CA2059199A1 - Method for producing mature reconstituted mammalian eggs by nuclear transplantation - Google Patents
Method for producing mature reconstituted mammalian eggs by nuclear transplantationInfo
- Publication number
- CA2059199A1 CA2059199A1 CA 2059199 CA2059199A CA2059199A1 CA 2059199 A1 CA2059199 A1 CA 2059199A1 CA 2059199 CA2059199 CA 2059199 CA 2059199 A CA2059199 A CA 2059199A CA 2059199 A1 CA2059199 A1 CA 2059199A1
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- CA
- Canada
- Prior art keywords
- eggs
- egg
- cell
- enucleated
- vitro
- 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.)
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Abstract
ABSTRACT OF THE DISCLOSURE
A method for producing mature reconstituted unfertilized mammalian eggs by transferring to enucleated ovulated or in vitro matured eggs nuclei from other sources, includes the following steps: 1) production of the enucleated mature egg or -egg fragment. 2) isolation of the cell or cell fragment containing the nucleus to be transferred. 3) fusion of the enucleated egg or -egg fragment with the cell or cell fragment containing the nucleus to be transferred. Nuclei from a variety of sources may be used including immature and mature eggs, and cells from embryos. The reconstituted eggs may be fertilized by exposure to sperm in vitro or by activation and pronuclear transplantation.
A method for producing mature reconstituted unfertilized mammalian eggs by transferring to enucleated ovulated or in vitro matured eggs nuclei from other sources, includes the following steps: 1) production of the enucleated mature egg or -egg fragment. 2) isolation of the cell or cell fragment containing the nucleus to be transferred. 3) fusion of the enucleated egg or -egg fragment with the cell or cell fragment containing the nucleus to be transferred. Nuclei from a variety of sources may be used including immature and mature eggs, and cells from embryos. The reconstituted eggs may be fertilized by exposure to sperm in vitro or by activation and pronuclear transplantation.
Description
205~199 METHOD FOR PRODUCING MATURE RECONSTITUTED MAMMALIAN EGGS BY
NUCLEAR TRANSPLANTATION
The present invention relates to the production by nuclear transplantation of mature reconstituted mammalian eggs to be used for the production of embryos by subsequent in vitro fertilization or pronuclear transplantation.
The ovaries of mammals contain large numbers of eggs, the vast majority of which are immature, arrested at the modified diplotene stage of the prophase of the first meiotic division. In vivo, only a miniscule proportion of these immature eggs will ever be recruited to undergo final ~o maturation and ovulation. The rest are either retained in the arrested state or degenerate in the course of follicular atresia.
In vivo, those immature eggs that are recruited for final maturation and ovulation resume Meiosis and within a short period of time - in cattle 16 to 24 hours - reach the metaphase of the second meiotic division. The egg is ovulated with its nucleus arrested in metaphase of the second meiotic divisionl and it is thought that in vivo the egg nucleus will generally only resume meiosis and, after expulsion of the second polar body, progress to form a female pronucleus if and when the mature egg is fertilized.
The ovulated egg with its nucleus arrested in metaphase of the second meiotic division is a "mature egg" in the context of the present application. Similarly, in the context of the present application, the resumption of meiosis and its progress to the arrest in metaphase of the second meiotic division constitutes "maturation .
Oocytes released from tertiary ovarian follicles tend to spontaneously undergo maturation during culture in vitro ~- following a time table very similar to that observed in vivo. In a number of mammalian species, including cattle, in vitro matured oocytes have been shown to be able to undergo fertilization, and give rise to live offspring. However, the success of in vitro maturation of oocytes, and the viability of the embryos to which they give rise, vary widely. Among the factors that have been suspected or identified as sources of this variation are 1) the follicular source of the oocyte, 2) the condition of the cumulus oophorus, 3) the condition of the oocyte itself, 4) the isolation procedure and 5) the culture conditions.
By applying stringent morphological criteria to the examination of oocytes both before and after in vitro maturation it is possible to select those that are most J
NUCLEAR TRANSPLANTATION
The present invention relates to the production by nuclear transplantation of mature reconstituted mammalian eggs to be used for the production of embryos by subsequent in vitro fertilization or pronuclear transplantation.
