5% C0 2 . After the tissue was digested with the enzymes, it was centrifuged at 1,500 rpm for 2 minutes, and suspended in DMEM (Dulbecco' s modified Eagle's medium, Gibco BRL, Life Technologies, USA) supplemented with 10% FBS, 1% NEAA (non- essential amino acids) and 1% penicillin-streptomycin.
  • DMEM Dulbecco' s modified Eagle's medium, Gibco BRL, Life Technologies, USA
  • the suspension was transferred to dishes for cell culture and incubated under an environment of 39 ° C, 5% C0 2 to give a somatic cell line. After that, the cells were trypsinized in solution containing 0.25% trypsin and ImM EDTA, and the cell number was adjusted to be 2 X 10 4 cells/ml to aliquot the cells in effendorf-tubes .
  • Figure 1 depicts the somatic cells isolated as single cells for nucleus donor.
  • follicles of which size was about 2 to 6 mm in diameter were aspirated from ovaries of Korean cows with a 10ml syringe having an 18G needle. Then, the follicular fluid were transferred into a 100mm dish with a grid (the length between lines was 1 cm) drawn on its bottom, and oocytes with homogeneous cytoplasm and sufficient number of cumulus cell layers around them were screened. The selected oocytes were washed three times with 2 ml of TCM199 washing medium (s_e_e_: Table 6) in 35mm dishes, and subsequently, once with TCM199-1 culture medium (s_e_£: Table 1) .
  • TCM199 washing medium s_e_e_: Table 6
  • oocytes were cultured in TCM199 culture medium containing 0.1% estradiol solution (s_e_e_: Table 7), 2.5% follicle stimulating hormone solution (s_e_e_: Table 8) and 10% FBS to give recipient oocytes.
  • the recipient oocytes prepared in Example 1 were washed once with TCM199 washing medium and transferred in 0.1% hyaluronidase (Sigma Chemical Co., U.S.A.) solution prepared by mixing 1ml of TCM199 washing medium with lll ⁇ l of hyaluronidase stock solution (lOmg/ml in TCM199 washing medium) . After cumulus cells were removed from the oocytes in the presence of 0.1% hyaluronidase, the denuded oocytes were washed three times and incubated in TCM199 washing medium.
  • 0.1% hyaluronidase Sigma Chemical Co., U.S.A.
  • cytochalasin B Sigma Chemical Co., U.S.A.
  • cytochalasin B Sigma Chemical Co., U.S.A.
  • TCM199 washing medium containing 10% FBS l ⁇ l of cytochalasin stock solution (7.5mg/ml in DMSO)
  • zona pellucida of each oocyte was cut by employing micromanipulator to make a slit through which 10 to 15% of cytoplasm can be squeezed out of the oocyte to give an enucleated oocyte.
  • the enucleation step is more specifically illustrated as following: a working dish was put on the micromanipulator plate, and the micromanipulator was equipped with a holding pipette on its left arm and a cutting pipette on its right arm. Then, the holding pipette and cutting pipette were placed in the direction of 9 o'clock and 3 o'clock, respectively, and adjusted to move freely in all directions by placing a pipette controller in the middle. These two pipettes were further adjusted to let them not touch the working dish and their tips placed to the middle of a microdroplet by moving them up and down over the microdroplet.
  • the oocytes were transferred from TCM199 washing medium to cytochalasin B solution by employing washing mouth pipettes (>200 ⁇ m inner diameter).
  • the micromanipulator was first focused on the oocyte by using its coarse adjustment knob and fine adjustment knob, and the focus was further adjusted by moving the two pipettes up and down.
  • the oocyte was placed with its first polar body oriented toward the direction of 12 o'clock, and the holding pipette was placed close to the oocyte in the direction of 9 o'clock of the oocyte to fix the oocyte by applying hydraulic pressure.
  • Figure 2 shows the process of cutting zona pellucida of the oocyte with the holding pipette and cutting pipette.
  • the oocyte was penetrated by the cutting pipette (2) from the direction of 1 o'clock to the direction of 11 o'clock with special care not to damage the cytoplasm of the oocyte.
  • hydraulic pressure was applied to the holding pipette (1) to separate the oocyte (3), and the holding pipette was contacted with the cutting pipette penetrating the zona pellucida bordering on the upper part of the first polar body to cut the portion of zona pellucida by rubbing the two pipettes.
  • the slit on the oocyte made above was used for both enucleation and donor cell injection.
