Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Rodent cell – per se
Reexamination Certificate
1999-11-15
2001-02-20
Leguyader, John L. (Department: 1633)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Rodent cell, per se
C435S325000, C435S352000, C424S582000
Reexamination Certificate
active
06190910
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel embryonic stem cells derived from mice.
2. Description of the Related Art
Embryonic stem cells, sometimes referred to as ES cells, are derived from inner cell mass (ICM) of fertilized eggs in blastocyst phase, and can be cultured and maintained in vitro while being kept in an undifferentiated state. Embryonic stem cells are extremely useful biological materials for preparing transgenic animals. For example, a gene knockout mouse in which a specific gene is inactivated can be produced by replacing an active gene in an embryonic stem cell chromosome with an inactivated gene by means of a homologous recombination system.
Embryonic stem cells derived from mice, hamsters, and pigs were previously reported. However, processes for establishing embryonic stem cells have not been sufficiently developed, and the sorts of embryonic stem cells are undesirably limited compared to the numbers of mouse strains that have been developed for wide variety of numerous purposes. Currently, researchers most widely use the cells derived from mouse strain 129/Sv as embryonic stem cells. However, the mice of 129/Sv are not always sufficiently bred, and accordingly, if a gene knockout mouse is established, the mouse must be disadvantageously made into a hybrid by crossbreeding with other strains. There is also a problem that the origin of embryonic stem cells established from the strain 129/Sv cannot be completely specified from a genetic viewpoint, since various kinds of substrains are involved in the strain.
Function of a certain gene is generally influenced by other genetic backgrounds in a whole-body level. Accordingly, for close comparative researches by means of gene knockout mice, it is indeed ideal to establish embryonic stem cells from an inbred mouse strain, whose basic data has sufficiently been accumulated, and compare the influence of the knockout of a gene of interest with results obtained from original strain. For these reasons, it has been desired to establish novel embryonic stem cells from inbred mouse strains. However, this class of embryonic stem cells has not been reported yet. As embryonic stem cells derived from mouse strains, embryonic undifferentiated cells disclosed in Japanese Patent Unexamined Publication (KOKAI) No. (Hei) 5-328878/1993 and the like are known. However, these cells are characterized as embryonic stem cells derived from F1 hybrid, and are not those established from inbred strains.
Accordingly, an object of the present invention is to provide novel embryonic stem cells. More specifically, the object of the present invention is to provide embryonic stem cell established from inbred mouse strains.
SUMMARY OF THE INVENTION
The inventors of the present invention conducted various studies to achieve the foregoing object, and as a result, they succeeded in establishing novel embryonic stem cells from inbred mouse strains such as C3H/HeN.
The present invention thus provide embryonic stem cells derived from mouse strains C3H/HeN, C57BL6N, DBA/1J, and BALB/c.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The embryonic stem cell of the present invention were established from mouse inbred strains of C3H/HeN, C57BL/6N, DBA/1J, and BALB/c. Among these inbred strains, C3H/HeN, C57BL/6N, and DBA/1J are genetically complete inbred strains. As for BALB/c strain, some substrains, e.g., BALB/cA and BALB/cCr, are known; however, the substrains are genetically very close to each other. These mouse strains are widely and conventionally used and can easily be obtained.
For example, the embryonic stem cells of the present invention can be established as follows: Male and female mice of the above strains are allowed to naturally mate, and on the next day, the female mice having a vaginal plug are assigned as day zero (0) of pregnancy. Blastocysts are removed from uteri of the female mice on day three (3) of pregnancy and then cultured. After hatching, the cells are transferred on feeder cells and cultivation is continued for 2 or 3 days. The feeder cells can be prepared, for example, by treating fibroblasts of 14 day old embryos of mouse BALB/cA strain with Mitomycin C. Trophectoderm adhered onto the surface of the culture dish and spread. An inner cell mass (ICM) became rising on the sheet of trophectoderm. This rising cell mass is mechanically exfoliated by fine scalpel and dissociated into a single cell in approximately 0.25% trypsin droplets. The small cell masses are transferred on the feeder cells in a 24-well plate as the primary culture. The successive treatments include the following steps: The culture medium is changed every day, and individual colonies are separately picked up after 2 to 5 days and made into single cell masses with trypsin treatments. These dissociated cells are then placed again on the feeder cells. This procedure is repeated up to tenth subculture to allow the establishment of embryonic stem cells.
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Kamon Toshio
Kusakabe Moriaki
Kerr Janet H.
LeGuyader John L.
The Institute of Physical and Chemical Research
Wenderoth , Lind & Ponack, L.L.P.
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