Multicellular living organisms and unmodified parts thereof and – Nonhuman animal
Reexamination Certificate
1998-08-26
2001-04-10
Hauda, Karen M. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
C800S013000, C800S014000
Reexamination Certificate
active
06215039
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to animals harboring a non-native germ cell, to corresponding animal lines and germ cells, and to methods for obtaining the same.
2. Discussion of the Background
There have been many attempts to influence differentiation of developing cells by modifying the genotype of an embryo and then observing its effect on the phenotypic development pattern in the progeny. These techniques included transgenic methods (Brinster et al, Harvey Lectures, 80:1-38 (1986) nuclear transfer (McGrath & Solter,
Science
(1983) 220:1300-1302) and cell-egg fusions (Graham, “Heterospecific Genome Interaction,” 1969 Wistar Institute Press, pp. 19-35, ed. Defendi). Of these, the latter two have had limited success.
Another approach might be to add a stem cell(s) to an early embryo and determine its effect on development. For example, one might imagine that a stem cell from bone marrow would contribute to the population of, and thus modify the differentiation of the evolving bone marrow cells in the host embryo. To pilot these experiments, older embryo cells were introduced into young embryos resulting in modest success with colonization (Moustafa & Brinster,
J. Exp. Zool.
(1972) 181:193).
These initial experiments were followed by studies using bone marrow stem cells and teratocarcinoma stem cells (also called embryonal carcinoma cells). There was evidence that both these cells colonized the developing embryo (Brinster,
J. Exp. Med.
(1974) 140:1049-1056). In the case of the embryonal carcinoma (EC) cell, the colonization was demonstrated dramatically by a change in the color of hairs in the coat of the mouse. This was an exciting result which stimulated a great deal of interest among scientists in the field, because it showed the possibility of colonizing an animal with non-embryo cells. This would provide a means to introduce new genetic information through the DNA of the colonizing cells.
The next year these results were confirmed and extended by two other laboratories (for work done in one of these laboratories, see Mintz & Illmensee,
Proc. Nat. Acad. Sci.
(
USA
) (1975) 72:3585-3589) and it was demonstrated that the introduced EC cells may colonize numerous tissues including germ cells (sperm and eggs). Thus, a gene that was mutated, modified, or added to the cell in vitro could eventually end up in sperm or eggs of an animal, creating a new genetic strain of mice.
Unfortunately EC cells colonized the germline poorly and a better cell line was sought. In 1981 two scientists, Gail Martin and Martin Evans, independently described a more efficient cell designated the embryonic stem (ES) cell (Martin,
Proc. Nat. Acad. Sci.
(
USA
) (1981) 78:7634; Evans & Kaufman,
Nature
(1981) 292:154). These cells colonize the germline better than EC cells. However, it seems likely that they arise from the same pool of primitive cells in the embryo and are quite similar in biological characteristics.
Embryonic stem cells can be modified in vitro (in culture flasks) by adding genes or changing endogenous genes and then the modified cells introduced into a blastocyst where they participate in development and can become sperm. This technique allows very specific modification of the mouse genome and perhaps other species.
Techniques for obtaining non-human transgenic animals through the injection of DNA into eggs are also known. See e.g., Gordon et al,
Proc. Nat. Acad. Sci.
(
USA
), (1980) 77:7380-7384. These techniques however, as well as the use of ES cells noted above, are very labor intensive.
Gene therapy is by definition the insertion of genes for the purpose of medicinal therapy. The principle underlying gene therapy is to, rather than deliver doses of one or more pharmacologic molecule, deliver a functional gene whose RNA or protein product will produce the desired biochemical effect in the target cell or tissue. The genes may be delivered into endogenous cells or within new cells delivered to the animal. There are several potential advantages of gene therapy over classical biochemical pharmacology, including the fact that inserted genes can produce extremely complex molecules, including RNA and proteins, which can be extraordinarily difficult or impossible to administer and deliver themselves.
The many applications of gene therapy, particularly via stem cell genetic insertion, have been extensively reviewed (see, Boggs et al., Int. J. Cell Cloning (1990) 8, 80; Kohn et al., Cancer Invest. (1989) 7, 179; Lehn, Bone Marrow Transpl. (1990) 5. 287; and Verma et al., Scientific Amer. (November 1990) 68). Genetically transformed human stem cells have wide potential application in clinical medicine, as agents of gene therapy.
Methods and compositions are known for the ex vivo replication and stable genetic transformation of animal, including human, stem cells and for the optimization of such stem cell cultures. The applications of gene therapy could be advantageously expanded if techniques were available for the application of gene therapy to animal primitive germ cells.
SUMMARY OF THE INVENTION
Accordingly, it is an object of this invention to provide a facile method for obtaining animals harboring a biologically functional non-native, preferably male, germ or stem cell, and for obtaining corresponding resultant germ or stem cells.
It is another object of this invention to provide a facile method for obtaining new animal lines, and for obtaining corresponding resultant germ or stem cells.
It is another object of this invention to provide animals harboring a biologically functional non-native germ or stem cell, their progeny, and corresponding resultant animal lines and germ or stem cells.
It is another object of this invention to use as said non-native germ cell, one or more animal stem cells which has been cultured maintained and/or expanded in culture ex vivo.
It has been discovered by the inventor that the above objects and other objects which become apparent from the description of the invention given hereinbelow are satisfied by the following method. Primitive germ cells are obtained and are optionally expanded and/or modified ex vivo. The testis (or testes) of a male animal host is (are) repopulated with at least one primitive cell (e.g. a totipotent stem cell) which is not native to the host. Because the donor cell(s) is (are) stem cells, they can expand to repopulate the seminiferous tubules of the recipient testis. The recipient animal may then be bred to obtain a novel animal line in which every cell of the descendant animal is genetically non-native to the original recipient host animal. The methods of the present invention have now been successfully used to produce progeny that contain genetic information different from the biological sire (father). That is, the genetic information in the progeny is that of the donor stem cell and not that of the recipient male which is the natural (biological) sire.
REFERENCES:
Kuroda et. al., Journal of Cellular Physiology 139:329-334, May 1989.*
Theiler, The House Mouse-Atlas of Embryonic Development; Springer-Verlag, New York, 1989.*
J. Wayne Streilein, “Unraveling Immune Privilege”, Science, vol. 270, pp. 1158-1159, Nov. 17, 1995.
D.N.J. Hart Et Al, “Major Histocompatibility Complex Antigens in the Rat Pacreas, Isolated Pacreatic Islets, Thyroid, and Adrenal”, Transplantation, vol. 36, No. 4, pp. 431-435, Oct. 1983.
Judith R. Head Et Al, “Immune Privilege in the Testis”, Transplantation, vol. 36, No. 4, pp. 423-432, Oct. 1983.
J.H. Dinsmore, “Immunoprivileged Sites for Allo- and Xenotransplantation”, Xenotransplantation: The Transplantation of Organs and Tissues Between Species, Second Edition, pp. 198-205.
Clyde F. Barker Et Al, “Immunologically Privileged Sites”, Advances in Immunology, vol. 25, Eds: Kunkel HG. & Dixon F.J., pp. 1-54, 1977.
Hauda Karen M.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
The Trustees of the University of Pennsylvania
Woitach Joseph T.
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