  • Figure 3 depicts the process of enucleation removing the first polar body and nucleus from the oocyte.
  • the oocyte (3) was placed with its slit oriented vertically, held with the holding pipette (1) on its lower part to prevent it from moving, and squeezed mildly on its upper part with the cutting pipette (2) to give an enucleated oocyte.
  • the enucleated oocyte was washed three times with TCM199 washing medium and incubated in TCM199 culture medium.
  • donor cells prepared in advance were transferred to enucleated oocytes by employing micromanipulator.
  • 4 ⁇ l of injection microdroplet was made on the middle of the working dish by using PHA-P solution prepared by mixing 400 ⁇ l of TCM199 washing solution and lOO ⁇ l of PHA-P (phytohemagglutinin) stock solution (0.5mg/ml in TCM199 washing solution).
  • PHA-P phytohemagglutinin
  • two microdroplets for donor cells were made with one above and the other below the injection microdroplet on the same working dish by using PBS containing 1% FBS. After these microdroplets were spread over with mineral oil, the working dish was placed on the micromaniulator plate.
  • FIG. 4 shows the process of transferring a somatic cell into an enucleated oocyte.
  • the enucleated oocyte was placed with its slit oriented toward the direction of 1 o'clock, fixed by using the holding pipette, ana injected with the donor cell through the slit by employing the injection pipette and hydraulic pressure to give a reconstructed embryo.
  • the embryo was washed three times with and incubated in TCM199 washing medium.
  • the reconstructed embryos were subjected to electrofusion employing an Electrocell Manipulator (ECM 2001, BTX, USA), followed by activation. 15 ⁇ l of mannitol solution containing 0.28M mannitol, 0.5mM HEPES (pH 7.2), O.lmM MgS0 4 and 0.05% BSA was added to TCM199 culture medium containing the reconstructed embryos by employing a mouth pipette for washing. After 1 minute's incubation in the said medium, the embryos were incubated for 1 minute in mannitol solution supplemented with TCM199 washing solution, and finally transferred into mannitol solution by employing the mouth pipette for washing.
  • the chamber (3.2mm chamber No.
  • Electrocell Manipulator was filled with mannitol solution supplemented with TCM199 washing medium, and then the embryos were placed in the chamber with their donor cell part facing the cathode. After the embryos were electrofused by applying DC pulse of 0.75 to 2.00kV/cm twice with one second's interval for 15 ⁇ s each time, they were transferred into and washed three times with TCM199 washing medium by way of mannitol solution. To activate the electrofused embryos, they were incubated in the dark for 4 minutes in ionomycin (Sigma Chemical Co., USA) solution which was TCM199 washing medium containing 5 ⁇ M ionomycin and 1% BSA.
  • ionomycin Sigma Chemical Co., USA
  • the ionomycin stock solution was prepared by dissolving lmg of ionomycin m 1.34 ml of DMSO.
  • the activated embryos were incubated for 5 minutes in a 35mm dish containing TCM199 washing medium supplemented with 10% FBS to remove ionomycin from the embryos .
  • the activated embryos were postactivated for 4hrs in 25 ⁇ l of cycloheximide (Sigma Chemical Co., USA) solution prepared by adding cycloheximide stock solution (lOmg/ml in ethanol) to an in vitro culture medium, mTALP (.s_e_e_: Table 3) in a final concentration of 10 ⁇ g/ml. Then, the embryos were screened, and the selected embryos were incubated for 7 days under an environment of 39 ° C, 5% C0 2 .
  • cycloheximide Sigma Chemical Co., USA
  • the present inventors produced an embryo, SNU5 (Korean Tiger NT Embryo), by using ear cells of tiger and oocytes of Korean cow as nuclear donors and recipient oocytes, respectively.
  • the embryo was deposited with an international depositary authority, KCTC (Korean Collection for Type Cultures; KRIBB #52, Oun-dong, Yusong-ku, Taejon, 305-333, Republic of Korea) on Mar. 10, 2000, under an accession number of KCTC 0752BP.
  • Example 5 Production of embryos employing the oocytes obtained from cat
  • the present inventors produced and cultured the embryos by employing the same method described in Examples 1 to 4 except using the oocytes obtained from cat as recipient oocytes.
  • Example 6 Freeze and thaw of embryos and transplantation
