Rat embryonic stem cell

• US 8,137,966 B2
• Filed: 03/01/2005
• Issued: 03/20/2012
• Est. Priority Date: 03/04/2004
• Status: Active Grant

• Current Assignee: Sumitomo Chemical Company Limited
• Original Assignee: National Cancer Center, Dainippon Sumitomo Pharma Company Limited (Sumitomo Chemical Company Limited)
• Inventors: Teratani, Takumi, Ochiya, Takahiro
• Primary Examiner(s): Nguyen, Quang
• Application Number:
  US10/591,407
• Publication Number:
  US 20070186293A1
• Time in Patent Office: 2,576 Days
• Field of Search: None
• US Class Current: 435/353
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/0393 Animal model comprising a reporter system for screening tests
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

The present invention provides a rat embryonic stem cell characterized by having the following properties of (a) expressing Oct3/4 gene and Nanog gene, (b) positive for alkaline phosphatase activity, (c) having an embryoid body forming ability, (d) expressing SSEA (Stage-Specific Embryonic Antigen)-1 and SSEA-4, (e) having the same number of chromosomes as does a normal rat cell, (f) capable of being subcultured and holding the undifferentiated state, (g) having in vitro pluripotency, (h) having a potential to differentiate for cells of three embryonic germ lineages, (i) having teratoma formation ability, and (j) having an ability to produce a chimeric rat, a method of establishing the aforementioned rat embryonic stem cell and the like.

Claims

1. A method of producing a rat embryonic stem cell, which method consists essentially of the following steps (A)-(E) performed using a culture medium with 2% or less serum concentration:

(A) culturing a rat blastocyst on inactivated mouse embryonic fibroblasts in a leukemia inhibitory factor (LIF)-free culture medium to form an inner cell mass in the blastocyst,

(B) dissociating the inner cell mass, wherein the dissociated inner cell mass is in a cell aggregate state,

(C) culturing primary embryonic stem cells resulting from a culture of the dissociated inner cell mass on inactivated mouse embryonic fibroblasts until the primary embryonic stem cells can be passaged,

(D) dissociating the primary embryonic stem cells, which can be passaged, wherein the dissociated primary embryonic stem cells are in a cell aggregate state, and

(E) culturing the dissociated primary embryonic stem cells on inactivated mouse embryonic fibroblasts to establish an embryonic stem cell,

wherein a rat leukemia inhibitory factor (rLIF)-containing culture medium is used in steps (C)-(E).

2. The method of claim 1, wherein the culture medium comprises a serum replacement reagent.

3. The method of claim 1, wherein step (B) comprises a step for mechanically dissociating the inner cell mass.

4. The method of claim 1, wherein step (D) comprises a step for mechanically dissociating the embryonic stem cells.

5. The method of claim 1, wherein a leukemia inhibitory factor (LIF)-free culture medium is used in step (B).