Multicellular living organisms and unmodified parts thereof and – Method of making a transgenic nonhuman animal
Reexamination Certificate
1996-09-20
2001-02-27
Chambers, Jasemine (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of making a transgenic nonhuman animal
C800S017000, C435S325000, C435S455000
Reexamination Certificate
active
06194635
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the area of undifferentiated cells and methods of producing such cells. More specifically, the invention relates to pluripotent ungulate cells, particularly porcine cells, and to transgenic and chimeric ungulates produced from such cells.
BACKGROUND OF THE INVENTION
Embryonic stem cells (ES cells) were first cultured from mouse embryos using a feeder layer of mouse fibroblasts or media conditioned with buffalo rat liver cells. The established ESC lines from mouse embryos have a characteristic phenotype consisting of a large nucleus, a prominent nucleolus, and relatively little cytoplasm. Such cells can be grown relatively indefinitely using the appropriate culture conditions. They can be induced to differentiate in vitro using retinoic acid or spontaneously by removal of the feeder layer or conditioned media. In addition, these cells can be injected into a mouse blastocyst to form a somatic and germ line chimera. This latter property has allowed mouse ESCs to be used for the production of transgenic mice with specific changes to the genome. See M. Evans et al.,
Nature
292, 154 (1981); G. Martin,
Proc. Natl. Acad. Sci. USA
78, 7638 (1981); A. Smith et al.,
Developmental Biology
121, 1 (1987); T. Doetschman et al.,
Developmental Biology
127, 224 (1988)(; A. Handyside et al.,
Roux's Arch Dev. Biol.
198, 48 (1989).
The active compound that allows the culture of murine embryonic stem cells has been identified as differentiation inhibiting activity (DIA), also known as leukemia inhibitory factor (LIF). See A. Smith,
J. Tiss. cult. Meth.
13, 89 (1991); J. Nichols et al.,
Development
110, 1341 (1990). Recombinant forms of LIF can be used to obtain ESCs from mouse embryos. See S. Pease et al.,
Developmental Biology
141, 344 (1990). Also see U.S. Pat. No. 5,166,065 issued Nov. 24, 1992 to Williams, et al.
Subsequent to the work with mouse embryos, several groups have attempted to develop stem cell lines from sheep, pig and cattle. A few reports indicate that a cell line with a stem cell-like appearance has been cultured from porcine embryos using culture conditions similar to that used for the mouse. See M. Evans et al., PCT Application W090/03432; E. Notarianni et al.,
J. Reprod. Fert., Suppl.
41, 51 (1990); J. Piedrahita et al.,
Theriogenology
34, 879 (1990); E. Notarianni et al.,
Proceedings of the
4th World Congress on Genetics Applied to Livestock Productions, 58 (Edinburgh, July 1990).
Attempts have been made regarding the culture of embryonic stem cells from avian embryos. It is difficult to establish a continuous line of chicken cells without viral or chemical transformation, and most primary chicken lines do not survive beyond 2-3 months. The culture of cells from the unincubated embryo is difficult, and under reported conditions such cells do not survive beyond two weeks. See E. Mitrani et al.,
Differentiation
21, 56-61 (1982); E. Sanders et al.,
Cell Tissue Res.
220, 539 (1981).
In U.S. Pat. No. 5,340,740 Petille et al. cultured chicken embryo cells on a mouse feeder layer in the presence of conditioned media and obtained the cultured stem cells.
Embryonic stem (ES) cells, the pluripotent outgrowths of blastocysts, can be cultured and manipulated in vitro and then returned to the embryonic environment to contribute normally to all tissues including the germline (for review see Robertson, E. G. (1986) Trends in Genetics 2:9-13). Not only can ES cells propagated in vitro contribute efficiently to the formation of chimeras, including germline chimeras, but in addition, these cells can be manipulated in vitro without losing their capacity to generate germ-line chimeras (Robertson, E. J., et al. (1986)
Nature,
323:445-447).
ES cells thus provide a route for the generation of transgenic animals such as transgenic mice, a route which has a number of important advantages compared with more conventional techniques, such as zygote injection and viral infection (Wagner and Stewart (1986) in
Experimental Approaches to Embryonic Development.
J. Rossant and A. Pedersen eds. Cambridge; Cambridge University Press), for introducing new genetic material into such animals.
However, it is known that ES cells and certain EC (embryonal carcinoma) cell lines will only retain the stem cell phenotype in vitro when cultured on a feeder layer of fibroblasts (such as murine STO cells, e.g., Martin, G. R. and Evans, M. J. (1975)
Proc. Natl. Acad. Sci. USA
72:1441-1445) or when cultured in medium conditioned by certain cells (e.g. Koopman, P. and Cotton, R. G. H. (1984)
Exp. Cell Res.
154:233-242; Smith, A. G. and Hooper, M. L. (1987)
Devel. Biol.
121:1-91). In the absence of feeder cells or conditioned medium, the ES cells spontaneously differentiate into a wide variety of cell types, resembling those found during embryogenesis and in the adult animal. The factors responsible for maintaining the pluripotency of ES cells have, however, remained poorly characterized.
The above methods involve the use of ES cells as starting materials. Very limited numbers of such cells are available. Any method which would allow for producing large numbers of ES cell would be very desirable.
SUMMARY OF THE INVENTION
A method of producing ungulate cells (porcine cells in particular) exhibiting an embryonic stem cell phenotype is disclosed as are the resulting pluripotent cells and chimeric ungulates (e.g., porcine) produced from the cells. Primordial germ cells are isolated from gonadal ridges of an ungulate embryo at a particular stage in development e.g., day-25 porcine embryos. The stage of development at which primordial germ cells are preferably extracted from an embryo of a particular species will vary with the species. For example, primordial germ cells are preferably extracted from a day 34-40 bovine embryo. Determination of the appropriate embryonic developmental stage for such extraction is readily performed using the guidance provided herein and ordinary skill in the art. The PG cells were cultured on inactivated STO cells under growth inducing conditions in long term cell culture (over 30 days). The resulting cells resembled ES cells in morphology including a large nucleus, prominent nucleoli and reduced cytoplasm as compared with differentiated adult cells. The cells can be passed several times in culture, be maintained for several months in culture, and survive cryopreservation in liquid nitrogen.
An object of the invention is to provide a method for producing ungulate cells (e.g., porcine cells) which exhibit an ES cell phenotype.
Another object is to provide pluripotent cells using germ cells as a starting material.
Another object is to provide chimeric ungulates (e.g., porcine, and bovine) using pluripotent cells of the invention.
Yet another object is to provide useful pharmaceutical products from the chimeric or transgenic ungulates produced with the cells of the invention.
An advantage of the invention is that large numbers of pluripotent cells can be quickly and efficiently produced from cells of an embryo thought to have developed too far to provide a source for pluripotent cells.
Another advantage is that the pluripotent cells of the invention can be used to produce a wide range of different chimeric ungulates (e.g., porcine) via homologous recombination methodology.
Yet another advantage of the invention is that thousands of pluripotent cells can be quickly and efficiently produced from germ cells extracted from a single ungulate embryo.
A feature of the invention is that the starting material is primordial germ cells isolated from gonadal ridges of ungulate embryo (e.g., day-25 porcine embryos or 34-40 day bovine embryos).
These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the ungulate cells expressing embryonic stem cell phenotype, method of making same, and chimeric and transgenic ungulates as more fully described below.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Before the present ungulate cells expr
Anderson Gary B.
Shim Hosup
Borden Paula A.
Bozicevic Karl
Bozicevic, Field & Francis
Chambers Jasemine
Martin Jill D.
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