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
1997-07-03
2001-05-22
Grouch, 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...
C800S017000
Reexamination Certificate
active
06235969
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to cloning procedures in which cell nuclei derived from differentiated pig cells are transplanted into enucleated mammalian oocytes of the same species as the donor nuclei. The nuclei are reprogrammed to direct the development of cloned embryos, which can then be transferred into recipient females to produce fetuses and offspring, or used to produce cultured inner cell mass cells (CICM). The cloned embryos can also be combined with fertilized embryos to produce chimeric embryos, fetuses and/or offspring.
BACKGROUND OF THE INVENTION
The use of ungulate inner cell mass (ICM) cells for nuclear transplantation has also been reported. For example, Collas et al.,
Mol. Reprod. Dev.,
38:264-267 (1994) discloses nuclear transplantation of bovine ICMs by microinjection of the lysed donor cells into enucleated mature oocytes. Collas et al. disclosed culturing of embryos in vitro for seven days to produce fifteen blastocysts which, upon transferral into bovine recipients, resulted in four pregnancies and two births. Also, Keefer et al.,
Biol. Reprod.,
50:935-939 (1994), disclosed the use of bovine ICM cells as donor nuclei in nuclear transfer procedures, to produce blastocysts which, upon transplantation into bovine recipients, resulted in several live offspring. Further, Sims et al.,
Proc. Natl. Acad. Sci., USA,
90:6143-6147 (1993), disclosed the production of calves by transfer of nuclei from short-term in vitro cultured bovine ICM cells into enucleated mature oocytes.
The production of live lambs following nuclear transfer of cultured embryonic disc cells has also been reported (Campbell et al.,
Nature,
380:64-68 (1996)). Still further, the use of bovine pluripotent embryonic cells in nuclear transfer and the production of chimeric fetuses has been reported (Stice et al.,
Biol. Reprod.,
54:100-110 (1996); Collas et al,
Mol. Reprod. Dev.,
38:264-267 (1994)). Collas et al demonstrated that granulosa cells (adult cells) could be used in a bovine cloning procedure to produce embryos. However, there was no demonstration of development past early embryonic stages (blastocyst stage). Also, granulosa cells are not easily cultured and are only obtainable from females. Collas et al did not attempt to propagate the granulosa cells in culture or try to genetically modify those cells. Wilmut et al (
Nature,
365:810-813 (1997)) produced nuclear transfer sheep offspring derived from fetal fibroblast cells, and one offspring from a cell derived from an adult sheep.
Cloning pig cells is more difficult in comparison with cells of other species. This phenomenon is illustrated by the following table:
SPECIES (from hardest
OFFSPRING
to easiest to clone)
CELL TYPE CLONED
PRODUCED
Pig (Prather, 1989)
2 and 4 cell stage
yes
embryo
Pig (Prather, 1989;
greater than 4
no
Liu et al., 1995)
cell stage
Mouse (Cheong et al.,
2, 4 and 8 cell
yes
1993)
stage embryo
Mouse (Tsunoda et
greater than 8
no
al., 1993)
cell stage
Cattle (Keefer et
64 to 128 cell
yes
al., 1994)
stage (ICM)
Cattle (Stice et al.,
embryonic cell
no
1996)
line from ICM
Sheep (Smith et al.,
64 to 128 cell
yes
1989)
stage (ICM)
Sheep (Campbell et
embryonic cell
yes
al., 1996)
line from ICM
Sheep (Wilmut et al.,
fetal and adult
yes
1997)
cells
There also exist problems in the area of producing transgenic pigs. By current methods, heterologous DNA is introduced into either early embryos or embryonic cell lines that differentiate into various cell types in the fetus and eventually develop into a transgenic animal. However, many early embryos are required to produce one transgenic animal and, thus, this procedure is very inefficient. Also, there is no simple and efficient method of selecting for a transgenic embryo before going through the time and expense of putting the embryos into surrogate females. In addition, gene targeting techniques cannot be easily accomplished with early embryo transgenic procedures.