The ovaries of mammals contain large numbers of eggs, the vast majority of which are immature, arrested at the modified diplotene stage of the prophase of the first meiotic division. In vivo, only a miniscule proportion of these immature eggs will ever be recruited to undergo final ~o maturation and ovulation. The rest are either retained in the arrested state or degenerate in the course of follicular atresia.
In vivo, those immature eggs that are recruited for final maturation and ovulation resume Meiosis and within a short period of time - in cattle 16 to 24 hours - reach the metaphase of the second meiotic division. The egg is ovulated with its nucleus arrested in metaphase of the second meiotic divisionl and it is thought that in vivo the egg nucleus will generally only resume meiosis and, after expulsion of the second polar body, progress to form a female pronucleus if and when the mature egg is fertilized.
The ovulated egg with its nucleus arrested in metaphase of the second meiotic division is a "mature egg" in the context of the present application. Similarly, in the context of the present application, the resumption of meiosis and its progress to the arrest in metaphase of the second meiotic division constitutes "maturation .
Oocytes released from tertiary ovarian follicles tend to spontaneously undergo maturation during culture in vitro ~- following a time table very similar to that observed in vivo. In a number of mammalian species, including cattle, in vitro matured oocytes have been shown to be able to undergo fertilization, and give rise to live offspring. However, the success of in vitro maturation of oocytes, and the viability of the embryos to which they give rise, vary widely. Among the factors that have been suspected or identified as sources of this variation are 1) the follicular source of the oocyte, 2) the condition of the cumulus oophorus, 3) the condition of the oocyte itself, 4) the isolation procedure and 5) the culture conditions.
By applying stringent morphological criteria to the examination of oocytes both before and after in vitro maturation it is possible to select those that are most J
2~
likely ~o undergo, or have undergone, normal rnaturatiom, and thus have the best chances to give rise to viable embryos.
There is evidence to show that while the nuclear events of in vitro maturation appear in most cases to proceed in a normal manner, cytoplasmatiG maturation is very often incomplete or abnormal in vitro.
The present invention uses transplantation of nuclei from oocytes, and mature eggs, unlikely to undergo, or unlikely to have undergone, normal cytoplasmatic maturation .o to enucleated eggs that have matured normally. This allows the ability of virtually all oocyte nucclei to undergo normal maturation and hence to supply the maternal chromosome complement o~ an embryo, irrespective of the quality of non-nuclear components of the oocyte from which they origiate, to be exploited more effectively -for the purpose of breeding animals.
In addition, the present invention uses the ability of enucleated mature eggs not only to maintain meiotic behaviour in transplanted oocyte nuclei, but also to induce meiotic behaviour in somatic cell nuclei transplanted to them. The latter use in effect allows somatic nuclei to supply the chromosome complement for mature unfertilized eggs.
In mammals, the principle of nuclear transplantation has so far been used in two forms involving eggs and embryos: experimental transplantation or exchange of pronuclei from fertilized or activated eggs with a view to examining differences between the paternally and maternally derived part of the genome, and transplantation o-f nuclei 9~ from embryos up to and including the blastocyst stage, with a view to cloning of animals.
There has been no report so far of atternpts to use transfer of oocyte nuclei with the aim of producing mature unfertilized eggs.
Nor has there been any report of transfer of somatic nuclei with the aim of having them sypply the nuclei of mature unfertilized eggs.
The discovery made by the author is that the cytoplasm of normal mature eggs has the ability to maintain and indeed ~o induce meiotic behaviour in transferred nuclei of somatic origin as well as nuclei from cells of the germline. Based on this discovery, the present invention may be used to shortcircuit the normal paths of rnamalian repoduction.
2 ~ 9 The invention is described as follows:
1. Production of enucleated mature egg or -egg fragment.
The eggs to be used as enucleated recipients for transferred nuclei may be matured in vivo or in vitro or by a combination of the two. Typically, they are selected from among the best quality eggs matured in vitro for 24 to 28 hours using one of the standard combinatios of culture conditions ( Wiemer & al, 1991).
