Multicellular living organisms and unmodified parts thereof and – Method of making a transgenic nonhuman animal – Via microinjection of a nucleus into an embryo – egg cell – or...
Reexamination Certificate
1999-11-02
2004-08-24
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of making a transgenic nonhuman animal
Via microinjection of a nucleus into an embryo, egg cell, or...
C800S008000, C800S014000, C435S377000
Reexamination Certificate
active
06781030
ABSTRACT:
BACKGROUND OF THE INVENTION
The production of desired proteins is useful in drug development and treatment of diseases. Several traditional methods for producing proteins, especially in high volume, are often inadequate for several reasons. Transgenic technology or cloning technology can lead to several advancements in medicine, including the production of useful proteins. Transgenic or cloning technology allows for the introduction of a transgenic nucleotide sequence into a host animal, thereby allowing for the expression of this transgenic nucleotide sequence, and production of the protein.
Accordingly, few reliable methods exist for producing transgenic or cloned animals, especially those methods that are able to produce useful proteins. Hence, a need exists for producing transgenic or cloned animals, and in particular, animals that make such desirable proteins.
SUMMARY OF THE INVENTION
The present invention provides effective methods for producing transgenic or cloned animals, and for obtaining useful proteins. The invention includes methods for cloning an animal by combining a genome from an activated donor cell with an activated, enucleated oocyte to thereby form a nuclear transfer embryo, and impregnating an animal with the nuclear transfer embryo in conditions suitable for gestation of the cloned animal. The activated donor cell is in a stage of the mitotic cell cycle such as G
1
phase, S phase, or G
2
/M phase. The activated donor cell can be a variety of cells such as a somatic cell (e.g., an adult somatic cell or an embryonic somatic cell), a germ cell or a stem cell. Types of somatic cells include fibroblast cells or epithelial cells. The activated, enucleated oocyte is in a stage of the meiotic cell cycle, such as metaphase I, anaphase I, anaphase II or telophase II. The oocyte can be enucleated chemically, by X-ray irradiation, by laser irradiation or by physical removal of the nucleus.
The invention also includes a method of producing a transgenic animal by combining a genetically engineered genome from an activated donor cell with an activated, enucleated oocyte to thereby form a transgenic nuclear transfer embryo; and impregnating an animal with the transgenic nuclear transfer embryo in conditions suitable for gestation of the transgenic animal. The stages of the cell cycle for the activated donor cell and the activated, enucleated oocyte are described above. The types of activated donor cell are also described above. The oocyte can be enucleated chemically, by X-ray irradiation, by laser irradiation or by physical removal of the nucleus.
The present invention also relates to methods of producing a nuclear transfer embryo, comprising combining a genome from an activated donor cell with an activated, enucleated oocyte. The oocyte is activated by exposing the oocyte to increased levels of calcium, and/or decreasing phosphorylation in the oocyle. Compounds or conditions that activate the oocyte are, for example, ethanol, ionophore or electrical stimulation in the presence of calcium. Increases of calcium can be between above 10% and 60% above baseline levels of calcium. The donor cell is activated by reducing the nutrients in the serum of the donor cell (e.g., 0.5% Fetal Bovine Serum) for a period of time, and then exposing the donor cell to serum having an increased amount of nutrients (10% Fetal Bovine Serum). Combining a genome from an activated donor cell with an activated oocyte can include fusing the activated donor cell with the activated oocyte, or microinjecting the nucleus of the activated donor cell into the activated oocyte.
The present invention also pertains to methods of producing a protein of interest in an animal, comprising combining a genome from an activated donor cell with an activated, enucleated oocyte to thereby form a nuclear transfer embryo, wherein the genome from the activated donor cell encodes the protein of interest; impregnating an animal with the nuclear transfer embryo in conditions suitable for gestation of a cloned animal; and purifying the protein of interest from the cloned animal. Purification of the protein of interest can be expressed in tissue, cells or bodily secretion of the cloned animal. Examples of such tissue, cells or bodily secretions are milk, blood, urine, hair, mammary gland, muscle, viscera (e.g., brain, heart, lung, kidney, pancreas, gall bladder, liver, stomach, eye, colon, small intestine, bladder, uterus and testes).
The present invention further encompasses a method of producing a heterologous protein in a transgenic animal comprising combining a genetically engineered genome from an activated donor cell with an activated, enucleated oocyte to thereby form a nuclear transfer embryo, wherein the genome from the activated donor cell encodes the heterologous protein; impregnating an animal with the nuclear transfer embryo in conditions suitable for gestation of the nuclear transfer embryo into a cloned animal; and recovering the heterologous protein from the cloned animal. The genetically engineered genome includes an operatively linked promoter (e.g., a host endogenous promoter, an exogenous promoter and a tissue-specific promoter). Examples of tissue-specific promoters are mammary-specific promoter, blood-specific promoter, muscle-specific promoter, neural-specific promoter, skin-specific promoter, hair-specific promoter and urinary-specific promoter.
The present invention also embodies methods of enucleating an oocyte having a meiotic spindle apparatus, by exposing the oocyte with a compound that destabilizes the meiotic spindle apparatus. Destabilizing the meiotic spindle apparatus results in destabilizing microtubules, chromosomes, or centrioles. Compounds that can destabilize the meiotic spindle apparatus are, for example, demecolcine, nocodazole, colchicine, and paclitaxel. To further enhance destabilization of the mieotic spindle apparatus, the temperature, osmolality or composition of medium which surrounds the oocyte can be altered.
Additionally, the invention includes methods of preparing an oocyte for nuclear transfer, comprising: exposing the oocyte to ethanol, ionophore, or to electrical stimulation, to thereby obtain an activated oocyte, and subjecting the activated oocyte to a compound that destabilizes meiotic spindle apparatus, to thereby enucleate the activated oocyte. The compounds described above destabilize the meiotic spindle apparatus. The activated oocyte can be in a stage of a meiotic cell cycle, such as metaphase I, anaphase I, anaphase II and telophase II.
The present invention advantageously allows for more efficient cloning methods. By fusing or combining an activated oocyte with the genome from an activated donor cell, the resulting nuclear transfer embryo is more competent to develop. This developmentally competent nuclear transfer embryo results in improved pregnancy rates of an animal impregnated with the nuclear transfer embryo. These animals give birth to cloned animals.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods of cloning an animal by combining an activated oocyte with the genome from an activated donor cell. “Cloning an animal” refers to producing an animal that develops From an oocyte containing genetic information or the nucleic acid sequence of another animal, the animal being cloned. The cloned animal has substantially the same or identical genetic information as that of the animal being cloned. “Cloning” also refers to cloning a cell, which includes producing an oocyte containing genetic information or the nucleic acid sequence of another animal. The resulting oocyte having the donor genome is referred to herein as a “nuclear transfer embryo.”
The present invention encompasses the cloning of a variety of animals. These animals include mammals (e.g., human, canines, felines), murine species (e.g., mice, rats), and ruminants (e.g., cows, sheep, goats, camels, pigs, oxen, horses, llamas). In particular, goats of Swiss origin, for example, the Alpine, Saanen and Toggenburg bread goats, were used in the Examples described herein. Th
Baguisi Alexander
Overstrom Eric W.
Beattie Ingrid A.
Crouch Deborah
Mintz Levin Cohn Ferris Glovsky and Popeo P.C.
Trustee of Tufts College, Ballou Hall
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