Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Avian cell – per se
Reexamination Certificate
1999-08-09
2001-12-25
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Avian cell, per se
C435S325000, C435S347000, C435S373000, C435S377000
Reexamination Certificate
active
06333192
ABSTRACT:
TECHNICAL FIELD
The present invention relates to undifferentiated avian cells expressing an embryonic stem cell phenotype in general, and particularly relates to avian primordial germ cells and undifferentiated avian cells expressing an embryonic stem cell phenotype.
Table of Abbreviations
ATCC
American Type Culture Collection
bFGF
basic fibroblast growth factor
BRL
buffalo rat liver
DMEM
Dulbecco's modified Eagle's medium
EDTA
ethylenediamine tetraacetate
ES
embryonic stem
ESCs
embryonic stem cells
FBS
fetal bovine serum
FGF
fibroblast growth factor
H&H
Hanburger & Hamilton staging system
IGF
insulin-like growth factor
LIF
leukemia inhibitory factor
PBS
phosphate buffered saline
PGCs
primordial germ cells
SSEA-1
stage-specific mouse embryonic antigen-1 marker
for PGCs and ESCs
STO
mouse fibroblast cell line
SCF
stem cell factor (also called steel factor or SF)
TGF-&bgr;1
transforming growth factor-&bgr;1
BACKGROUND ART
In mammals, primordial germ cells cultured from the genital ridge have the ability to give rise to pluripotent embryonic stem cells. For example, U.S. Pat. Nos. 5,690,926 issued Nov. 25, 1997 to Hogan; 5,670,372 issued Sep. 23, 1997 to Hogan; and 5,537,357 issued Sep. 26, 1995 to Hogan each disclose pluripotential mammalian embryonic stem cells and methods of making the same. The disclosure of these patents is limited to mammalian embryonic stem cells and particularly to the culturing of murine and other mammalian embryonic stem cells using a combination of growth factors consisting of SCF, FGF and LIF.
Current prior art reports on the culture of avian primordial germ cells (PGCs) have concentrated on efforts to maintain a PGC-phenotype and to stimulate proliferation. See e.g., Chang, I. K. et al.,
Cell. Biol. Int.
1997 August; 21(8): 495-9; Chang, I. K. et al.,
Cell. Biol. Int.
1995 February; 19(2): 143-9; Allioli, N. et al.,
Dev. Biol.
1994 September; 165(1): 30-7 and PCT Publication No. WO 99/06533, published Feb. 11, 1999 (Applicant—University of Massachusetts; Inventors—Ponce de Leon et al.).
Undifferentiated avian cells expressing an embryonic stem cell (ESC) phenotype and processes for producing the same are disclosed in U.S. Pat. Nos. 5,340,740 issued Aug. 23, 1994 to Petitte et al.; 5,656,479 issued Aug. 12, 1997 to Petitte et al.; and 5,830,510 issued Nov. 3, 1998 to Petitte et al. Undifferentiated avian cells expressing an ESC phenotype are useful, among other things, as a tool for the study of embryological development (i.e., by labeling the cells with a marker gene and observing their distribution after injection in vivo) and the production of transgenic poultry. They are useful in allowing the application of homologous recombination to the production of transgenic poultry. In view of such uses, the development of additional methods for obtaining undifferentiated avian cells expressing an ESC phenotype represents a continuing need in the art.
SUMMARY OF THE INVENTION
A method of producing a sustained culture of undifferentiated avian cells expressing an embryonic stem cell phenotype is disclosed. A preferred embodiment of the method comprises: (a) collecting avian cells comprising primordial germ cells from an avian embryo after formation of the primitive streak; (b) depositing the avian cells in contact with a preconditioned feeder matrix; and (c) growing the avian cells on the feeder matrix in the presence of media for a time sufficient to produce an avian cell culture consisting essentially of undifferentiated avian cells expressing an embryonic stem cell phenotype.
The avian cells are collected from an embryo later than stage 14 (H&H), preferably from a stage 14 to stage 45 embryo, more preferably from a stage 15 to stage 31 embryo, and even more preferably from a stage 27 to stage 30 embryo. More preferably, the avian cells are deposited in contact with the feeder matrix in a number ranging from about 10,000 to about 20,000 cells. Even more preferably, about 20,000 avian cells are deposited in contact with the feeder matrix.
The feeder matrix may comprise fibroblast cells. The fibroblast cells can be present in the feeder matrix in a number ranging from about 40,000 to about 60,000 cells, preferably from about 50,000 to about 60,000 cells, even more preferably, from about 55,000 to about 60,000 cells. Most preferably, about 60,000 fibroblast cells are present in the feeder matrix.
Preferably, the fibroblast cells comprise mouse fibroblast cells, more preferably mouse STO fibroblast cells. In a preferred embodiment, the mouse fibroblast cells form a mouse fibroblast feeder layer.
The avian embryo is a embryo optionally selected from the group including but not limited to chicken, turkey, duck, goose, quail and pheasant embryo. In a preferred embodiment, the avian embryo is a chicken embryo.
Accordingly, it is an object of the present invention to provide a novel process for the culturing of undifferentiated avian cells expressing an embryonic stem cell phenotype.
It is another object of the present invention to provide a process for the culturing of undifferentiated avian cells expressing an embryonic stem cell phenotype from avian cells comprising primordial germ cells.
It is a further object of the present invention to provide a feeder matrix for use in preparing a culture of undifferentiated avian cells expressing an embryonic stem cell phenotype using avian primordial germ cells.
It is yet a further object of the present invention to characterize an optimal number of avian cells comprising primordial germ cells for use in establishing a culture of undifferentiated avian cells expressing an embryonic stem cell phenotype.
Some of the objects of the invention having been stated herein above, other objects will become evident as the description proceeds, when taken in connection with the accompanying Laboratory Examples and Drawings as best described herein below.
REFERENCES:
patent: 5166065 (1992-11-01), Williams et al.
patent: 5340740 (1994-08-01), Petitte et al.
patent: 5453357 (1995-09-01), Hogan
patent: 5656479 (1997-08-01), Petitte et al.
patent: 5670372 (1997-09-01), Hogan
patent: 5690926 (1997-11-01), Hogan
patent: 5830510 (1998-11-01), Petitte et al.
patent: 5985659 (1999-11-01), Kusakabe et al.
patent: WO96/12793 (1996-05-01), None
patent: WO99/06533 (1999-02-01), None
patent: WO99/06534 (1999-02-01), None
Pettite, J.N., “The Origin of the Avian Germ Line and Transgenesis in Birds,” Poultry Science, PSA Excellence in Science Publishing (San Diego), vol. 76 (No. 8), pp. 1084-1092, (Aug. 9, 1997).
Chang, I.K., “Production of Germline Chimeric Chickens By Transfer of Cultured Primordial Germ Cells,” Cell Biology International, Seoul National University (Suweon, Korea), vol. 21 (No. 8), pp. 495-499, (Aug. 1997).
Chang, I.K., “Germ Line Chimera Produced By Transfer of Cultured Chick Primordial Germ Cells,” Cell Biology International, Institute of Physical and Chemical Research (Tsukuba Science City, Japan), vol. 19 (No 7), pp. 569-576, (Jul. 1995).
Chang Il-Kuk
Petitte James N.
Jenkens & Wilson, P.A.
Lankford , Jr. Leon B.
North Carolina State University
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