The method found most suitable for producing enucleated ~- eggs and -egg fragments is the one described in a previous patent application by the author ( Willadsen, 1991) as it applies to non-activated eggs 24 to 28 hours after the resumption of Meiosis:
After the eggs have been denuded of corona radiata cells, they are placed in a saline medium at a pH of 6 to 8 ( typically 7.3) and with an osmolarity in the 250 to 350 mosm range ( typically 290 mosm. at a temperature above 20C
( typically 38-39C). Typical incubation media are PBI ( phosphate buffered saline medium with organic substances t~ added) or Hepes or bicarbonate buffered TCM 199( Tissue culture medium 199 ), with organic additions.
It is advantageous to either open or completely remove the zona pellucida prior to incubating the eggs.
After 30 to 90 minutes' incubation at 38-39C, the ' position of the metaphase plate will be revealed in a very ! high proportion of the eggs by the appearance of a small 1 clear hemispherical bulge of the cell membrane and underlieing cytoplasm. The bulge indicates the precise location of the metaphases plate of egg chromosomes.
likely ~o undergo, or have undergone, normal rnaturatiom, and thus have the best chances to give rise to viable embryos.
There is evidence to show that while the nuclear events of in vitro maturation appear in most cases to proceed in a normal manner, cytoplasmatiG maturation is very often incomplete or abnormal in vitro.
The present invention uses transplantation of nuclei from oocytes, and mature eggs, unlikely to undergo, or unlikely to have undergone, normal cytoplasmatic maturation .o to enucleated eggs that have matured normally. This allows the ability of virtually all oocyte nucclei to undergo normal maturation and hence to supply the maternal chromosome complement o~ an embryo, irrespective of the quality of non-nuclear components of the oocyte from which they origiate, to be exploited more effectively -for the purpose of breeding animals.
In addition, the present invention uses the ability of enucleated mature eggs not only to maintain meiotic behaviour in transplanted oocyte nuclei, but also to induce meiotic behaviour in somatic cell nuclei transplanted to them. The latter use in effect allows somatic nuclei to supply the chromosome complement for mature unfertilized eggs.
In mammals, the principle of nuclear transplantation has so far been used in two forms involving eggs and embryos: experimental transplantation or exchange of pronuclei from fertilized or activated eggs with a view to examining differences between the paternally and maternally derived part of the genome, and transplantation o-f nuclei 9~ from embryos up to and including the blastocyst stage, with a view to cloning of animals.
There has been no report so far of atternpts to use transfer of oocyte nuclei with the aim of producing mature unfertilized eggs.
Nor has there been any report of transfer of somatic nuclei with the aim of having them sypply the nuclei of mature unfertilized eggs.
The discovery made by the author is that the cytoplasm of normal mature eggs has the ability to maintain and indeed ~o induce meiotic behaviour in transferred nuclei of somatic origin as well as nuclei from cells of the germline. Based on this discovery, the present invention may be used to shortcircuit the normal paths of rnamalian repoduction.
2 ~ 9 The invention is described as follows:
1. Production of enucleated mature egg or -egg fragment.
The eggs to be used as enucleated recipients for transferred nuclei may be matured in vivo or in vitro or by a combination of the two. Typically, they are selected from among the best quality eggs matured in vitro for 24 to 28 hours using one of the standard combinatios of culture conditions ( Wiemer & al, 1991).
The method found most suitable for producing enucleated ~- eggs and -egg fragments is the one described in a previous patent application by the author ( Willadsen, 1991) as it applies to non-activated eggs 24 to 28 hours after the resumption of Meiosis:
After the eggs have been denuded of corona radiata cells, they are placed in a saline medium at a pH of 6 to 8 ( typically 7.3) and with an osmolarity in the 250 to 350 mosm range ( typically 290 mosm. at a temperature above 20C
( typically 38-39C). Typical incubation media are PBI ( phosphate buffered saline medium with organic substances t~ added) or Hepes or bicarbonate buffered TCM 199( Tissue culture medium 199 ), with organic additions.
It is advantageous to either open or completely remove the zona pellucida prior to incubating the eggs.
After 30 to 90 minutes' incubation at 38-39C, the ' position of the metaphase plate will be revealed in a very ! high proportion of the eggs by the appearance of a small 1 clear hemispherical bulge of the cell membrane and underlieing cytoplasm. The bulge indicates the precise location of the metaphases plate of egg chromosomes.
