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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- 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.)
- Abandoned
Links
- 235000013601 eggs Nutrition 0.000 title claims abstract description 73
- 238000004519 manufacturing process Methods 0.000 title abstract description 9
- 238000010449 nuclear transplantation Methods 0.000 title description 5
- 239000012634 fragment Substances 0.000 claims abstract description 29
- 210000004027 cell Anatomy 0.000 claims abstract description 27
- 238000000338 in vitro Methods 0.000 claims abstract description 20
- 210000002257 embryonic structure Anatomy 0.000 claims abstract description 14
- 230000004913 activation Effects 0.000 claims abstract description 5
- 230000004927 fusion Effects 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 15
- 230000031864 metaphase Effects 0.000 claims description 13
- 241000283690 Bos taurus Species 0.000 claims description 9
- 230000004720 fertilization Effects 0.000 claims description 8
- 210000004340 zona pellucida Anatomy 0.000 claims description 6
- 238000012546 transfer Methods 0.000 claims description 5
- 241001494479 Pecora Species 0.000 claims description 3
- 210000002459 blastocyst Anatomy 0.000 claims description 3
- 210000001161 mammalian embryo Anatomy 0.000 claims description 2
- 241000283725 Bos Species 0.000 claims 1
- 241000283699 Bos indicus Species 0.000 claims 1
- 241000283705 Capra hircus Species 0.000 claims 1
- 238000006467 substitution reaction Methods 0.000 claims 1
- 238000002054 transplantation Methods 0.000 abstract description 7
- 238000002955 isolation Methods 0.000 abstract description 3
- 210000004940 nucleus Anatomy 0.000 description 21
- 210000000287 oocyte Anatomy 0.000 description 14
- 230000035800 maturation Effects 0.000 description 11
- 210000000349 chromosome Anatomy 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 230000021121 meiosis Effects 0.000 description 5
- 230000000295 complement effect Effects 0.000 description 4
- 210000004681 ovum Anatomy 0.000 description 4
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 230000007159 enucleation Effects 0.000 description 3
- 230000003325 follicular Effects 0.000 description 3
- 230000000392 somatic effect Effects 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 210000001109 blastomere Anatomy 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000016087 ovulation Effects 0.000 description 2
- 210000001082 somatic cell Anatomy 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 210000000719 corona radiata cell Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000030699 diplotene Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000002394 ovarian follicle Anatomy 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 210000004508 polar body Anatomy 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000031877 prophase Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
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- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
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- 239000003104 tissue culture media Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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 |
| US7326824B2 (en) | 1995-08-31 | 2008-02-05 | Roslin Institute (Edinburgh) | Unactivated ungulate oocytes to produce a transgenic ungulate by nuclear transfer |
| US6525243B1 (en) | 1995-08-31 | 2003-02-25 | Roslin Institute | Unactivated oocytes as cytoplast recipients of quiescent cell nuclei while maintaining correct ploidy |
| US7232938B2 (en) | 1995-08-31 | 2007-06-19 | Roslin Institute | Cloning ungulates from a quiescent donor cell |
| US7304204B2 (en) | 1995-08-31 | 2007-12-04 | Roslin Institute | Ungulates produced by nuclear transfer of G1 cells |
| US7307198B2 (en) | 1995-08-31 | 2007-12-11 | Roslin Institute | Ungulates produced by nuclear transfer of G1 cells |
| US7321075B2 (en) | 1995-08-31 | 2008-01-22 | Roslin Institute (Edinburgh) | Serial nuclear transfer of ungulate embryos |
| US7321076B2 (en) | 1995-08-31 | 2008-01-22 | Roslin Institute | Unactivated ungulate oocytes to produce a cloned ungulate by nuclear transfer |
| 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 |
| US7329796B2 (en) | 1995-08-31 | 2008-02-12 | Roslin Institute (Edinburgh) | Serial nuclear transfer of ungulate embryos |
| 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 |
| 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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