Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal to manufacture... – The protein is isolated or extracted from milk
Reexamination Certificate
1999-07-15
2002-05-28
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal to manufacture...
The protein is isolated or extracted from milk
C800S015000, C800S016000, C800S017000, C800S024000
Reexamination Certificate
active
06395958
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the cloning of animals.
BACKGROUND OF THE INVENTION
The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Researchers have been developing methods for cloning mammalian animals over the past two decades. These reported methods typically include the steps of (1) isolating a cell, most often an embryonic cell; (2) inserting the cell or nucleus isolated from the cell into an enucleated oocyte (e.g., the oocyte's nucleus was previously extracted), and (3) allowing the embryo to mature in vivo.
The first successful nuclear transfer experiment using mammalian cells was reported in 1983, where the pronuclei isolated from a murine (mouse) zygote were inserted into an enucleated oocyte and resulted in like offspring(s). McGrath & Solter, 1983
, Science
220:1300-1302. Subsequently, others described the production of chimeric murine embryos (e.g., embryos that contain a subset of cells having significantly different nuclear DNA from other cells in the embryo) using murine primordial germ cells (PGC). These cells are and can give rise to pluripotent cells (e.g., cells that can differentiate into other types of cells but do not differentiate into a grown animal). Matsui et al., 1992
, Cell
70:841-847 and Resnick et al., 1992
, Nature
359:550; Kato et al., 1994
, Journal of Reproduction and Fertility Abstract Series
, Society For the Study of Fertility, Annual Conference, Southampton, 13:38.
Some publications related to murine pluripotent cells stress the importance of steel factor for converting precursor cells into pluripotent cells. U.S. Pat. Nos. 5,453,357 and 5,670,372, entitled “Pluripotent Embryonic Stem Cells and Methods of Making Same,” issued to Hogan. These same publications indicate that murine pluripotent cells exhibit strong, uniform alkaline phosphatase staining.
Although murine animals were never clearly cloned from nuclear transfer techniques using embryonic cells, some progress was reported in the field of cloning ovine (sheep) animals. One of the first successful nuclear transfer experiments utilizing ovine embryonic cells as nuclear donors was reported in 1986. Willadsen, 1986
, Nature
320:63-65. A decade later, others reported that additional lambs were cloned from ovine embryonic cells. Campbell et al., 1996
, Nature
380:64-66 and PCT Publication WO 95/20042. Recently, another lamb was reported to be cloned from ovine somatic mammary tissue. Wilmut et al., 1997
, Nature
385:810-813. Some methods for cloning ovine animals focused upon utilizing serum deprived somatic ovine cells and cells isolated from ovine embryonic discs as nuclear donors. PCT Publications WO 96/07732 and WO 97/07669. Other methods for cloning ovine animals involved manipulating the activation state of an in vivo matured oocyte after nuclear transfer. PCT Publication WO 97/07668.
While few lambs were produced, publications that disclose cloned lambs report a cloning efficiency that is, at best, approximately 0.4%. Cloning efficiency, as calculated for the previous estimate, is a ratio equal to the number of cloned lambs divided by the number of nuclear transfers used to produce that number of cloned lambs.
Despite the slower progress endemic to the field of cloning bovine animals, a bovine animal was cloned using embryonic cells derived from 2-64 cell embryos. This bovine animal was cloned by utilizing the nuclear transfer techniques set forth in U.S. Pat. Nos. 4,994,384 and 5,057,420. Others reported that cloned bovine embryos were formed by nuclear transfer techniques utilizing the inner cell mass cells of a blastocyst stage embryo. Sims & First, 1993
, Theriogenology
39:313 and Keefer et al., 1994
, Mol. Reprod. Dev
. 38:264-268. In addition, another publication reported that cloned bovine embryos were prepared by nuclear transfer techniques that utilized PGCs isolated from fetal tissue. Delhaise et al., 1995
, Reprod. Fert. Develop
. 7:1217-1219; Lavoir 1994
, J Reprod. Dev
. 37:413-424; and PCT application WO 95/10599 entitled “Embryonic Stem Cell-Like Cells.” However, the reports demonstrated that cloned PGC-derived bovine embryos never clearly developed past the first trimester during gestation. Similarly, embryonic stem cell (e.g., cell line derived from embryos which are undifferentiated, pluripotent, and can establish a permanent cell line which exhibits a stable karyotype), ESC, derived bovine embryos never developed past fifty-five days, presumably due to incomplete placental development. Stice et al., 1996
, Biol. Reprod
. 54: 100-110.
Despite the progress of cloning ovine and bovine animals, there remains a great need in the art for methods and materials that increase cloning efficiency. In addition there remains a great need in the art to expand the variety of cells that can be utilized as nuclear donors, especially expanding nuclear donors to non-embryonic cells. Furthermore, there remains a long felt need in the art for karyotypically stable permanent cell lines that can be used for genome manipulation and production of transgenic cloned animals.
SUMMARY
The present invention relates to cloning technologies. The invention relates in part to immortalized, totipotent cells useful for cloning animals, the embryos produced from these cells using nuclear transfer techniques, animals that arise from these cells and embryos, and the methods and processes for creating such cells, embryos, and animals.
The present invention provides multiple advantages over the tools and methods currently utilized in the field of mammalian cloning. Such features and advantages include:
(1) Production of cloned animals from virtually any type of cell. The invention provides materials and methods for reprogramming non-totipotent cells into totipotent cells. These non-totipotent cells may be of non-embryonic origin. This feature of the invention allows for the ability to assess the phenotype of an existing animal and then readily establish a permanent cell line for cloning that animal.
(2) Creation of permanent cell lines from virtually any type of cell. Permanent cell lines provide a nearly unlimited source of genetic material for nuclear transfer cloning techniques. In one aspect of the invention, non-totipotent precursor cells can be reprogrammed into totipotent and permanent cells. These non-totipotent precursor cells may be non-embryonic cells. Permanent cell lines provide the advantage of enhancing cloning efficiency due to the lower cellular heterogeneity within the cell lines (e.g., permanent cells that have lower rates of differentiation than primary culture cell lines currently used for cloning). In addition, the permanent cell lines can be manipulated in vitro to produce cells, embryos, and animals whose genomes have been manipulated (e.g., transgenic). Furthermore, permanent cell lines can be more easily stored, transported, and re-established in culture than other types of cell lines.
(3) Enhancement of the efficiency for cloning embryos as a result of utilizing asynchronous, permanent, and karyotypically stable cell lines in a complete in vitro embryo production system.
Cloning efficiency can be expressed by the ratio between the number of embryos resulting from nuclear transfer and the number of nuclear transfers performed to give rise to the embryos. Alternatively, cloning efficiency can be expressed as the ratio between the number of live born animals and the number of nuclear transfers performed to give rise to these animals.
Immortalized and Totipotent Cells of the Invention
In a first aspect, the invention features a totipotent mammalian cell. Preferably, the totipotent mammalian cell is (1) a cultured cell; (2) a cell cultured in a cell line; and (3) an immortalized cell. In addition, the mammalian cell is preferably an ungulate cell and more preferably a bovine cell.
The term “mammalian” or “mammal” as used herein refers to any animal of the class Mammalia. A mammalian animal o
Betthauser Jeffrey M.
Bishop Michael D.
Jurgella Gail L.
Pace Marvin M.
Strelchenko Nikolai S.
Crouch Deborah
Foley & Lardner
Infigen, Inc.
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