Gene transfer in chickens by introduction of DNA into muscle...

Multicellular living organisms and unmodified parts thereof and – Method of making a transgenic nonhuman animal

Reexamination Certificate

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C800S019000, C800S023000, C514S04400A, C119S006800

Reexamination Certificate

active

06395961

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the methods of altering the phenotype of birds by introducing foreign DNA into the muscle of birds.
BACKGROUND OF THE INVENTION
Commercial poultry is an extremely important source of food. However, there has been comparatively little attention given to methods of producing useful changes in the phenotype of birds through genetic engineering techniques. This is unfortunate, because such techniques offer a much more rapid technique for introducing desirable phenotypic traits into birds than classical breeding techniques.
Currently, the most widely investigated method of gene transfection in poultry employs retroviral vectors. Exemplary is Souza et al.,
J. Exptl. Zool
. 232, 465-473 (1984), in which a retroviral vector encoding growth hormone was injected into the vascularized portion of the yolk sac of 9 day old embryos. See also Shuman and Shoffer,
Poult. Sci
. 65, 1437-1444 (1986); Salter et al.,
Poultry Sci
. 65, 1445-1468 (1986); Salter et al.,
Virology
157, 236-240 (1987); Bosselman et al.,
Science
243, 533-535 (1989); and U.S. Pat. No. 5,162,215 to Bosselman et al.
Nabel et al.,
Science
249 1285-1288 (1990), and Wolff et al.,
Science
247, 1445-1468 (1990), state that transient expression of 2-5 months may be obtained from direct microinjection of DNA, but do not suggest how these techniques may be applied to genetically engineering poultry. Nabel et al. note that the expression of DNA encoding &bgr;-galactosidase injected into porcine arterial segments was limited to the microinjection site. Acsadi et al.,
New Biologist
3, 71-81 (1991) state that myocardial cells were able to transiently express injected foreign genes.
Simkiss et al.,
Protoplasma
151, 164-166 (1989) indicate that primordial germ cells of Stage XVII embryos containing endogenous retroviral sequences can be transferred to comparable recipient Stage XVI embryos that lack the retroviral marker by cardiac puncture. At day 17 of incubation, dot blots on recipient birds showed donor DNA to be present in the gonads, and traces of donor DNA to be present in the liver and heart tissues. The expression of the injected DNA molecules was not reported.
PCT patent application Ser. No. US90/01515 discloses a method of delivering a nucleic acid sequence to the interior of a vertebrate cell. Injection of a DNA molecule into poultry was not reported.
In view of the foregoing, an object of the present invention is to provide methods of changing the phenotype of birds through genetic engineering procedures.
An additional object of the present invention is to provide a method of changing the phenotype of birds in which expression of an exogenous DNA sequence is sufficient produce the phenotypic change.
Another object of the present invention is to provide a method of changing the phenotype of birds which is rapid and convenient.
SUMMARY OF THE INVENTION
A first aspect of the present invention is a method of altering the phenotype of a bird. The method comprises introducing a DNA molecule into the cells of a bird contained within an egg during in ovo incubation, with the DNA molecule being effective to cause a change in phenotype in the bird after hatch (e.g., a change in growth rate, feed efficiency, disease resistance, or a combination of all of these factors). Introduction of the DNA may be carried out by any suitable means, including injecting the DNA molecule in ovo into any compartment of the egg including the body of the embryo.
Preferably, the egg into which the DNA is introduced is incubated to hatch, and the bird so produced raised to at least an age at which the change in phenotype is expressed.
A second aspect of the present invention is a bird produced by the foregoing methods.
In an illustrative embodiment of the foregoing, the DNA molecule is introduced into muscle tissue of the bird in ovo, preferably by direct microinjection during late embryonic development.
A third aspect of the present invention is a method for altering the phenotype of a bird comprising introducing a DNA molecule into the muscle tissue of a bird contained within an egg during in ovo incubation, wherein the DNA molecule is effective in causing a change in phenotype in the bird after hatch.
A fourth aspect of the present invention is a method for immunizing a bird comprising introducing a DNA molecule into the muscle tissue of a bird contained within in an egg during in ovo incubation, wherein the DNA molecule is effective in inducing an immune response in the bird.
A fifth aspect of the present invention is a method for treating a bird comprising introducing a DNA molecule encoding for an antigen into the muscle tissue of a bird contained within an egg during in ovo incubation in an amount sufficient to neutralize maternal antibodies. In a preferred embodiment, the DNA molecule is introduced at or after the development of immunocompetence by the bird.
A sixth aspect of the present invention is the use of a DNA molecule for the preparation of a medicament for carrying out any of the foregoing methods.
A seventh aspect of the present invention is an apparatus for the introduction of a DNA molecule in an egg during in ovo incubation for carrying out any of the foregoing methods.