3~ The clear bulge and a smaller or larger part of the neighbouring ooplasm, typically one eighth to one sixteenth of the total cell volume of the egg, may now be easily pinched off with the aid of a suction pipette of appropriately small bore with smooth edges. This operation typically takes place at room temperature. The small clear hemispherical bulge tends to disappear within 15 to 30 minutes at room temperature. If this happens before enucleation can take place, it is necessary to reincubate the egg or egg fragment at elevated temperature until the bulge re-appears.
The enucleated egg may be further subdivided or reduced in size in a similar way.
If the zona has been completely removed prior to enucleation, it will be necessary to replace it with an empty zona as described by Willadsen (1979).
2~5~1~9 2. Isolation of cell or cell fragment containing the nucleus to be transferred.
Three categories of nuclei have so far been used successfully for transfer to enucleated mature eggs in the use of the present invention:
a) metaphase plates isolated from eggs in metaphase of the second meiotic division b) germinal vesicles isolated from immature follicular oocytes ,~ c) somatic cell nuclei isolated from cleaving embryos ad a): a membrane bounded egg fragment containing the metaphase chromosomes of the second meiotic division is isolated as described under "1. Production of enucleated mature egg or -egg fragment." above, only in this instance it is the nucleated egg fragment that is kept for further use.
ad b): after removal of granulosa-/ cumulus oophorus cells and zona pellucida the germinal vesicle is visible as a more or less sperical sparsely granulated region, with a diameter - of about 1/4 of the oocyte diameter, situated excentrically in the cell. A membrane-bounded oocyte fragment containing the germinal vesicle in a small amount of cytoplasm is easily pinched off with the aid of a suction pipette of appropriately small bore with smooth edges.
ad c) whole blastomeres are isolated mechanically, with the aid of suction pipettes of appropriate diameters, from embryos up to and including the early blastocyst stage, after the zona pellucida has been opened or removed.
Blastomeres with the diploid chromosome complement, i.e.
o~ cells resu1ting from recent divisions, should be chosen preferentially.
3. Fusion of the enucleated egg or -egg fragment with the cell or cell fragment containing the nucleus to be transferred.
The cell or cell fragment containing the nucleus to be transferred is placed with its cell membrane in close contact with that of an enucleated egg contained within its own or within a substitute zona pellucida.
For electrofusion, the following procedure is typically ~o used ( a variant of the one described by Willadsen, 1986):
The micromanipulated eggs are placed in an aquous solution of 0.3 M mannitol and Zimmerman's cell fusion medium 25 : 1 for 15-30 min.. They are then transferred, in the same medium to the chamber ( electrode distance 200 m) of an ' ~.
~, . . .
2 0 ~
electrofusion apparatus ( Zimmerman; CGA, Chicago) and exposed to the following fusion conditions: cell alignment 600 kHz 8 V for 8 seconds followed by one 24 V d.c. pulse lasting 0.1 second. Immediately afterwards the eggs are transferred to a buffered saline holding medium, such as PBI
or TCM 199, pending further processing.
The enucleated egg may be further subdivided or reduced in size in a similar way.
If the zona has been completely removed prior to enucleation, it will be necessary to replace it with an empty zona as described by Willadsen (1979).
2~5~1~9 2. Isolation of cell or cell fragment containing the nucleus to be transferred.
Three categories of nuclei have so far been used successfully for transfer to enucleated mature eggs in the use of the present invention:
a) metaphase plates isolated from eggs in metaphase of the second meiotic division b) germinal vesicles isolated from immature follicular oocytes ,~ c) somatic cell nuclei isolated from cleaving embryos ad a): a membrane bounded egg fragment containing the metaphase chromosomes of the second meiotic division is isolated as described under "1. Production of enucleated mature egg or -egg fragment." above, only in this instance it is the nucleated egg fragment that is kept for further use.
ad b): after removal of granulosa-/ cumulus oophorus cells and zona pellucida the germinal vesicle is visible as a more or less sperical sparsely granulated region, with a diameter - of about 1/4 of the oocyte diameter, situated excentrically in the cell. A membrane-bounded oocyte fragment containing the germinal vesicle in a small amount of cytoplasm is easily pinched off with the aid of a suction pipette of appropriately small bore with smooth edges.
ad c) whole blastomeres are isolated mechanically, with the aid of suction pipettes of appropriate diameters, from embryos up to and including the early blastocyst stage, after the zona pellucida has been opened or removed.
