Method of subculturing culturing avian cells at pH 7.8 or above

Details Method of subculturing culturing avian cells at pH 7.8 or above Method of subculturing culturing avian cells at pH 7.8 or above

1997-05-01

2001-01-30

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S349000, C435S375000, C435S378000, C435S395000

Reexamination Certificate

active

06180400

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a method for culturing avian cells and cell lines that are obtained from said culture method.
BACKGROUND OF THE INVENTION
Since a “super-mouse” was created in 1982 by introducing a growth-hormone gene of a rat into a fertilized egg of a mouse (R. D. Palmiter et al.: Nature, 300, 611-615, 1982) and demonstrated that exogenous genes can be introduced into mammals, various attempts have been made to produce transgenic animals by introducing exogenous genes. Among such attempts, transgenic livestock are intensively produced to promote the improvement of breed, to provide donors for organ transplantation, and to construct animal factories that produce useful substances (bioreactors).
Especially, production of pharmaceutical and useful proteins by transgenic livestock should form a new field of industry for producing useful proteins since in animal cells sugar-chain modification that is indispensable for expressing pharmaceutical activities can be conducted in higher levels than in bacteria or yeast and since the cost for production of transgenic live stock and their maintenance is much lower than protein production in mass culture facilities for animal cells. Physiological effects of such useful proteins may be harmful for the animals that produces them. To eliminate such an adverse effect, attempts are made to secrete such products outside the body. Two practicable production systems are proposed.
The first system is production and secretion of useful proteins into the milk of a mammal. Implemented examples are a transgenic sheep which produces and secretes high concentrations of &agr;-1-antitrypsin in the milk (G. Wright et al.: Bio/Technology, 9, 830-834, 1991) and a transgenic goat which produces and secretes human tissue-type plasminogen activator in the milk (K. M. Ebert et al.: Bio/Technology, 9, 835-838, 1991).
The other system is production of useful substances into the albumen of an avian egg. Such a system with domestic fowls should be more useful than transgenic livestock since a large number of fowls can be kept under a closed and fully controlled environment, the generation interval is only half a year until sexual maturation, eggs are constantly obtained, and the breeding cost is much lower than the cost for raising livestock.
Three methods are presently used for introducing genes into avian fertilized eggs (embryos) to produce transgenic birds.
(1) Method for introducing genes by infecting blastoderm cells of a fertilized and oviposited egg of a fowl (the number of cells is already above 60,000) with retrovirus vectors to which genes to be introduced are connected. This method is now widely used. As the first transgenic chicken, Bosselman et al. obtained sperms of male that hatched from the eggs of which blastoderm cells were infected with avian retrovirus vectors lacking reproducing ability, and artificially fertilized a female with the sperms. And they confirmed that the vector DNA's were transmitted to their F
1
(Bosselman, R. A. et al.: Science, 243, 533-535, 1989).
(2) Method for obtaining transgenic birds by introducing genes into blastoderm cells of a oviposited egg, which are dissociated in advance with trypsin, injecting these cells into the subgerminal cavity of other blastoderm-stage eggs to create chimera birds, and crossing the chimeras. Since the technology to produce blastoderm chimera is already established (Petitte, J. N. et al.: Development, 108, 185-189, 1990), there should be a possibility of producing transgenic birds with this method if foreign genes could be introduced into injected cells.
(3) Method by microinjecting a gene into a one-cell-stage fertilized egg immediately after ovulation and externally cultivating the egg until it hatches. Unlike Methods (1) and (2), which introduce genes into blastoderm cells of eggs after oviposition, this method is only possible with the method developed by Perry et al. in 1988 for taking fertilized eggs out of a fowl immediately after ovulation and cultivating the egg until it develops into a chicken (Perry, M. M.: Nature, 331, 70-72, 1988). However, this method should be improved in many points to be used for producing transgenic fowls since it is difficult to inject genes into the nucleus or pronucleus because the nucleus of a fertilized egg is difficult to identify and the egg is polyspermy, and thus genes are mostly injected into the cytoplasm, producing a mosaic transgenic fowl.
Besides these methods, the method that injects genes into cells of the following types and produces chimeras should be useful for producing transgenic birds.
Corresponding cell types are 1) cells that exist within an embryo at the initial stage, such as blastoderm cells, primordial germ cells (PGC), and gonia (a general term for oogonia and spermatogonia), 2) embryonic stem cells (ES cells), which are cell lines established in vitro from blastoderm cells, and 3) embryonic germ cells (EG cells), which are cell lines derived from primordial germ cell gonia.
Since ES and EG cells keep growing without differentiating in vitro for a long period of time, it is possible to introduce genes into these cells under culture, select specific cell clones, produce germline chimeras with the technology for producing blastoderm chimeras, mutually cross the chimeras, and produce an animal that derives from an ES or EG cell. An ES cell lines that have the ability to form germline chimeras have been established for mouse (A. Bradley et al.: Nature, 309, 255, 1984). For birds, however, a cell line that may be ES has been reported to be established (WO93/23528) but the ability to form chimeras is not confirmed.
Since primordial germ cells and gonia, which develop into sperms or eggs, grow actively, gene manipulation is possible under culture as for ES cells if culture conditions for primordial germ cells and gonia are established. A report of a method for producing a fowl from primordial germ cells and gonia by transplanting these cells to another embryo (Kuwana, T. Jikken Igaku, Vol. 12, No.2 (special number), 260-265, 1994) suggests the possibility of producing transgenic birds from primordial germ cells and gonia.
EG cells were first established by Matsui et al. in mouse. Matsui et al. cultured the primordial germ cells of a mouse in the presence of stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and STO cells (Kawase et al., Jikken Igaku, Vol. 10, No. 13 (special number), 1575-1580, 1992) as feeder cells and established EG cell lines that was derived from the primordial germ cells, which formed colonies of the ES-cell-like morphology, grew, and had the ability to form chimeras (Matsui, Y., Zsebo, K. & Hogan, B. L. M.: Cell, 70: 841-847, 1992). This method may be applied to avian primordial germ cells to establish EG cell lines of birds.
These three types of cells either exist within an avian embryo or have been established from them. A method for stably and long culturing the cells from birds containing these cells in order to enable cell and gene manipulations, such as isolation of cells, establishment of cell lines and introduction of genes.
Prolonged passage cultures of avian cells may be used for producing vaccines for preventing virus diseases, such as Marek's disease of fowls. Cell culture is more appropriate for producing vaccines than fowl eggs since it can avoid contamination by other antigens.
However, avian cells could not be subcultured since cells grow and divide actively in the primary culture but stop growing and die several weeks after a number of vacuoles are observed within the cytoplasm of the cultured cells. Carrel reported that he succeeded in culturing for 34 years the connective tissue of a fowl embryo (Kuroda, Y., Dobutsu Soshiki Baiyo-ho, p. 2, Kyoritsu Shuppan k.k., 1974), but no one succeeded in confirming his experiment, which is now denied by the academic world. The fibroblast of fowl embryos was the only normal cells that can be subcultured for approximately 34 generations.
This invention aims to provide a

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