Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-08-10
2002-04-23
Clark, Deborah J. R. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S013000, C800S014000, C800S021000, C435S325000, C435S455000
Reexamination Certificate
active
06376743
ABSTRACT:
BACKGROUND OF THE INVENTION
Transgenic animals are important for scientific, pharmaceutical and agricultural purposes. Production of foreign proteins in milk using genetically engineered livestock is believed to be a suitable system for making therapeutic recombinant proteins. Moreover, the insertion of human genes into the genomes of animals, such as pigs, could enable such animals to act as living organ or cell “factories” for human organs or cells that will not be rejected by the human immune system.
There are several reported methods of obtaining transgenic mammals by introducing foreign DNA into their somatic and germinal cells. One of these methods, pronuclear microinjection, has become widely used and was first developed in a mouse model in the early 1980s. Pronuclear microinjection entails injection of transgene (tg) DNA into a pronucleus of a one-cell embryo [J. W. Gordon, et al.,
Proc. Natl. Acad. Sci. U.S.A.
77, 7380 (1980); J. W. Gordon and F. H. Ruddle,
Science
214, 1244 (1981); R. D. Palmiter and R. L. Brinster,
Annu. Rev. Genet.
20, 465 (1986); and J. W. Gordon,
Int. Rev. Cytol.
115, 171 (1989)]. Whereas the generation of pronuclear zygotes has been straightforward in the mouse, this is not necessarily true for species exemplified by the large commercial animal breeds. For example, zygotes are difficult substrates for pronuclear injection when their lipid richness renders them opaque, as in cattle and pigs; in contrast, mouse zygotes are translucent.
Transgenic embryonic stem (ES) cells, obtained by transfection with DNA constructs, have been used to obtain chimeric animals in cattle, sheep, and the like. This method involves the injection of genetically engineered ES cells harboring a desired mutation into fertilized embryos which are at the morula stage (about 20 to 50 cells) or the blastocyst stage (about 100 cells) of embryonic development. Upon implantation, such embryos often give rise to chimeric animals, whose subsequent breeding with wild-type animals results in germ line transmission of the ES cell-derived genome at variable frequencies (often equal to zero). Because the efficiency of gene transfer is low and because large numbers of recipient animals are required for embryo transfer, production of transgenic large animals by this method has been difficult.
Neither the pronuclear microinjection method nor the ES cell transfection method, described above, as yet permits the outcome of tg insertion to be controlled or predicted because the introduction of heterologous DNA into the cell often results in detrimental “position” or copy number effects caused by the quasi-random manner in which the transgene, or multiple copies thereof, integrate into the host genome (J. W. Gordon, supra). Therefore, the efficiency of these methods in producing transgenic large animals has been low.
It has been reported that greater control over the outcome of transgene integration can be achieved by using mouse ES cell lines transfected with DNA constructs capable of homologous recombination [M. J. Evans and M. H. Kaufman,
Nature
292, 154 (1981); M. Kuehn, et al., ibid. 326, 295 (1987)]. These “gene targeted” ES cells are those in which one or more specific genes are knocked out or modified in a very precise manner that does not affect any other locus, genome-wide. “Immortalized” transgenic ES cell lines have been established and well characterized in vitro to confirm the construct integration site. However, gene targeting is currently restricted to the one species for which established, germline-contributing ES cell lines exist—the mouse.
Limitations in the available strategies for modifying mammalian germ lines have fueled a search for alternative methods, including the use of recombinant retroviruses to infect oocytes or preimplantation embryos [D. Jähner, et al.,
Proc. Natl. Acad. Sci. U.S.A.
82, 6927 (1985); A. W. S. Chan, et al., ibid., 95, 14028 (1998)] and the use of replication-deficient adenovirus-mediated delivery systems [Y. Kanegae, et al,
Nucleic Acids Res.
23, 3816 (1995)]. However, viral protocols imply extra steps in cloning, necessitating specialized containment facilities for the recombinant adenoviruses and retroviruses that must be engineered. Delivery of the virus by these methods still requires either microinjection equipment or removal of the zona pellucida of the oocyte.
It has also been reported that spermatozoa may be used as vehicles for DNA delivery during in vitro fertilization (IVF) [M. Lavitrano, et al.,
Cell
57, 717 (1989)]. In this approach, live spermatozoa are used as a vector for introducing recombinant DNA into the oocyte in vitro. Although sperm-mediated DNA transfer to offspring has the potential to markedly simplify the generation of transgenic animals, there has been considerable controversy about the efficacy of the live spermatozoa method in promoting transgenesis because of its unreliability in consistently producing transgenic animals [M. Lavitrano, et al., 1989, supra; R. N. Brinster, et al.,
Cell
59, 239 (1989); B. Maione, et al.,
Mol. Reprod. Dev.
50, 406 (1998)]. In one report, exogenous DNA has been demonstrated to decorate intact spermatozoa in a reversible fashion [M. Lavitrano, et al.,
Mol. Reprod. Dev.,
31, 161 (1992)], indicating that membrane structures may act as a barrier to the stable association of sperm heads with extraneous, recombinogenic DNA. In another report, live mouse spermatozoa incubated in vitro for two hours with a plasmid DNA showed some uptake of the exogenous DNA into the nucleus, as well as the plasma membrane. Sperm from the vas deferens into which plasmid DNA had been injected six hours previously, also showed some nuclear uptake. However, none of these spermatozoa were used to fertilize oocytes [E. Huguet and P. Esponda,
Mol. Reprod. Dev.
51, 42 (1998)].
Therefore, there is still a need for an efficient transgene transfer method that can reliably be used to produce transgenic animals. More particularly, there is a need for an efficient method of obtaining genetically engineered livestock or other large animals for use as pharmaceutical “factories” and as a source of human organs or cells for xenotransplantation.
SUMMARY OF THE INVENTION
The invention provides a method for obtaining a transgenic embryo, comprising the steps of coinserting an exogenous nucleic acid and a membrane-disrupted sperm head or a demembranated sperm head into the cytoplasm of an unfertilized oocyte to form a transgenic fertilized oocyte, and allowing the transgenic fertilized oocyte to develop into a transgenic embryo and, if desired, into a live offspring. The coinserting step preferably comprises the substep of preincubating the membrane-disrupted or demembranated sperm head with the exogenous nucleic acid for a time period of about 30 seconds to about 5 minutes, typically about 45 seconds to about 3 minutes, more typically about 1 minute to about 2 minutes. The coinsertion of the sperm head and exogenous nucleic acid into the oocyte is by microinjection, preferably by piezo electrically-actuated microinjection. The exogenous nucleic acid mixed with the membrane-disrupted or demembranated sperm heads may comprise more than one transgene, to produce an embryo that is transgenic for more than one transgene.
Membrane-disrupted sperm heads suitable for use in the invention can be obtained from frozen-thawed spermatozoa or rehydrated freeze-dried spermatozoa. A method for preserving spermatozoa by freeze-drying and using the resulting reconstituted freeze-dried spermatozoa to fertilize oocytes in vitro to produce embryos and live offspring is the subject of our copending U.S. patent application, Ser. No. 09/177,391, filed Oct. 23, 1998, the disclosure of which is hereby incorporated by reference. Demembranated sperm heads suitable for use in the invention, comprising the nucleus and perinuclear materials, can be obtained by detergent-treatment of fresh spermatozoa, as described below.
The method of the invention may be use
Clark Deborah J. R.
Jones Day Reavis & Pogue
Paras, Jr. Peter
University of Hawai'i
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