Blastomeres with the diploid chromosome complement, i.e.
o~ cells resu1ting from recent divisions, should be chosen preferentially.
3. Fusion of the enucleated egg or -egg fragment with the cell or cell fragment containing the nucleus to be transferred.
The cell or cell fragment containing the nucleus to be transferred is placed with its cell membrane in close contact with that of an enucleated egg contained within its own or within a substitute zona pellucida.
For electrofusion, the following procedure is typically ~o used ( a variant of the one described by Willadsen, 1986):
The micromanipulated eggs are placed in an aquous solution of 0.3 M mannitol and Zimmerman's cell fusion medium 25 : 1 for 15-30 min.. They are then transferred, in the same medium to the chamber ( electrode distance 200 m) of an ' ~.
~, . . .
2 0 ~
electrofusion apparatus ( Zimmerman; CGA, Chicago) and exposed to the following fusion conditions: cell alignment 600 kHz 8 V for 8 seconds followed by one 24 V d.c. pulse lasting 0.1 second. Immediately afterwards the eggs are transferred to a buffered saline holding medium, such as PBI
or TCM 199, pending further processing.
4. Post electro-pulsing treatment.
Whatever the category the transferred nucleus belonged to, the eggs produced by the procedure must ultimately be fertilized, either by in vitro fertilization or by a combination of activation and pronuclear transfer, for viable embryos to be produced. Where the transplanted nucleus was not at metaphase of the second meiotic division or equivalent, the egg should be cultured under conditions allowing the typical metaphase configuration to be established before in vitro fertilization or activation is attempted.
a) In vitro fertilization:
When mature eggs, or fragments of mature eggs, are exposed to spermatozoa,under.the conditions of one of the standard procedures of in vitro fertilization used in cattle,after the zona pellucida has been opened or removed, they are easily penetrated by sperm the chance that they will become polyspermic is high ( Willadsen, unpublished observation, 1991). This problem may be overcome by lowering the sperm concentration to which the egg is exposed, and by shortening the period during which the egg is exposed to active sperm ( Willadsen, unpublished observation, 1991).
b) Activation and pronuclear transplantation:
o Electro-pulsing can be used to artificially activate mature unfertilized eggs. This is most easily achieved when the egg is at a stage of 32 hours or more after resumption of meiosis. Pronuclei for transplantation may be isolated from a) in vitro fertilized eggs ( male and female pronuclei),b) artificially activated eggs or artificially activated nucleated egg fragments ( female pronuclei), or c) eggs or egg fragments fertilized in vitro after removal of the female chromosome complement ( Willadsen, unpublished observation, 1991). It can be advantageous when enucleated eggs or - egg fragments are used as the source of male pronuclei to centrifuge and divide the eggs into agranular and granular fragments prior to enucleation and exposure to sperm ( Willadsen, unpublished observation, 1991) . ~ ~
2 ~ 9 REFERENCES:
Wiemer, K.E., Watson, A.J., Polanski, V., McKenna, A.I., Fick, G.H., and Schultz, G.A., 1991: Effects of maturation and co-culture treatments on the developmental capacity of early bovine embryos. Molecular Reproduction and Development, 30: 330 - 338.
Willadsen, S.M., 1979: A method for culture of micromanipulated sheep embryos and its use to produce monozygotic twins. Nature (London),277: 298 - 300.
Willadsen, S.M., 1986: Nuclear transplantation in sheep embryos. Nature (London), 320: 63 - 65.
Willadsen, S.M., 1991: Method for producing enucleated and nucleated fragments of mammalian eggs. Patent application to the Commissioner of Patents, Canada. Filing date 8 Nov, 1 99 1 .
..