  • a thawing medium containing PBS supplemented with 20% FBS was prepared m 35mm dishes, and added with glycerol to give thawing media each having 0%, 3% and 6% glycerol (.s_e_e_: Tables 9 and 11). Then, the frozen straw was taken out from the liquid N 2 , held in the air for 5 seconds, and thawed m a container (>20cm in diameter) containing warm water(30 ° C). After thawing, the straw was cut on the air layers at both ends, and the medium containing the embryos was collected. The embryos were examined under the microscope. To remove the freezing medium from the embryos, they were consecutively incubated in the thawing media containing 6% glycerol, 3% glycerol and 0% glycerol, each for 5 minutes.
  • the thawed embryo was placed in PBS containing 20% FBS, and drawn up into a straw. And then, it was transferred into the uterus of a surrogate mother.
  • Example 7 Comparison of embryos employing various donor cells
  • the embryos produced in Examples 4 and 5 were implanted to surrogate mothers, and compared regarding the following terms: number of electrofused oocytes, electrofusion rate(%), division rate(%), number (%) of 8-cell embryos, number (%) of 16-cell embryos, number (%) of 32-cell embryos, number (%) of morulae/blastocysts developed, number of transferred embryos and number of pregnancies following the embryo transfer (s_e_e_: Table 12). Number(%) of morulae/blastocysts represents the ratio of embryos developed by in vitro culture to the stage right before implantation over the total embryos produced by nuclear transfer. Table 12: Comparison of cloned tiger embryos
  • the present invention provides a method for producing cloned tigers by inter-species nuclear transplantation technique involving the fusion of somatic cells of tiger with the oocytes obtained from cow or cat. It also provides cloned tiger embryos and cloned tigers developed from the said embryos.
  • genetic characters of rare or endangered species can be preserved permanently by employing the inter-species nuclear transplantation to produce their cloned embryos as a way to preserve wild animals.
  • the method of the present invention is also expected to be employed for development of many related applications involving inter-species nuclear transplantation technique.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Cette invention se rapporte à un procédé permettant de produire des tigres clonés en utilisant une technique de transplantation de noyau inter-espèces. Ce procédé de production de tigres clonés consiste: à préparer des lignées de cellules somatiques donneuses, prélevées sur un tigre; à faire mûrir des ovocytes prélevés de l'ovaire d'une vache ou d'un chat in vitro; à retirer les cellules cumuliques entourant les ovocytes, à découper une partie de la membrane pellucide des ovocytes mûris, pour pratiquer une fente, et à faire sortir par pression une partie du cytoplasme contenant le premier corps polaire par ladite fente, pour donner des ovocytes receveurs énucléés; à transférer le noyau dans l'ovocyte receveur par injection des cellules donneuses dans les ovocytes receveurs énucléés, puis à pratiquer une électrofusion ultérieure et une activation des cellules ainsi électrofusionnées, pour produire des embryons; à post-activer et cultiver les embryons in vitro; et à transférer des embryons ainsi cultivés dans des mères porteuses, en vue de produire des tigres clonés. Cette invention peut être largement appliquée à la conservation des caractères génétiques d'espèces rares ou en danger de façon permanente sous la forme d'embryons de transfert de noyau.
PCT/KR2000/000706 1999-06-30 2000-06-30 Procede de production de tigres clones utilisant une technique de transplantation de noyau inter-especes Ceased WO2001000794A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP00941006A EP1117763A4 (fr) 1999-06-30 2000-06-30 Procede de production de tigres clones utilisant une technique de transplantation de noyau inter-especes
CA002334954A CA2334954A1 (fr) 1999-06-30 2000-06-30 Procede de production de tigres clones utilisant une technique de transplantation de noyau inter-especes
NZ508739A NZ508739A (en) 1999-06-30 2000-06-30 Method for producing cloned tigers by employing inter-species nuclear transplantation technique
AU55777/00A AU753209B2 (en) 1999-06-30 2000-06-30 Method for producing cloned tigers by employing inter-species nuclear transplantation technique
JP2001506788A JP2003503045A (ja) 1999-06-30 2000-06-30 異種間核移植技術を用いたクローントラの生産方法

