EP1117763A1 - Method for producing cloned tigers by employing inter-species nuclear transplantation technique - Google Patents
Method for producing cloned tigers by employing inter-species nuclear transplantation techniqueInfo
- Publication number
- EP1117763A1 EP1117763A1 EP00941006A EP00941006A EP1117763A1 EP 1117763 A1 EP1117763 A1 EP 1117763A1 EP 00941006 A EP00941006 A EP 00941006A EP 00941006 A EP00941006 A EP 00941006A EP 1117763 A1 EP1117763 A1 EP 1117763A1
- Authority
- EP
- European Patent Office
- 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.)
- Withdrawn
Links
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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.
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Applications Claiming Priority (15)
Application Number | Priority Date | Filing Date | Title |
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KR9926163 | 1999-06-30 | ||
KR9926166 | 1999-06-30 | ||
KR19990026166 | 1999-06-30 | ||
KR19990026164 | 1999-06-30 | ||
KR19990026163 | 1999-06-30 | ||
KR19990026165 | 1999-06-30 | ||
KR9926164 | 1999-06-30 | ||
KR9926165 | 1999-06-30 | ||
KR9936373 | 1999-08-30 | ||
KR19990036373 | 1999-08-30 | ||
KR2000004381 | 2000-01-28 | ||
KR1020000004381A KR20010069167A (en) | 1999-08-30 | 2000-01-28 | Embryo from wild animal with inter-species nuclear transplantation and method for production thereof |
KR2000004382 | 2000-01-28 | ||
KR1020000004382A KR20010076941A (en) | 2000-01-28 | 2000-01-28 | Embryo from wild animal with inter-species nuclear transplantation and method for production thereof |
PCT/KR2000/000706 WO2001000794A1 (en) | 1999-06-30 | 2000-06-30 | Method for producing cloned tigers by employing inter-species nuclear transplantation technique |
Publications (2)
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EP1117763A1 true EP1117763A1 (en) | 2001-07-25 |
EP1117763A4 EP1117763A4 (en) | 2004-12-01 |
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EP00941006A Withdrawn EP1117763A4 (en) | 1999-06-30 | 2000-06-30 | Method for producing cloned tigers by employing inter-species nuclear transplantation technique |
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EP (1) | EP1117763A4 (en) |
JP (1) | JP2003503045A (en) |
CN (1) | CN1304445A (en) |
AU (1) | AU753209B2 (en) |
CA (1) | CA2334954A1 (en) |
NZ (1) | NZ508739A (en) |
WO (1) | WO2001000794A1 (en) |
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DE10047272B4 (en) * | 2000-09-14 | 2008-01-24 | Eppendorf Ag | Process for the fusion of dendritic cells with diseased tissue cells, in particular tumor cells |
AU2002217606A1 (en) * | 2001-12-29 | 2003-11-03 | Woo Suk Hwang | Gfp-transfected clon pig, gt knock-out clon pig and methods for production thereof |
KR100733012B1 (en) * | 2005-07-26 | 2007-06-28 | 재단법인서울대학교산학협력재단 | Cloned Caninds And Method For Producing Thereof |
KR102289987B1 (en) | 2015-03-26 | 2021-08-17 | 삼성디스플레이 주식회사 | Color filter panel and liquid crystal display including the same |
CN113450876A (en) * | 2017-05-23 | 2021-09-28 | 毕节市农业科学研究所 | Method for finding out advantageous genetic material deleted in each individual organism and recovery method in endangered population |
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WO1999001163A1 (en) * | 1997-07-03 | 1999-01-14 | University Of Massachusetts | Cloning using donor nuclei from non-serum starved, differentiated cells |
WO1999005266A2 (en) * | 1997-07-26 | 1999-02-04 | Wisconsin Alumni Research Foundation | Trans-species nuclear transfer |
WO2002028164A2 (en) * | 2000-10-06 | 2002-04-11 | Advanced Cell Technology, Inc. | Cloning endangered and extinct species |
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GB9517780D0 (en) * | 1995-08-31 | 1995-11-01 | Roslin Inst Edinburgh | Biological manipulation |
GB9517779D0 (en) * | 1995-08-31 | 1995-11-01 | Roslin Inst Edinburgh | Biological manipulation |
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 |
NZ337495A (en) * | 1997-03-06 | 2001-06-29 | Infigen Inc | Bovine primordial germ cells and their use in cloning |
US6011197A (en) * | 1997-03-06 | 2000-01-04 | Infigen, Inc. | Method of cloning bovines using reprogrammed non-embryonic bovine cells |
-
2000
- 2000-06-30 EP EP00941006A patent/EP1117763A4/en not_active Withdrawn
- 2000-06-30 CA CA002334954A patent/CA2334954A1/en not_active Abandoned
- 2000-06-30 WO PCT/KR2000/000706 patent/WO2001000794A1/en not_active Application Discontinuation
- 2000-06-30 CN CN 00800651 patent/CN1304445A/en active Pending
- 2000-06-30 JP JP2001506788A patent/JP2003503045A/en active Pending
- 2000-06-30 NZ NZ508739A patent/NZ508739A/en not_active IP Right Cessation
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Patent Citations (3)
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WO1999001163A1 (en) * | 1997-07-03 | 1999-01-14 | University Of Massachusetts | Cloning using donor nuclei from non-serum starved, differentiated cells |
WO1999005266A2 (en) * | 1997-07-26 | 1999-02-04 | Wisconsin Alumni Research Foundation | Trans-species nuclear transfer |
WO2002028164A2 (en) * | 2000-10-06 | 2002-04-11 | Advanced Cell Technology, Inc. | Cloning endangered and extinct species |
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SHIN S J; LEE B C; PARK J I; LIM J M; HWANG W S: "A separate procedure of fusion and activation in an ear fibroblast nuclear transfer program improves preimplantation development of bovine reconstituted oocytes" THERIOGENOLOGY, vol. 55, 1 May 2001 (2001-05-01), pages 1697-1704, XP002294950 * |
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CN1304445A (en) | 2001-07-18 |
JP2003503045A (en) | 2003-01-28 |
CA2334954A1 (en) | 2001-01-04 |
AU753209B2 (en) | 2002-10-10 |
AU5577700A (en) | 2001-01-31 |
WO2001000794A1 (en) | 2001-01-04 |
NZ508739A (en) | 2002-11-26 |
EP1117763A4 (en) | 2004-12-01 |
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