Method for selecting cell lines to be used for nuclear transfer in mammalian species
First Claim
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1. A method for cloning a non-human mammal through a nuclear transfer process comprising:
- (i) obtaining desired differentiated mammalian cells to be used as a source of donor nuclei;
(ii) obtaining at least one oocyte from a mammal of the same species as the cells which are the source of donor nuclei;
(iii) enucleating said at least one oocyte;
(iv) transferring the desired differentiated cell or cell nucleus into the enucleated oocyte;
(v) simultaneously fusing and activating the cell couplet to form a first transgenic embryo;
(vi) activating a cell-couplet to create a transgenic embryo that is activated after an initial electrical shock;
(vii) culturing said activated first and/or second transgenic embryo(es) until greater than the 2-cell developmental stage; and
(viii) transferring said first and/or second transgenic embryo into a host mammal such that the embryo develops into a fetus;
(ix) wherein wherein the desired differentiated mammalian cell line to be used as a karyoplast is selected according to the objective parameters of cleavage and/or fusion patterns.
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Abstract
The present invention provides data to demonstrate that the fusion performance of a cell-line in procedures involving fusion and cleavage indices either alone or in combination are a means for selecting a cell lines that will be successful in a nuclear transfer or microinjection program. This technique and method of selecting a cell line offers an additional alternative and improvement in the creation of activated and fused nuclear transfer-capable embryos for the production of live offspring in various mammalian non-human species including goats, pigs, rodents, primates, rabbits and cattle.
6 Citations
53 Claims
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1. A method for cloning a non-human mammal through a nuclear transfer process comprising:
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(i) obtaining desired differentiated mammalian cells to be used as a source of donor nuclei;
(ii) obtaining at least one oocyte from a mammal of the same species as the cells which are the source of donor nuclei;
(iii) enucleating said at least one oocyte;
(iv) transferring the desired differentiated cell or cell nucleus into the enucleated oocyte;
(v) simultaneously fusing and activating the cell couplet to form a first transgenic embryo;
(vi) activating a cell-couplet to create a transgenic embryo that is activated after an initial electrical shock;
(vii) culturing said activated first and/or second transgenic embryo(es) until greater than the 2-cell developmental stage; and
(viii) transferring said first and/or second transgenic embryo into a host mammal such that the embryo develops into a fetus;
(ix) wherein wherein the desired differentiated mammalian cell line to be used as a karyoplast is selected according to the objective parameters of cleavage and/or fusion patterns. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
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24. A method for producing cultured inner cell mass cells, comprising:
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(i) obtaining desired differentiated mammalian cells to be used as a source of donor nuclei;
(ii) obtaining at least one oocyte from a mammal of the same species as the cells which are the source of donor nuclei;
(iii) enucleating said at least one oocyte;
(iv) transferring the desired differentiated cell or cell nucleus into the enucleated oocyte;
(v) simultaneously fusing and activating the cell couplet to form a first transgenic embryo;
(vi) activating a cell-couplet to create a first transgenic embryo that is activated after an initial electrical shock; and
(vi) culturing cells obtained from said cultured activated embryo to obtain cultured inner cell mass cells;
(vii) wherein the desired differentiated mammalian cell line to be used as a karyoplast is selected according to the objective parameters of cleavage and/or fusion patterns - View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49)
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50. A method for cloning a non-human mammal through a nuclear transfer process comprising:
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(i) obtaining desired differentiated mammalian cells to be used as a source of donor nuclei;
(ii) obtaining at least one oocyte from a mammal of the same species as the cells which are the source of donor nuclei;
(iii) enucleating said oocytes;
(iv) transferring the desired differentiated cell or cell nucleus into the enucleated oocyte;
employing at least two electrical shocks to a cell-couplet to initiate fusion and activation of said cell-couplet into an activated and fused embryo. (vii) culturing said activated and fused embryo until greater than the 2-cell developmental stage;
(viii) transferring said first and/or second transgenic embryo into a host mammal such that the embryo develops into a fetus;
wherein the second of said at least two electrical shocks is administered at least 15 minutes after an initial electrical shock;
wherein a desired gene is inserted, removed or modified in said differentiated mammalian cell or cell nucleus prior to insertion of said differentiated mammalian cell or cell nucleus into said enucleated oocyte; and
wherein the desired differentiated mammalian cell line to be used as a karyoplast is selected according to the objective parameters of cleavage and/or fusion patterns.
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51. An improved method of cloning a non-human mammal by nuclear transfer comprising the introduction of a non-human mammalian donor cell or a non-human mammalian donor cell nucleus into a non-human mammalian enucleated oocyte of the same species as the donor cell or donor cell nucleus to form a nuclear transfer (NT) unit, implantation of the NT unit into the uterus of a surrogate mother of said species, and permitting the NT unit to develop into the cloned mammal, wherein the improvement comprises utilizing a pre-screened differentiated mammalian cell line as a karyoplast, said karyoplast being selected according to successful cleavage patterns.
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52. An improved method of cloning a non-human mammal by nuclear transfer comprising the introduction of a non-human mammalian donor cell or a non-human mammalian donor cell nucleus into a non-human mammalian enucleated oocyte of the same species as the donor cell or donor cell nucleus to form a nuclear transfer (NT) unit, implantation of the NT unit into the uterus of a surrogate mother of said species, and permitting the NT unit to develop into the cloned mammal, wherein the improvement comprises utilizing a pre-screened differentiated mammalian cell line as a karyoplast, said karyoplast being selected according to successful fusion patterns.
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53. An improved method of cloning a non-human mammal by nuclear transfer comprising the introduction of a non-human mammalian donor cell or a non-human mammalian donor cell nucleus into a non-human mammalian enucleated oocyte of the same species as the donor cell or donor cell nucleus to form a nuclear transfer (NT) unit, implantation of the NT unit into the uterus of a surrogate mother of said species, and permitting the NT unit to develop into the cloned mammal, wherein the improvement comprises utilizing a pre-screened differentiated mammalian cell line as a karyoplast, said karyoplast being selected according to successful cleavage and fusion patterns.
Specification