REFERENCES:
patent: 5162215 (1992-11-01), Bosselman et al.
patent: 2223755 (1988-09-01), None
patent: WO 90/03439 (1990-04-01), None
patent: WO90/11092 (1990-10-01), None
Verma et al., Gene therapy-promises, problems and prospects, 1997, Nature, vol. 389, pp. 239-242.*
Ross et al., Gene therapy in the United States: a five year status report, 1996, Human Gene Therapy, vol. 7, pp. 1781-1790.*
Souza et al. Expt. Zool., vol. 232, pp. 465-473, 1984.*
Antin et al. Devel. Biol., vol. 143, pp. 122-129, 1991.*
Nabel et al. Science, vol. 247, pp. 1285-1288, Sep. 14, 1990.*
Verma Nature, vol. 289, pp. 239-241, Sep. 1997.*
Ross Human Gene Therapy, vol. 7, pp. 1781-1790, Sep. 1996.*
Al Moustafa et al.; “Targets of v-myc tumorigenesis in the avian embryo depend on time and not on site of retroviral infection, ”Cell Differentiation and Development25:119-134 (1988).
Chen et al.; “Vectors, promoters, and expression of genese in chick embryos,”J. Reprod. Fert. Suppl.41:173-182 (1990).
Demeneix et al.; “Gene transfer into intact vertebrate embryos,”Int. J. Dev. Biol.35:4481-484 (1991).
Yuko Ando et al.,Ultrastructural Evidence that Chick Primordial Germ Cells Leave the Blood-Vascular System Prior to Migrating to the Gonadal Anlagen,Develop., Growth and Differ., 25:345-352 (1983).
Robert A. Bosselman et al.,Germline Transmission of Exogenous Genes in the Chicken, Science, 243:533-535 (1989).
C.L. Brazolot et al.,Efficient Transfection of Chicken Cells by Lipofection, and Introduction of Transfected Blastodermal Cells Into the Embryo, Molecular Reproduction and Development, 30:304-312 (1991).
Andy Coghlan,New Scientist19 (Oct. 31, 1992).
Elisabeth Dupin,Cell Division in the Ciliar Ganglion of Quail Embroys in Situ and after Back-Transplantation into the Neural Crest Migration Pathways of Chick Embryos, Developmental Biology, 105:288-299 (1984).
R.S. Goldstein et al.,The Microenvironment Created by Grafting Rostral Half-Somites is Mitogenic for Neural Crest Cells, Proc. Natl.Acad. Sci., 87:4476-4480 (1990).
M. Bagnall et al.,The Contribution Made by Cells from a Single Somite to Tissues Within a Body Segment and Assessment of their Integration with Similar Cells from Adjacent Segments, Development, 107:931-943 (1989).
E.G. Nabel,Site-Specific Gene Expression in Vivo by Direct Gene Transfer into the Arterial Wall, Science, 249:1285-1288.
D.W. Salter et al,Transgenic Chickens; Insertion of Retroviral Genes into the Chicken Germ Line, Virology, 157:236-240 (1987).
D.W. Salter et al.,Gene Insertion into the Chicken Germ Line by Retroviruses, Symposium, 1445-1458 (1985).
R.M. Shuman et al.,Gene Transfer by Avian Retroviruses, Symposium, 1437-1444 (1985).
K. Simkiss et al.,Transfer of Promordial Germ Cell DNA Between Embryos, Protoplasma, 151:164-166 (1989).
L.M. Souza et al,Application of Recombinant DNA Technologies to Studies on

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