Applications Claiming Priority (14)

Application Number Priority Date Filing Date Title
KR1999/26166 1999-06-30
KR19990026165 1999-06-30
KR19990026163 1999-06-30
KR1999/26165 1999-06-30
KR19990026166 1999-06-30
KR1999/26163 1999-06-30
KR1999/26164 1999-06-30
KR19990026164 1999-06-30
KR1999/36373 1999-08-30
KR19990036373 1999-08-30
KR1020000004382A KR20010076941A (ko) 2000-01-28 2000-01-28 이종간 핵이식 방법 및 그를 이용한 야생동물 복제수정란과 그 생산 방법
KR2000/4382 2000-01-28
KR2000/4381 2000-01-28
KR1020000004381A KR20010069167A (ko) 1999-08-30 2000-01-28 이종간 핵이식 방법 및 그를 이용한 야생동물 복제수정란과 그 생산 방법

Publications (1)

Publication Number Publication Date
WO2001000794A1 true WO2001000794A1 (fr) 2001-01-04

Family

ID=27567133

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2000/000706 Ceased WO2001000794A1 (fr) 1999-06-30 2000-06-30 Procede de production de tigres clones utilisant une technique de transplantation de noyau inter-especes

Country Status (7)

Country Link
EP (1) EP1117763A4 (fr)
JP (1) JP2003503045A (fr)
CN (1) CN1304445A (fr)
AU (1) AU753209B2 (fr)
CA (1) CA2334954A1 (fr)
NZ (1) NZ508739A (fr)
WO (1) WO2001000794A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1465986A4 (fr) * 2001-12-29 2005-01-26 Woo Suk Hwang Porc clone transfecte par gfp, porc clone a gt inactive et methodes de production
WO2007013763A1 (fr) * 2005-07-26 2007-02-01 Seoul National University Industry Foundation Canides clones et leur procede de production
DE10047272B4 (de) * 2000-09-14 2008-01-24 Eppendorf Ag Verfahren zur Fusion von dendritischen Zellen mit erkrankten Gewebezellen, insbesondere Tumorzellen
US9864229B2 (en) 2015-03-26 2018-01-09 Samsung Display Co., Ltd. Color filter panel and liquid crystal display device including the same

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107190059A (zh) * 2017-05-23 2017-09-22 毕节市农业科学研究所 一种拯救濒危生物的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997007669A1 (fr) * 1995-08-31 1997-03-06 Roslin Institute (Edinburgh) Populations de cellules quiescentes pour transfert de noyau
WO1997007668A1 (fr) * 1995-08-31 1997-03-06 Roslin Institute (Edinburgh) Ovocytes inactives utilises en tant que receveurs de cytoplastes aux fins de transfert nucleaire
US5945577A (en) * 1997-01-10 1999-08-31 University Of Massachusetts As Represented By Its Amherst Campus Cloning using donor nuclei from proliferating somatic cells
US6011197A (en) * 1997-03-06 2000-01-04 Infigen, Inc. Method of cloning bovines using reprogrammed non-embryonic bovine cells

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002512510A (ja) * 1997-03-06 2002-04-23 インフィジェン・インコーポレーテッド 動物のクローニング方法
JP2001509361A (ja) * 1997-07-03 2001-07-24 ユニヴァーシティー オヴ マサチューセッツ 分化した無血清飢餓細胞からの供与核を用いるクローニング
AU8587598A (en) * 1997-07-26 1999-02-16 Wisconsin Alumni Research Foundation Trans-species nuclear transfer
WO2002028164A2 (fr) * 2000-10-06 2002-04-11 Advanced Cell Technology, Inc. Clonage d'especes disparues ou en voie de disparition

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997007669A1 (fr) * 1995-08-31 1997-03-06 Roslin Institute (Edinburgh) Populations de cellules quiescentes pour transfert de noyau
WO1997007668A1 (fr) * 1995-08-31 1997-03-06 Roslin Institute (Edinburgh) Ovocytes inactives utilises en tant que receveurs de cytoplastes aux fins de transfert nucleaire
US5945577A (en) * 1997-01-10 1999-08-31 University Of Massachusetts As Represented By Its Amherst Campus Cloning using donor nuclei from proliferating somatic cells
US6011197A (en) * 1997-03-06 2000-01-04 Infigen, Inc. Method of cloning bovines using reprogrammed non-embryonic bovine cells