Whatever the category the transferred nucleus belonged to, the eggs produced by the procedure must ultimately be fertilized, either by in vitro fertilization or by a combination of activation and pronuclear transfer, for viable embryos to be produced. Where the transplanted nucleus was not at metaphase of the second meiotic division or equivalent, the egg should be cultured under conditions allowing the typical metaphase configuration to be established before in vitro fertilization or activation is attempted.
a) In vitro fertilization:
When mature eggs, or fragments of mature eggs, are exposed to spermatozoa,under.the conditions of one of the standard procedures of in vitro fertilization used in cattle,after the zona pellucida has been opened or removed, they are easily penetrated by sperm the chance that they will become polyspermic is high ( Willadsen, unpublished observation, 1991). This problem may be overcome by lowering the sperm concentration to which the egg is exposed, and by shortening the period during which the egg is exposed to active sperm ( Willadsen, unpublished observation, 1991).
b) Activation and pronuclear transplantation:
o Electro-pulsing can be used to artificially activate mature unfertilized eggs. This is most easily achieved when the egg is at a stage of 32 hours or more after resumption of meiosis. Pronuclei for transplantation may be isolated from a) in vitro fertilized eggs ( male and female pronuclei),b) artificially activated eggs or artificially activated nucleated egg fragments ( female pronuclei), or c) eggs or egg fragments fertilized in vitro after removal of the female chromosome complement ( Willadsen, unpublished observation, 1991). It can be advantageous when enucleated eggs or - egg fragments are used as the source of male pronuclei to centrifuge and divide the eggs into agranular and granular fragments prior to enucleation and exposure to sperm ( Willadsen, unpublished observation, 1991) . ~ ~
2 ~ 9 REFERENCES:
Wiemer, K.E., Watson, A.J., Polanski, V., McKenna, A.I., Fick, G.H., and Schultz, G.A., 1991: Effects of maturation and co-culture treatments on the developmental capacity of early bovine embryos. Molecular Reproduction and Development, 30: 330 - 338.
Willadsen, S.M., 1979: A method for culture of micromanipulated sheep embryos and its use to produce monozygotic twins. Nature (London),277: 298 - 300.
Willadsen, S.M., 1986: Nuclear transplantation in sheep embryos. Nature (London), 320: 63 - 65.
Willadsen, S.M., 1991: Method for producing enucleated and nucleated fragments of mammalian eggs. Patent application to the Commissioner of Patents, Canada. Filing date 8 Nov, 1 99 1 .
..
Claims (11)
1. A process for producing reconstituted mammalian eggs by fusion of nucleated cells or cell fragments with enucleated eggs or enucleated fragments of eggs.
2. A process as defined in Claim 1, in which the enucleated egg or egg fragment is produced from an egg that has reached metaphase of the second meiotic division.
3. A process as defined in Claim 1, in which the nucleated cell or cell fragment is isolated from an egg whose nucleus is in metaphase of the second meiotic division.
4. A process as defined in Claim 1, in which the nucleated cell or cell fragment is isolated from an egg whose nucleus is at the germinal vesicle stage.
5. A process as defined in Claim 1, in which the nucleated cell or cell fragment is isolated from an embryo at the blastocyst stage or younger.
6. A process as defined in Claims 1-4, in which any or all of the enucleated eggs or egg fragments, and the nucleated cells or cell fragments are derived from Bos taurus, Bos indicus, Bos bubalis, Ovis aries, and Capra hircus.
7. Eggs produced in accordance with the process of Claim 1.
8. A process for obtaining fertilization of bovine eggs in vitro involving exposure to sperm in vitro after opening, removal or substitution of the zona pellucida.
