Gene transfer in poultry by introduction of embryo cells in ovo

Details Gene transfer in poultry by introduction of embryo cells in ovo Gene transfer in poultry by introduction of embryo cells in ovo

1995-05-19

2003-02-04

Crouch, Deborah (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

Transgenic nonhuman animal

C800S013000, C800S024000

Reexamination Certificate

active

06515199

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the methods of altering the phenotype of birds by introducing avian embryo cells into an egg containing the bird prior to hatch, which embryo cells carry heterogenous genetic material.
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 Serial 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 sequence into somatic cells of a bird contained within an egg during in ovo incubation, with the DNA sequence being effective to cause a change in phenotype in said 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 sequence 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.
In an illustrative embodiment of the foregoing, the DNA sequence is introduced by first transfecting avian hematopoietic progenitor cells with the DNA sequence in vitro, and then injecting said transfected hematopoietic progenitor cells into the egg, preferably into the yolk sac or onto the chorioallantoic membrane, and preferably during early embryonic development.
A second aspect of the present invention is a method of altering the phenotype of a bird comprising introducing avian somatic tissue-specific stem cells to an egg containing a bird during in ovo incubation, wherein the avian somatic tissue-specific stem cells contain and are capable of expressing at least one DNA molecule in an amount effective to cause a change in the phenotype of the bird. Introduction of hematopoietic stem cells is a preferred embodiment, as it has been demonstrated that a DNA molecule contained therein persists and can express the protein encoded therefor at the introduction site, the bone marrow, and in the peripheral blood of the embryo. It is also preferred that the somatic tissue-specific stem cell be introduced to the bird during a developmental stage at which the cells responsible for the phenotypic expression desired to be altered are colonizing within the target tissue.
A third aspect of the present invention is a method of altering the phenotype of a bird comprising introducing avian embryo cells to the air cell of an egg containing a bird during in ovo incubation, wherein the avian progenitor cells contain and are capable of expressing at least one DNA molecule in an amount effective to cause a change in the phenotype of the bird. The inventors have demonstrated that embryo cells introduced into the air cell migrate across the air cell membrane and colonize the appropriate target tissue. Preferred cell types are embryonic stem cells and primordial germ cells.
A fourth aspect of the present invention is a method of altering the phenotype of a bird comprising introducing avian somatic tissue-specific stem cells to the air cell of an egg containing a bird during in ovo incubation, wherein the avian somatic tissue-specific stem cells containing and capable of expressing at least one DNA molecule in an amount effective to cause a change in the phenotype of the bird. Preferred cells for introduction are hematopoietic stem cells and neural crest stem cells.
A fifth aspect of the present invention is a bird produced by the foregoing methods.
A sixth aspect of the present invention is the use of a DNA sequence for the preparation of a medicament for producing a phenotypic change in a bird by introducing the medicament in ovo, as described above.
A seventh aspect of the present invention is an apparatus for the introduction of a DNA sequence in an egg during in ovo incubation, the DNA sequence capable of producing a phenotypic change in the bird carried by the egg after hatch, as described above.
DETAILED DESCRIPTION OF THE INVENTION
There are several aspects of avian embryonic development which make it an attractive target for DNA introduction by stem cell transfer. First, since the greatest period of embryonic development occurs in the egg outside the maternal reproductive tract, the embryo can be easily accessed for the introduction of exogenous DNA.
Second, the fact that the egg is a multi-compartmentalized unit can be exploited to deliver biological materials to specific embryonic sites. For example, the yolk sac in the early embryo functions to manufacture blood. Immediately prior to hatching, the yolk sac serves a primarily nutritional function and is taken into the intestinal tract and thereby transported to the cecal pouches during and after hatch. Therefore, yolk sac administration of materials can lead to both embryonic cecal or vascular system delivery. Vascular system delivery through administration of DNA into the yolk sac would be particularly desirable for administering DNA constructs capable of expressing physiologically active peptides in the bird, such as growth hormone, lymphokines such as interferon and interleukin-2, insulin-like growth factor, thyroid releasing hormone (TRH) or epidermal growth factor. In addition, administration of a peptide or DNA construct can be efficiently carried out by injection of the molecule onto the chorio-allantoic membrane or onto the air

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