Cloning using donor nuclei from proliferating somatic cells
First Claim
1. 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 using as the donor cell or donor cell nucleus a proliferating somatic cell that has been expanded in culture, or a nucleus isolated from said somatic cell.
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Abstract
An improved method of nuclear transfer involving the transplantation of donor differentiated cell nuclei into enucleated oocytes of the same species as the donor cell is provided. The resultant nuclear transfer units are useful for multiplication of genotypes and transgenic genotypes by the production of fetuses and offspring, and for production of isogenic CICM cells, including human isogenic embryonic or stem cells. Production of genetically engineered or transgenic mammalian embryos, fetuses and offspring is facilitated by the present method since the differentiated cell source of the donor nuclei can be genetically modified and clonally propagated.
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Citations
24 Claims
- 1. 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 using as the donor cell or donor cell nucleus a proliferating somatic cell that has been expanded in culture, or a nucleus isolated from said somatic cell.
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2. 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 using as the donor cell or donor cell nucleus a proliferating somatic cell that has been expanded in culture, or a nucleus isolated from said somatic cell, and wherein the donor cell or donor cell nucleus has been genetically transformed to comprise at least one addition, substitution or deletion of a nucleic acid sequence.
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3. A method of cloning a non-human mammal by nuclear transfer comprising the following steps:
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(i) inserting a desired non-human mammalian proliferating somatic cell that has been expanded in culture, or a nucleus isolated from said somatic cell, into a non-human mammalian enucleated oocyte of the same species under conditions suitable for the formation of a nuclear transfer (NT) unit; (ii) activating the resultant nuclear transfer unit; (iii) culturing said activated NT unit until greater than the 2-cell developmental stage; and (iv) transferring said cultured NT unit to a host non-human mammal of the same species such that the NT develops in to a non-human mammal.
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- 4. An improved method of cloning a non-human mammalian fetus 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 the same species, and permitting the NT unit to develop into the mammalian fetus, wherein the improvement comprises using as the donor cell or donor cell nucleus a proliferating somatic cell that has been expanded in culture, or a nucleus isolated from said somatic cell.
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5. An improved method of cloning a non-human mammalian fetus 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 the same species, and permitting the NT unit to develop into the mammalian fetus, wherein the improvement comprises using as the donor cell or donor cell nucleus a proliferating somatic cell that has been expanded in culture, or a nucleus isolated from said somatic cell, and wherein the donor cell or donor cell nucleus has been genetically transformed to comprises at least one addition, substitution or deletion of a nucleic acid sequence.
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6. A method of cloning a non-human mammalian fetus by nuclear transfer comprising the following steps:
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(i) inserting a desired non-human mammalian proliferating somatic cell that has been expanded in culture, or a nucleus isolated from said somatic cell, into a non-human mammalian enucleated oocyte of the same species under conditions suitable for the formation of a nuclear transfer (NT) unit; (ii) activating the resultant nuclear transfer unit; (iii) culturing said activated NT unit until greater than the 2-cell developmental stage; and (iv) transferring said cultured NT unit to a host non-human mammal of the same species such that the NT develops into a fetus.
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Specification