Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2000-05-22
2004-01-20
Falk, Anne-Marie (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S325000
Reexamination Certificate
active
06680199
ABSTRACT:
FIELD OF THE INVENTION
The present invention is generally directed to an improved process for cloning or multiplying mammalian embryonic cells and to an improved process for transferring the nuclei of donor embryonic cells into enucleated recipient oocytes. The present invention is specifically directed to a process for parthenogenically activating mammalian oocytes and to the use of the oocytes.
CITATION OF REFERENCES
A full citation of the references appearing in this disclosure can be found in the section preceding the Claims.
DESCRIPTION OF THE PRIOR ART
Advanced genetic improvement and selection techniques continue to be sought in the field of animal husbandry. With specific reference to dairy cattle, for example, significant increases in milk production have been made with the wide scale use of genetically superior sires and artificial insemination. Dairy cows today produce nearly twice as much milk as they did 30 years ago. Further genetic improvement can be accomplished by the multiplication of superior or genetically manipulated animals by cloning using embryonic cells. For purposes of the present invention, the term “embryonic cell” refers to embryos and cells cultured from embryos including embryonic stem cells.
It has now become an accepted practice to transplant embryonic cells in cattle to aid in the production of genetically superior stock. The cloning of embryonic cells together with the ability to transplant the cloned embryonic cells makes it possible to produce multiple genetically identical animals. Embryonic cell cloning is the process of transferring the nucleus of an embryonic donor cell to an enucleated recipient ovum or oocyte. The clone then develops into a genetically identical offspring to the donor embryonic cell.
Nuclear Transfer
The ability to produce multiple copies of genetically identical individuals from embryonic cells derived from a single embryo provides a means for embryonic cell selection where the cloned lines descending from one embryo could be selected by progeny testing for further clonal multiplication. Nuclear transfer creates the possibility of permitting rapid changes in animal characteristics such as meat and milk production. Nuclear transfer is one process for producing multiple copies of an embryo. Reference is made to
First and Prather
(1991) and U.S. Pat. No. 4,994,384 to
Prather et al.
, which are incorporated herein by reference, for a description of nuclear transfer.
Briefly, nuclear transfer involves the transfer of an embryonic cell or nucleus from an embryonic cell. Either entity is derived from a multicellular embryo (usually 20 to 64-cell stage) into an enucleated oocyte, an oocyte with the nucleus removed or destroyed. The oocyte then develops into a multi-cellular stage and is used to produce an offspring or as a donor in serial recloning.
Cloning by nuclear transfer has great potential for the multiplication of genotypes of superior economic value (
Gray et al.
, 1991). Nuclear transfer to produce identical offspring has many advantages over embryo splitting or embryonic cell aggregation to produce fetal placental chimeras: 1) multiple copies of superior, genetically identical animals are possible; 2) embryonic cell sexing and cryopreservation may be applied to the cloning scheme allowing all clones to be of preselected sex; and 3) embryonic cells from different genetic strains can be frozen and can be multiplied after testing.
Oocyte Activation
Cattle ovulate spontaneously approximately every 21 days, about 24-36 hours after a surge of luteinizing hormone (LH). In vivo and in vitro matured oocytes are activated by entry of sperm into the oocyte. Activation by sperm can occur in bovine oocytes matured in vitro as early as 15 hours. However, currently oocytes must be matured for more than about 28 hours to respond to parthenogenic activation stimuli. This datum implies that either the sperm provide a factor necessary for oocyte activation (
Whitaker and Irvine
, 1984
; Stice and Robl
, 1990
; Swann
, 1990) or that processes that increase intracellular calcium alone are not sufficient in the bovine oocyte to overcome the cytostatic factor(s).
The stage of maturation of the oocyte at enucleation and nuclear transfer is important (
First and Prather
, 1991). In general, successful mammalian embryonic cell cloning practices use the metaphase II stage oocyte as the recipient oocyte. At this stage, it is believed the oocyte is sufficiently “activatable” to treat the introduced nucleus as it does a fertilizing sperm.
Activation of mammalian oocytes involves exit from meiosis and reentry into the mitotic cell cycle by the secondary oocyte and the formation and migration of pronuclei within the cell. Viable oocytes prepared for maturation and subsequent activation are required for nuclear transfer techniques.
Activation requires cell cycle transitions. The maturation Promoting Factor complex becomes essential in the understanding of oocyte senescence and age dependent responsiveness to activation. MPF activity is partly a function of calcium (Ca
2+
). A major imbalance in the components of the multi-molecular complex which is required for cell cycle arrest may be responsible for the increasing sensitivity of oocytes to activation stimuli during aging.
Parthenogenetic Activation
Parthenogenic activation of oocytes may be used instead of fertilization by sperm to prepare the oocytes for nuclear transfer. Parthenogenesis is the “production” of embryonic cells, with or without eventual development into an adult, from a female gamete in the absence of any contribution from a male gamete (
Kaufman
1981).
Parthenogenetic activation of mammalian oocytes has been induced in a number of ways. Using an electrical stimulus to induce activation is of particular interest because electrofusion is part of the current nuclear transfer procedure.
Tarkowski, et al.
(1970) reported successful use of electric shock to activate the mouse ova while in the oviduct. Parthenogenetic activation in vitro by electrical stimulation with electrofusion apparatus used for embryonic cell-oocyte membrane fusion has been reported (
Stice and Robl
, 1990
; Collas and Robl
, 1990);
Onodera and Tsunoda
, 1989). In the rabbit, with the combined AC and DC pulse 80 to 90 percent of freshly ovulated oocytes have been activated (
Yang, et al.
, (1990, 1991).
Ozil
(1990) used multiple electrical pulses to induce adequate activation of rabbit oocytes. Adapting this for nuclear transfer,
Collas and Robl
(1990) obtained improved development to term.
It is believed that the most effective activating stimulus would be one that mimicked the response of mammalian oocytes to fertilization. One such response of rabbit oocytes is characterized by repetitive transient elevations in intracellular Ca
2+
levels followed by rapid return to base line (
Fissore and Robl
, 1992), which may explain the improved development with activation by multiple electrical pulses.
Parthenogenic activation of metaphase II bovine oocytes has proven to be more difficult than mouse oocytes. Mouse oocytes have been activated by exposure to Ca
+2
-Mg
+2
free medium (
Surani and Kaufman
, 1977), medium containing hyaluronidase (
Graham
, 1970), exposure to ethanol (
Cuthbertson
, 1983), Ca
+2
ionophores or chelators (
Steinhardt et al.
, 1974;
Kline and Kline
, 1992), inhibitors of protein synthesis (
Siracusa et al.
, 1978) and electrical stimulation (
Tarkowski et al.
, 1970). These procedures that lead to high rates of parthenogenic activation and development of mouse oocytes do not activate young bovine oocytes and/or lead to a much lower development rate. Fertilization and parthenogenic activation of mouse oocytes is also dependent on post ovulatory aging (
Siracusa et al.
, 1978).
Activation of bovine oocytes has been reported by ethanol (
Nagai
, 1987), electrical stimulation (
Ware et al.
, 1989), exposure to room temperature (
Stice and Keefer
, 1992), and a combination of electrical stimulation and cycloheximide (
First et al.
, 1992
; Yang et al.
, 1992).
Leibfried-Rutledge M. Lorraine
Northey David L.
Stice Steven L.
Susko-Parrish Joan L.
Falk Anne-Marie
Foley & Lardner
Infigen, Inc.
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