Multicellular living organisms and unmodified parts thereof and – Method of making a transgenic nonhuman animal – Via microinjection of dna into an embryo – egg cell – or...
Reexamination Certificate
1998-08-05
2002-12-24
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of making a transgenic nonhuman animal
Via microinjection of dna into an embryo, egg cell, or...
C800S017000
Reexamination Certificate
active
06498285
ABSTRACT:
BACKGROUND OF THE INVENTION
The first successful production of a transgenic mammal was accomplished using mice (Gordon et al., “Genetic Transformation of Mouse Embryos by Microinjection of Purified DNA”
Proc. Nat;. Acad. Sci. USA,
77:7380-7384 (1980)). Transgenic large mammals (i.e., mammals of a species in which normal mature adults of either sex may attain a body mass of at least one kilogram) have also been produced, albeit with greater difficulty, and with a much lower frequency of transgenic offspring being obtained. The production of transgenic large mammals, including agriculturally valuable livestock, has thus been hindered by a low frequency of success (e.g., generally less than 3% in pigs) in obtaining transgenic offspring following introduction of exogenous nucleic acid molecules into zygotes, one celled embryos, or, in some cases, two celled embryos. This relatively low frequency increases the difficulty of obtaining any particular desired transgenic large mammal, with concomitant increases in time and expense.
The successful production of a variety of different transgenic large mammals has been reported. For example, transgenic large mammals have been made in which the animal produces an exogenous protein in milk, for example, tissue plasminogen factor expressed in goats and human anti hemophilic factor IX in sheep. (See, for review, Ebert, K. M. and J. P. Selgrath, “Changes in Domestic Ungulates through Genetic Engineering” in
Animal Applications in Mammalian Development,
Cold Spring Harbor Laboratory Press, 1991.
Transgenic Animal Technology: A Laboratory Handbook,
Carl A. Pinkert, ed., Academic Press (1994). Transgenic swine have been produced which express porcine growth hormone under the control of promoter/enhancer elements originally isolated from Moloney murine leukemia virus or from cytomegalovirus (Ebert, K. M. et al.,
Animal Biotechnology
1:145-159 (1990).
Transgenic mammals are useful not only as improved agricultural stock or as a means of production for a protein, but are also valuable sources of altered cells, tissues, and organs, as well as valuable research tools, e.g., for discovering the mechanisms by which gene expression is controlled. (See, for example, Low, M. J. et al.,
Molecular Endocrinology
3:2028-2033 (1989), U.S. Pat. Nos. 5,573,940, 5,624,837, and 5,627,264, and PCT patent publications No. WO 95/04756, WO 95/23512, and WO 95/34202, which are incorporated herein by reference).
Transgenic mammals have been produced successfully by several methods, including introducing foreign DNA into the male pro-nucleus of a zygote (each pro-nucleus comprises only one haploid chromosomal complement); see, for example, Wagner et al., U.S. Pat. No. 4,873,191 (1989), disclosing transgenic mice). See also, Brinster, R. L. et al.,
Proc. Natl Acad. Sci. USA
82:4438-4442 (1985); Leder et al., U.S. Pat. No. 4,736,866 (1988)).
Transgenic large mammals have been obtained following injection of a transgene into both one and two celled embryos (Hammer, R E et al.,
Nature
315:680-683 (1985); Hammer, R E et al.,
J Anim Sci
63:269-278 (1986); Pursel et al.,
Proc Intl Cong Anim Reprod Artif Insem
11:480a-480c (1988); Pursel et al.,
J Reprod Fert Supl
40:235-245(1990); Pursel et al.,
J Reprod Fertil Suppl
41:77-87 (1990); Mullins J J et al.,
Hypertension
22:630-633 (1993); Martin et al., “Production of Transgenic Swine” pp 315-388 in
Transgenic Animal Technology: A Laboratory Handbook,
Carl A Pinkert, ed., Academic Press (1994); Seamark, R F
Reprod Fertil Dev,
6:653-657 (1994); copending U.S. application Ser. No. 08/668,703, filed in the name of Karl M. Ebert on Jun. 24, 1996.
As a consequence of the low frequency with which transgenic large mammals have been obtained, new techniques that will allow a higher frequency of success in obtaining transgenic large mammals would be of great value, and are actively being sought.
SUMMARY OF THE INVENTION AND DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention provides methods of making transgenic large mammals that generate a higher frequency of transgenic offspring than has been heretofore achievable. These methods involve procedures in which at least one embryo is prepared and genetically modified. The genetic modification is accomplished by the introduction of at least one isolated nucleic acid molecule, and preferably at least one clone (i.e., a plurality of identical copies) of isolated nucleic acid molecules into an embryo.
Large Mammals: Preferred large mammals for use in the method of the invention are herbivores, and include ungulates (i.e., hoofed mammals such as pigs, cows, goats, sheep, horses, donkeys, deer, antelopes and the like) and more generally, livestock (i.e., mammals raised for agricultural purposes such as pigs, cows, goats, sheep, horses, rabbits and the like, and/or as beasts of burden such as donkeys, horses, elephants, camels, llamas, and the like). More preferable large mammals for use in the method of the invention are pigs, goats, sheep, and bovine cattle. Particularly preferred large mammals for use in the method of the invention are members of the genus Sus, with domestic pigs being the most highly preferred of these.
Embryos: In accordance with a preferred embodiment of the invention, embryos (or zygotes, which in accordance with certain aspects of the invention will be incubated in vitro and allowed to develop into embryos before genetic modification) are recovered from the reproductive tract of a donor female (e.g., a gilt or sow), typically from the oviduct or uterus of a mature, hormonally synchronized, ovulation induced female. The embryos are microscopically inspected to determine which are at the desired stage of development. Embryo development is typically measured and characterized in terms of the number of cells making up the embryo, which number is readily counted via microscopic inspection of a living embryo and increases throughout early embryonic development.
In accordance with the invention, embryos are prepared so as to provide at least one target embryo (i.e., an embryo into which at least one clone of isolated nucleic acid molecules is to be introduced). In accordance with the invention at least one of the at least one target embryos has at least three cells. Preferably the at least one target embryo that has at least three cells is obtained by preparing a group of embryos comprising the at least one target embryo that has at least three cells.
The genetic modification is accomplished by the introduction of at least one clone of isolated nucleic acid molecules into at least one blastomere of the at least one target embryo that has at least three cells. A blastomere is any of the cells of a multi-celled pre-gastrulation embryo, which cells each comprise a diploid nucleus. The at least one clone of isolated nucleic acid molecules preferably comprises at least one transgene (the at least one transgene-comprising clone being referred to hereinafter as a “transgene clone”)
Preferably at least one clone of isolated nucleic acid molecules is introduced into a plurality of the blastomeres of each multi-cellular embryo in the group of embryos. More preferably the at least one clone of isolated nucleic acid molecules is introduced into the diploid nucleus of a blastomere of a target embryo that has at least three cells.
In accordance with certain of the preferred embodiments of the invention, at least one target embryo in the group of embryos is a target embryo comprising at least four cells. Preferably the target embryo comprising at least four cells is modified by the introduction of a transgene clone into at least one blastomere of the target embryo.
A preferred target embryo is a porcine embryo comprising at least three or at least four cells.
In accordance with certain of the preferred embodiments of the invention, at least one target embryo in the group of embryos is a target embryo comprising at least five or at least six cells. Preferably the target embryo comprising at least five or at least six cells is modified by the introduction of a tran
Alexion Pharmaceuticals, Inc.
Crouch Deborah
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