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
J. REPROD. AND FERTILITY, vol. 107, 1996, pages 53 - 58 *
J. REPROD. FERT. SUPPL., vol. 47, 1993, pages 203 - 207 *
J. REPROD. FERT., vol. 96, 1992, pages 725 - 734 *
NATURE, vol. 380, March 1996 (1996-03-01), pages 64 - 66 *
NATURE, vol. 385, February 1997 (1997-02-01), pages 810 - 813 *
NATURE, vol. 394, 1998, pages 369 - 374 *
See also references of EP1117763A4 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10047272B4 (de) * 2000-09-14 2008-01-24 Eppendorf Ag Verfahren zur Fusion von dendritischen Zellen mit erkrankten Gewebezellen, insbesondere Tumorzellen
EP1465986A4 (fr) * 2001-12-29 2005-01-26 Woo Suk Hwang Porc clone transfecte par gfp, porc clone a gt inactive et methodes de production
WO2007013763A1 (fr) * 2005-07-26 2007-02-01 Seoul National University Industry Foundation Canides clones et leur procede de production
US9864229B2 (en) 2015-03-26 2018-01-09 Samsung Display Co., Ltd. Color filter panel and liquid crystal display device including the same

Also Published As

Publication number Publication date
AU753209B2 (en) 2002-10-10
CN1304445A (zh) 2001-07-18
EP1117763A1 (fr) 2001-07-25
EP1117763A4 (fr) 2004-12-01
AU5577700A (en) 2001-01-31
CA2334954A1 (fr) 2001-01-04
NZ508739A (en) 2002-11-26
JP2003503045A (ja) 2003-01-28

Similar Documents

Publication Publication Date Title
EP0739412B1 (fr) CELLULES d'ongules, SOUCHES D'EMBRYONS UTILISEES COMME DONNEUSES DE NOYAUX ET PROCEDES DE TRANSFERT DE NOYAUX POUR LA PRODUCTION D'ANIMAUX CHIMERIQUES ET TRANSGENIQUES
US5453366A (en) Method of cloning bovine embryos
De Sousa et al. Reprogramming of fibroblast nuclei after transfer into bovine oocytes
AU753207B2 (en) Method for producing cloned cows
JP2004529648A (ja) 核移植法
EP1109890A1 (fr) Procede d'obtention d'embryons humains clones au moyen de techniques de transplantation inter-especes de noyaux
Song et al. Modification of maturation condition improves oocyte maturation and in vitro development of somatic cell nuclear transfer pig embryos.
Du et al. The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus)
AU753209B2 (en) Method for producing cloned tigers by employing inter-species nuclear transplantation technique
AU7372500A (en) Cloning pigs using donor cells or nuclei from differentiated cells and production of pluripotent porcine.
Manik et al. Micromanipulation and cloning studies on buffalo oocytes and embryos using nucleus transfer
Eyestone et al. Nuclear transfer from somatic cells: applications in farm animal species
KR20070013432A (ko) 복제된 개과 동물 및 이의 생산 방법
Van Stekelenburg‐Hamers et al. Nuclear transfer and electrofusion in bovine in vitro‐matured/in vitro‐fertilized embryos: Effect of media and electrical fusion parameters
RU2205537C2 (ru) Способ получения клонированного тигра путем применения метода межвидовой трансплантации ядер
EP1284285A2 (fr) Méthode pour recueillir des cellules en phase M ou G1 et utilisation de ces cellules pour le transfert nucléaire
Cheong et al. Development of mouse embryonic nuclei transferred to enucleated oocytes and zygotes
Ushijima et al. Production of a calf from a nuclear transfer embryo using in vitro matured oocytes
Ushijima et al. Bovine nuclear transplantation using a parthenogenetically activated oocyte as the recipient cytoplast
KR100427217B1 (ko) 체세포 복제동물의 생산을 위한 세포융합방법 및수핵난자의 탈핵방법
Polejaeva et al. A double nuclear transfer technique for cloning pigs
Gręda et al. Developmental potential of selectively enucleated mouse zygotes reconstituted with embryonic cell, embryonic stem cell and somatic cell nuclei.
WO2001030970A2 (fr) Protocole ameliore d'activation d'oocytes
CA2043088A1 (fr) Methode de clonage d'embryons de mammiferes
JP2005508185A (ja) ウサギの卵母細胞を用いる異種間体細胞核移植

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 00800651.2

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 55777/00

Country of ref document: AU

Ref document number: IN/PCT/2000/603/KOL

Country of ref document: IN

WWE Wipo information: entry into national phase

Ref document number: 2000941006

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 508739

Country of ref document: NZ

ENP Entry into the national phase

Ref document number: 2334954

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 09719472

Country of ref document: US

AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWP Wipo information: published in national office

Ref document number: 2000941006

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWG Wipo information: grant in national office

Ref document number: 55777/00

Country of ref document: AU

WWW Wipo information: withdrawn in national office

Ref document number: 2000941006

Country of ref document: EP