9. A process for obtaining fertilization of bovine eggs involving activation in vitro and pronuclear transfer.
10. Bovine embryos obtained by the process of Claim 8.
11. Bovine embryos obtained by the process of Claim 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2059199 CA2059199A1 (en) | 1992-01-10 | 1992-01-10 | Method for producing mature reconstituted mammalian eggs by nuclear transplantation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2059199 CA2059199A1 (en) | 1992-01-10 | 1992-01-10 | Method for producing mature reconstituted mammalian eggs by nuclear transplantation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2059199A1 true CA2059199A1 (en) | 1993-07-11 |
Family
ID=4149075
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2059199 Abandoned CA2059199A1 (en) | 1992-01-10 | 1992-01-10 | Method for producing mature reconstituted mammalian eggs by nuclear transplantation |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2059199A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6147276A (en) * | 1995-08-31 | 2000-11-14 | Roslin Institute (Edinburgh) | Quiescent cell populations for nuclear transfer in the production of non-human mammals and non-human mammalian embryos |
| US6252133B1 (en) | 1995-08-31 | 2001-06-26 | Roslin Institute (Edinburgh) | Unactivated oocytes as cytoplast recipients of quiescent and non-quiescent cell nuclei, while maintaining correct ploidy |
| US7304204B2 (en) | 1995-08-31 | 2007-12-04 | Roslin Institute | Ungulates produced by nuclear transfer of G1 cells |
| US7361804B1 (en) | 1997-02-19 | 2008-04-22 | Roslin Institute (Edinburgh) | Unactivated oocytes in nuclear transfer to produce ungulates |
-
1992
- 1992-01-10 CA CA 2059199 patent/CA2059199A1/en not_active Abandoned
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7326825B2 (en) | 1995-08-31 | 2008-02-05 | Roslin Institute (Edinburgh) | Methods of ungulate nuclear transfer |
| US7304204B2 (en) | 1995-08-31 | 2007-12-04 | Roslin Institute | Ungulates produced by nuclear transfer of G1 cells |
| US6147276A (en) * | 1995-08-31 | 2000-11-14 | Roslin Institute (Edinburgh) | Quiescent cell populations for nuclear transfer in the production of non-human mammals and non-human mammalian embryos |
| US7232938B2 (en) | 1995-08-31 | 2007-06-19 | Roslin Institute | Cloning ungulates from a quiescent donor cell |
| US7326824B2 (en) | 1995-08-31 | 2008-02-05 | Roslin Institute (Edinburgh) | Unactivated ungulate oocytes to produce a transgenic ungulate by nuclear transfer |
| US7307198B2 (en) | 1995-08-31 | 2007-12-11 | Roslin Institute | Ungulates produced by nuclear transfer of G1 cells |
| US7321076B2 (en) | 1995-08-31 | 2008-01-22 | Roslin Institute | Unactivated ungulate oocytes to produce a cloned ungulate by nuclear transfer |
| US7329796B2 (en) | 1995-08-31 | 2008-02-12 | Roslin Institute (Edinburgh) | Serial nuclear transfer of ungulate embryos |
| US6525243B1 (en) | 1995-08-31 | 2003-02-25 | Roslin Institute | Unactivated oocytes as cytoplast recipients of quiescent cell nuclei while maintaining correct ploidy |
| US6252133B1 (en) | 1995-08-31 | 2001-06-26 | Roslin Institute (Edinburgh) | Unactivated oocytes as cytoplast recipients of quiescent and non-quiescent cell nuclei, while maintaining correct ploidy |
| US7321075B2 (en) | 1995-08-31 | 2008-01-22 | Roslin Institute (Edinburgh) | Serial nuclear transfer of ungulate embryos |
| US7332648B2 (en) | 1995-08-31 | 2008-02-19 | Roslin Institute | Unactivated oocytes in nuclear transfer to produce cultured inner cell mass cells and ungulates |
| US7355094B2 (en) | 1995-08-31 | 2008-04-08 | Roslin Institute (Edinburgh) | Methods of ungulate nuclear transfer |
| US7524677B2 (en) | 1995-08-31 | 2009-04-28 | Rosiin Institute (Edinburgh) | Mammalian cultured inner cell mass cell culture using a g1 cell as nuclear donor |
| US7432415B2 (en) | 1995-08-31 | 2008-10-07 | Roslin Institute (Edinburgh) | Unactivated oocytes as cytoplast recipients for nonprimate mammalian and pig nuclear transfer |
| US7514258B2 (en) | 1995-08-31 | 2009-04-07 | Roslin Institute (Edinburgh) | Mammalian cultured inner cell mass cell culture using a quiescent cell as nuclear |
| US7361804B1 (en) | 1997-02-19 | 2008-04-22 | Roslin Institute (Edinburgh) | Unactivated oocytes in nuclear transfer to produce ungulates |
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