Cloning using donor nuclei from proliferating somatic cells

• US 5,945,577 A
• Filed: 01/10/1997
• Issued: 08/31/1999
• Est. Priority Date: 01/10/1997
• Status: Expired due to Fees

• Current Assignee: University of Massachusetts A Public Institution of Higher Education of The Commonwealth of MA
• Original Assignee: University of Massachusetts As Represented By Its Amherst Campus
• Inventors: Cibelli, Jose, Stice, Steven L., Golueke, Paul, Robl, James, Jerry, D. Joseph, Ponce de Leon, F. Abel
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US08/781,752
• Time in Patent Office: 963 Days
• Field of Search: 800/2, 800/DIG. 1, 435/172.3, 435/325, 435/350, 435/351, 435/352, 435/363, 435/366
• US Class Current: 800/24
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/101 Bovine
• CPC Class Codes: A01K 2227/106 Primate
• CPC Class Codes: A01K 67/027 New or modified breeds of vertebrates
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 13/02 of urine or of the urinary tract, e.g. urine acidifiers
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: C12N 15/8771 Bovine embryos
• CPC Class Codes: C12N 15/8778 Swine embryos
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2517/02 : Cells from transgenic animals
• CPC Class Codes: C12N 2517/04 : Cells produced using nuclear transfer
• CPC Class Codes: C12N 5/0603 : Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies

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.

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.

8. The method of any of claims 1-6, wherein the donor cell or donor cell nucleus is from mesoderm.

9. The method of any of claims 1-6, wherein the donor cell or donor cell nucleus is from endoderm.

10. The method of any of claims 1-6, wherein the donor cell or donor cell nucleus is from ectoderm.

11. The method of any of claims 1-6, wherein the donor cell or donor cell nucleus is from a fibroblast.

12. The method of any of claims 1-6, wherein the donor cell or donor cell nucleus is from an ungulate.

13. The method of any of claims 1-6, wherein the donor cell or donor cell nucleus is from an ungulate selected from the group consisting of bovine, ovine, porcine, equine, caprine and buffalo.

14. The method of any of claims 1-6, wherein the donor cell or donor cell nucleus is from a non-human mammalian fetus.

15. The method of any of claims 1-6, wherein the donor cell or donor cell nucleus is from an adult non-human mammalian cell.

16. The method of any of claims 1-6, wherein the donor cell or donor cell nucleus is selected from the group consisting of epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-lymphocytes, T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts, muscle cells, and nuclei isolated therefrom.

17. The method of any of claims 1-6, wherein the donor cell or donor cell nucleus is from an organ selected from the group consisting of skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organ, bladder, kidney and urethra.

18. The method of any of claims 1-6, wherein the oocyte is matured in vitro or in vivo prior to enucleation.

19. The method of any of claims 1-6, wherein the oocyte is matured in vitro prior to enucleation.

20. The method of any of claims 1-6, wherein the oocyte is enucleated by micro-surgical methods.

21. The method of any of claims 1-6, wherein the oocyte is enucleated about 10 to 40 hours after initiation of in vitro maturation.

22. The method of any of claims 1-6, wherein the oocyte is matured in vivo prior to enucleation.

23. The method of any of claims 1-6, wherein the non-human mammal is bovine.

24. The method of any of claims 1-6, wherein the non-human mammal is bovine.

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.

3. A method of cloning a non-human mammal by nuclear transfer comprising the following steps:

(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.

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.

7. The method of any of claims 4-6, wherein the fetus develops into an offspring.

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.

6. A method of cloning a non-human mammalian fetus by nuclear transfer comprising the following steps:

(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.