Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-10-09
2002-11-19
Baker, Anne-Marie (Department: 1632)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024310
Reexamination Certificate
active
06482937
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to a method of isolating and/or enriching and/or selectively propagating pluripotential porcine cells, genetically modified porcine cells and pigs for use in said method, transgenic pigs providing a source of such cells and genetic selectable marker constructs for producing genetically modified cells and transgenic pigs.
Stem cells are progenitor cells which have the capacity both to self-renew and to differentiate into mature somatic cells. Embryonic stem cells are the archetypal stem cell, being capable of differentiating to form the whole gamut of cell types found in an adult animal. Such stem cells are described as “totipotential” or “pluripotential” as they are capable of differentiating into many cell types. Other types of stem cells, for example bone marrow stem cells and epidermal stem cells, persist in the adult animal. These stem cells have a more restricted capacity for differentiation.
In general, when required for research purposes or for medical use, stem cells have to be isolated from tissue samples by various fractionation procedures. However, even after careful segregation of cell types these stem cell preparations consist of mixed cell types, and while enriched for stem cells include high proportions of differentiated cells which are not categorized as stem cells.
Furthermore, most stem cells cannot be grown readily in culture. When attempts are made to culture stem cells, the cells being cultured (which ordinarily contain a mixed population of cell types) grow at different rates and stem cells rapidly become overgrown by non-stem cell types. An exception is that embryonic stem cells from two specific strains of mice (129 and Black 6) can be cultured in vitro (Evans et al. (1981) Nature 292:154-156). Thus, established lines of murine embryonic stem cells can be obtained by culturing early (3½ day) embryonic cells from murine strain 129 and Black 6, or hybrids thereof. Embryonic cell lines from species other than the mouse are not so easily propagated. For an extensive review of isolation and propagation of stem cells see PCT publication WO 94/24274, incorporated by reference herein.
There has developed a pressing need to isolate and maintain in vitro embryonic stem cells from species of animals other than murine, such as other laboratory animals and domesticated animals, and most especially, from pigs. However, prior to the present invention the problems associated with producing cultures of porcine (or pig) stem cells, including the problem of producing cell populations of a satisfactorily low degree of heterogeneity and the problem of overgrowth in culture of non-pluripotent porcine cells, have not been solved. A particular problem associated with the continuing presence of certain differentiated cell types is that these can cause elimination of stem cells from the culture by inducing their differentiation or programmed cell death.
Thus according to the present invention, there is provided a porcine cell capable of being cultured under appropriate selective culture conditions so as to enable selective propagation of pluripotential stem cells, characterized in that said pluripotent porcine cells contain a genetic selectable marker, whereby a gene product associated with the genetic selectable marker is produced and which under said culture conditions causes selective survival and/or division of the desired pluripotent cells to occur. “Selective culture conditions” are those conditions under which a population of cells is selectively grown. For example, to selectively grow cells that contain a gene which transfers resistance to a specific drug, the selective culture conditions would contain the drug so that all cells that did not express the drug resistance would be eliminated. Such selective culture conditions are well known in the art.
The invention further provides according to another aspect thereof, a transgenic animal, in this instance a transgenic pig, having genetic characteristics such that it or its progeny, during embryonic development or later life, constitute a source of porcine pluripotential cells as defined above. Such transgenic pigs may be produced according to the invention by introducing a genetic selectable marker into a fertilized oocyte or an embryonic cell, the genetic marker having the characteristics defined below, and utilizing the resulting transformed oocyte or embryonic cells as a progenitor cell for the desired transgenic animal.
A further aspect of the invention is vectors for use in producing an animal cell, for example a pig cell. Thus the invention further provides vectors for use in genetically modifying animal cells so as to produce transformed cells suitable for use as the source of cells for the method referred to below, said vector comprising a first genetic component corresponding to a genetic selectable marker and a second genetic component which, in the genetically modified porcine cell(s), results in the differential expression of the genetic selectable marker as a stably integrated construct. Such vectors may be in the form of expression vectors in which said second genetic component includes control sequences which are differentially activated in pluripotential stem cells and in cells other than the desired stem cells. The invention covers vectors which when used to transform porcine stem cells become integrated into the animal genome as well as vectors which do not become so integrated.
SUMMARY OF THE INVENTION
The present invention provides for a method of isolating and/or propagating porcine stem cells, more specifically pluripotential porcine embryonic stem cells. The pluripotential (or “pluripotent”) cells are isolated and/or propagated by the use of a selectable marker gene or nucleic acid sequence which is inserted into the genetic material of cells contained in a cell culture comprising porcine pluripotent embryonic stem cells, and which permits the survival and growth of said porcine embryonic stem cells. The selectable marker gene or nucleic acid sequence is inserted so as to be regulated by a control or promoter nucleotide sequence in said embryonic stem cells, for example the control sequence being the porcine Oct-4 promoter nucleotide sequence as described below. The terms “Oct-4 promoter nucleotide sequence” or “Oct-4 promoter sequence” refer to the promoter region of the Oct-4 gene, or any fragment of the promoter region that maintains promoter activity. The invention also provides for a transgenic pig which will constitute a source of said pluripotent cells.
By providing a sufficient and reliable source of porcine pluripotential embryonic stem cells, the present invention permits those skilled in the art to genetically modify the cells with a desired genetic modification. For example, said embryonic stem cells may be genetically altered so as to not express a cell surface membrane protein that may cause rejection of porcine cells after xenotransplantation. Said genetically altered cells are then useful in creating a transgenic pig, or line of transgenic pigs, which will not express said surface membrane protein and which, therefore, will contain organs that are less likely to be rejected upon xenotransplantation.
REFERENCES:
patent: 5817789 (1998-10-01), Heartlein et al.
patent: WO 94/24274 (1994-10-01), None
patent: WO 95/20042 (1995-07-01), None
patent: 2476 (1995-12-01), None
Thomas e.t al., Site-Directed Mutagenesis by Gene Targeting in Mouse Embryo-Derived Stem Cells, 1987, Cell, vol. 51: 503-512.*
Campbell and Wilmut. Totipotency or multipotentiality of cultured cells: Applications and progress. Theriogenology 47: 63-72, Jan. 1997.*
Gearhart, J. New potential for human embryonic stem cells. Science 282: 1061-1062, Nov. 1998.*
Iannaccone et al. Pluripotent embryonic stem cells from the rat are capable of producing chimeras. Dev. Biol. 163: 288-292, 1994.*
Shamblott et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. PNAS 95: 13726-13731, Nov. 1998.*
Stice et al. Pluripotent bovine embryonic cell lin
Akiyoshi Donna E.
Baetscher Manfred W.
Kaplan Ruth A.
Baker Anne-Marie
BioTransplant, Inc.
Grant Alan J.
Olstein Elliot M.
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