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Tissue specific expression of exogenous proteins in transgenic chickens

a technology of exogenous protein and chicken, which is applied in the field of protein expression in transgenic chickens, can solve the problems of affecting the promise of transgenic protein production by genetic engineering in transgenics is frustrated, and the ubiquitous expression of an exogenous protein is usually very damaging to the overall health and well-being of the animal. , to achieve the effect of facilitating tissue specific expression of an exogenous protein and facilitating concentration and collection of protein

Inactive Publication Date: 2006-03-09
SYNAGEVA BIOPHARMA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011] The present invention includes transgenic and chimeric birds exhibiting non-specific and tissue specific expression of exogenous proteins, transgene constructs that enable exogenous protein expression, isolated compositions of exogenous proteins, and related methods. The invention uses long term avian ES cell cultures and special techniques to produce chimeric and transgenic birds derived from prolonged embryonic stem cell cultures, wherein the genome of the ES cells have a stably integrated transgene expressing an exogenous protein such that progeny of the ES cells contain the transgene. In some embodiments, these genetic constructs modify the DNA of the ES cell to facilitate tissue specific expression of an exogenous protein. When combined with a host avian embryo, by the procedures described below, those modified ES cells produce chimeric birds that incorporate the transgene into specific, selected somatic tissue of the resulting animals. These chimeric or transgenic birds exhibit an ES-cell derived phenotype and express the foreign protein across all tissues or in a selected tissue. Preferably, a specific expression pattern focuses expression in a tissue or tissue type to the substantial exclusion of other tissues and facilitates concentration and collection of the protein.

Problems solved by technology

However, the production of genetically modified animals involves significant technical hurdles that have only been overcome for a few species.
For other species, the promise of genetic engineering in transgenics for protein production has been frustrated by the lack of sustainable long-term embryonic stem cell cultures.
However, the collection of a valuable protein from the tissues of an animal typically requires that the expression be limited to certain specific tissues that facilitate collection of the expressed protein.
Under such circumstances, it is unlikely that a meaningful quantity of the protein could be separated from the animal, and furthermore the ubiquitous expression of an exogenous protein is usually very damaging to the overall health and well being of the animal.
The transgenes that enable tissue specific expression are complex and the genetic manipulations that are necessary to incorporate the transgenes into an embryonic cell line require extensive manipulation of the embryonic stem cells and can threaten the pluripotency of the stem cells unless the culture conditions are optimized for transgenesis.
However, application of the full range of mammalian transgenic techniques to avian species has been unsuccessful.

Method used

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  • Tissue specific expression of exogenous proteins in transgenic chickens
  • Tissue specific expression of exogenous proteins in transgenic chickens
  • Tissue specific expression of exogenous proteins in transgenic chickens

Examples

Experimental program
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Effect test

example 1

Derivation of Chicken Embryonic Stem Cells (cES Cells)

[0038] Chicken ES cells were derived from one of two crosses: Barred Rock X Barred Rock or Barred Rock X Rhode Island Red. These breeds were selected to obtain a feather marker when testing the developmental potential of cES cells. The cES cells are injected into White Leghorn embryos, which are homozygous dominant at the dominant, white locus. Chimeric chickens resulting from injection of these ES cells display black feathers from the cES cells and white feathers from the recipient embryo.

[0039] Initial establishment of the cES cell culture was initiated according to the protocol described in U.S. Pat. No. 5,565,479. At stage X, the embryo is a small round disk, consisting of approximately 40,000-60,000 cells, situated on the surface of the yolk. To retrieve the embryo a paper ring is put on the yolk membrane, exposing the embryo in the middle. The yolk membrane is cut around the ring, which is then lifted off the yolk. The em...

example 2

Injection of Chicken Embryonic Stem Cells into Recipient Embryos

[0054] To permit access to the embryo in a freshly laid egg the shell must be breeched, inevitably leading to a reduction in the hatch rate at the end of the 21-day incubation period. The convention was to cut a small hole (less than 10 mm diameter) in the side of the egg, through which the embryo was manipulated, and re-seal with tape, a glass cover slip, shell membrane or a piece of shell. Though relatively simple to perform, this “windowing” method caused embryonic mortality between 70 and 100%. Improved access to the embryo and increased survival and hatchability can be achieved if the embryo is transferred to surrogate eggshells for incubation to hatching using two different shells and a method (adapted from Callebaut) (Callebaut, Poult. Sci 60: 723-725, 1981) and (Rowlett, K. and K. Simkiss, J. Exp. Biol. 143: 529-536, 1989), which are specifically incorporated herein by reference with this technique, the mean ha...

example 3

Somatic Chimeras from Chicken Embryonic Stem Cells (cES)

[0063] To demonstrate that pluripotency of the cES cells of the invention, cES cells are injected into White Leghorn recipient embryos. In the first round of experiments, a total of 14 cell lines in 28 experiments are injected into stage X recipient embryos (See Table 2). The cES cells have been propagated in culture between 4 and 106 days and some lines had been cryopreserved. Chicken ES cells are lightly trypsinized, resulting in small clumps of cES cells, and resuspended in DMEM supplemented with 25 mM HEPES+10% fetal calf serum. Three to five μl of the cell suspension, containing between 2000-5000 cells, are injected into the subgerminal cavity of the recipient embryos. All embryos that developed feathers are analyzed and twenty four percent of embryos (83 / 347) are chimeric as determined by feather color. Feather chimeras are obtained from 11 / 14 cell lines. The extent of chimerism varied from 1%-95% with a mean extent of 2...

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Abstract

Transgenes encoding exogenous proteins are stably integrated into embryonic stem cells and are present in the somatic tissue of transgenic or chimeric birds. The transgenes encode exogenous proteins and are expressed in any of endodermal, ectodermal, mesodermal, or extra embryonic tissue. Tissue specificity is provided by selecting the content of the transgene accordingly. Transgenic birds whose genome is comprised of trangene derived exogenous DNA express exogenous proteins with tissue specificity, and specifically express exogenous proteins in the tubular gland cells of the oviduct to concentrate exogenous proteins in egg white.

Description

FIELD OF THE INVENTION [0001] This invention relates to the field of protein expression in transgenic and chimeric animals, and enabling technologies such as genetic engineering, transgenics, and the long-term culture of embryonic stem cells. Embryonic stem cells are engineered with specially designed genetic constructs to introduce genetic modification into birds, including by the insertion of transgenes that yield tissue specific expression of exogenous proteins. Transgenic birds of the invention express the transgene-derived exogenous protein in the oviduct and the protein is deposited in large quantities in the egg. BACKGROUND OF THE INVENTION [0002] Genetically engineered animals offer the potential for tremendous advances in the production of valuable pharmaceutical products from the cells of such animals. However, the production of genetically modified animals involves significant technical hurdles that have only been overcome for a few species. The ability to incorporate gen...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027A61K35/54C12N15/09C12N15/85
CPCA01K67/0275C12N2840/203A01K2207/15A01K2217/00A01K2217/05A01K2227/30A01K2267/01C07K16/02C07K16/44C07K2317/21C12N15/8509C12N2830/008C12N2830/15C12N2830/42C12N2830/60C12N2830/90C12N2840/20A01K67/0278
Inventor ZHU, LEIWINTERS-DIGIACINTO, PEGGYETCHES, ROBERTJ
Owner SYNAGEVA BIOPHARMA CORP
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