Embryonic stem cells in mice have enabled researchers to select for transgenic cells and perform gene targeting. This allows more genetic engineering than is possible with other transgenic techniques. However, embryonic stem cell lines and other embryonic cell lines must be maintained in an undifferentiated state that requires feeder layers and/or the addition of cytokines to media. Even if these precautions are followed, these cells often undergo spontaneous differentiation and cannot be used to produce transgenic offspring by currently available methods. Also, some embryonic cell lines have to be propagated in a way that is not conducive to gene targeting procedures.
Methods for deriving embryonic stem (ES) cell lines in vitro from early preimplantation mouse embryos are well known. (See, e.g., Evans et al.,
Nature,
29:154-156 (1981); Martin,
Proc. Natl. Acad. Sci., USA,
78:7634-7638 (1981)). ES cells can be passaged in an undifferentiated state, provided that a feeder layer of fibroblast cells (Evans et al., Id.) or a differentiation inhibiting source (Smith et al.,
Dev. Biol.,
121:1-9 (1987)) is present.
ES cells have been previously reported to possess numerous applications. For example, it has been reported that ES cells can be used as an in vitro model for differentiation, especially for the study of genes which are involved in the regulation of early development. Mouse ES cells can give rise to germline chimeras when introduced into preimplantation mouse embryos, thus demonstrating their pluripotency (Bradley et al.,
Nature,
309:255-256 (1984)).
In view of their ability to transfer their genome to the next generation, ES cells have potential utility for germline manipulation of livestock animals by using ES cells with or without a desired genetic modification. Moreover, in the case of livestock animals, e.g., ungulates, nuclei from like preimplantation livestock embryos support the development of enucleated oocytes to term (Smith et al.,
Biol. Reprod.,
40:1027-1035 (1989); and Keefer et al.,
Biol. Reprod.,
50:935-939 (1994)). This is in contrast to nuclei from mouse embryos which beyond the eight-cell stage after transfer reportedly do not support the development of enucleated oocytes (Cheong et al,
Biol. Reprod.,
48:958 (1993)). Therefore, ES cells from livestock animals are highly desirable because they may provide a potential source of totipotent donor nuclei, genetically manipulated or otherwise, for nuclear transfer procedures.
Some research groups have reported the isolation of purportedly pluripotent embryonic cell lines. For example, Notarianni et al.,
J. Reprod. Fert. Suppl.,
43:255-260 (1991), reports the establishment of purportedly stable, pluripotent cell lines from pig and sheep blastocysts which exhibit some morphological and growth characteristics similar to that of cells in primary cultures of inner cell masses isolated immunosurgically from sheep blastocysts. Also, Notarianni et al.,
J. Reprod. Fert. Suppl.,
41:51-56 (1990) discloses maintenance and differentiation in culture of putative pluripotential embryonic cell lines from pig blastocysts. Gerfen et al.,
Anim. Biotech,
6(1):1-14 (1995) discloses the isolation of embryonic cell lines from porcine blastocysts. These cells are stably maintained in mouse embryonic fibroblast feeder layers without the use of conditioned medium, and reportedly differentiate into several different cell types during culture.
Further, Saito et al.,
Roux's Arch. Dev. Biol.,
201:134-141 (1992) reports cultured, bovine embryonic stem cell-like cell lines which survived three passages, but were lost after the fourth passage. Handyside et al.,
Roux's Arch. Dev. Biol.,
196:185-190 (1987) discloses culturing of immunosurgically isolated inner cell masses of sheep embryos under conditions which allow for the isolation of mouse ES cell lines derived from mouse ICMs. Handyside et al. reports that under such conditions, the sheep ICMs attach, spread, and develop areas of both ES cell-like and endoderm-like cells, but that after prolonged culture only endoderm-like cells are evident.
Recently, Cherny et a
Cibelli Jose
Golueke Paul
Robl James M.
Stice Steven L.
Grouch Deborah
Teskin Robin L.
University of Massachusetts
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