JPH05328878A - Embryo-undifferentiated cell - Google Patents

Abstract

PURPOSE:To provide an embryo-undifferentiated (ES) cell originated from a mouse of a strain other than 129 mouse and capable of efficiently producing descendant mice. CONSTITUTION:The objective embryo-undifferentiated (ES) cell originated from a 4-days embryo obtained by crossing a female of C57BL/6 mouse with a male of CBA mouse and characterized by the generation of chimera mouse from a 3-days embryo (8-cell embryo) and free from generation of chimera mouse from a 4-days embryo. The ES cell is e.g. TT2 embryo undifferentiated (ES) cell of FERM P-12971.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to embryonic undifferentiated (ES) cells used for producing chimeric mice.

[0002]

2. Description of the Related Art Undifferentiated cells (ES cells) considered to be close to the inner cells (ICM) of a 4-day mouse embryo can be normally cultured. When the cultured ES cells were re-injected into the vagina of the 4-day embryo, and were allowed to develop and grow in the pseudouterine uterus of the foster mother, cells derived from both the ES cells injected into each tissue of the body and the ICM of the 4-day embryo of the host. A chimeric mouse composed of Differentiation of ES cells into germ cells also occurs in chimeric mice, and thus ES cell-derived mice can be obtained by their progeny. [A. Bradley et al .: Nature 309 , 2
55 (1984)]. Unlike common somatic cells, ES cells probably divide indefinitely without showing hypomitosis-crisis [Y. Suda et al., J. Am. Cell. P
hysiol. , 133 , 197 (1987)]. ES
The cell means that each mouse individual can be treated as one cultured cell in the incubator.

However, not all undifferentiated cells cultured from 4-day embryos differentiate into germ cells, and still 12
Only a few limited cell lines derived from 9 mice are commonly used. Although the identification of the leukemia inhibitor facilitated its culturing,
Establishment of ES cells having good germ cell differentiation ability is not easy. In addition, careful culture is required to keep the germ cell differentiation potential of ES cells stable under the current culture conditions. The efficiency of ES cell differentiation into germ cells is also affected by the combination of ES cell-host embryo mouse strains in the production of chimeric mice, and whether ES cells efficiently differentiate into germ cells remains a major issue. is there.

Therefore, an object of the present invention is to provide an embryonic undifferentiated (ES) cell which is derived from a mouse of a strain other than 129 mouse and which efficiently produces a progeny mouse.

[0005]

The present invention provides a C57BL /
It is derived from a 4-day embryo obtained by mating a 6-mouse female with a CBA-male male, and produces a chimeric mouse in a 3-day embryo (8-cell embryo) and does not produce a chimeric mouse in a 4-day embryo (blastocyst embryo). An embryonic undifferentiated cell characterized by:

The present invention, in particular, has been entrusted to the Institute of Microbiology, Institute of Industrial Science, on May 25, 1992, with the contract number of Micro Engineering Research Institute No. 12
No. 971 (FERM P-12971) deposited TT2 embryonic undifferentiated cells.

The present invention, particularly the TT2ES cells, will be specifically described below. (A) Origin It is a 4-day embryo obtained by mating a C57BL / 6 mouse female and a CBA mouse male.

(B) Preparation method i) C57BL / 6 female mice and CBA male mice were crossed to obtain 4-day embryos. ii) 4-day embryos were cultured on feeder cells for 5 days (Yagi et al., Proc. Natl. Acad. Sci. USA).
87 , 9918-9922 (1990)). Trophectoderm-like cells were opportunistically removed from the grown cylindrical structure under a microscope, dispersed with trypsin, and cultured again on feeder cells. (Yagi et al., Proc. Natl. Acad.
Sci. USA 87 , 9918-9922 (199
0)). The feeder cells are BALB / c mouse 15
It was prepared by treating fibroblast-like cells cultured from the embryo of the day with mitomycin. iii) After culturing for 4 days, cell clusters showing an undifferentiated cell-like morphology were collected using a capillary under a microscope, dispersed with trypsin, and cultured again on feeder cells. iv) After culturing for 4 days, iii) was repeated. v) After culturing for 4 days, all the cells were subcultured, further subcultured for 3 days, and most of the cells were frozen at each subculture, and TT2 cells were preserved.

(C) Properties Cultured normal cells having properties close to undifferentiated cells / inner cell mass of 4-day embryo (undifferentiated cells capable of differentiating into all cells of 3 germ layers including germ cells) as a differentiation stage. It is an undifferentiated cell. It shows a morphology similar to that of previously reported ES cells, can be cultured under similar conditions, and differentiates in vitro or subcutaneously in syngeneic mice and nude mice, but previously reported ES cells (E14, D3,
It seems that ES cells are in a slightly earlier developmental stage than CCE, etc., and when injected into 3-day embryos (8-cell embryos), progeny mice are generated with extremely high efficiency.

Previously reported ES cells give rise to chimeric mice when injected into 4-day embryos (blastocyst embryos).
2 cells do not give rise to chimeric mice when injected into 4-day embryos.
In contrast, the former does not give rise to chimeric mice when injected into 3-day embryos, but TT2 cells are mainly TT2 when injected into 3-day embryos.
Mice composed of cells derived from the mouse are generated at a frequency of 70% or more, and the progeny from such a male mouse is almost 100% T.
It is a descendant derived from T2. Thus far, there has been no report of ES cells giving rise to progeny mice at a high rate.
There are cells, but AB cells are injected into 4-day embryos.

It is also an advantage of TT2 cells that ICR mouse embryos (3-day embryos) can be used as 4-day embryos to be injected (unless ES cells so far used C57BL / 6 mouse embryos (4-day embryos). However, the cost was high). All ES cells that give rise to conventional offspring mice are derived from 129 mice, but TT2 cells are the first ES cells to give rise to offspring mice derived from other strains of mice.

[0012]

[Examples] Preparation of chimeric mice using TT2E cells will be described. a) Superovulated ICR female mice were mated with male mice, and embryos were collected on the 3rd day. b) TT2 cells (or TT2-derived mutant cells) were dispersed with trypsin and kept under ice cooling (Yagi et al., Pro.Nt.
l. Acad. Su. USA 87 , 9918-9922.
(1990)). c) About 10 TT2 (or TT2-derived mutant cells) cells were injected under the zona pellucida on the 3rd day embryo using a micromanipulator. d) After injection, the embryos were cultured for 1 day and grown into blastocyst embryos, which were then transplanted into the uterus of a pseudopregnant temporary parent (ICR). e) Only male mice born from the foster mother and having a gray coat color of 50% or more in the mouse were mated with BALB / c female mice. f) A mouse with a murine coat color that had a genetic mutation was analyzed by Southern method after extracting DNA from its tail. Mutated mice were crossed with each other to obtain progeny mice. The mice were maintained by mating below.

The results are shown in Table 1. E14, D for comparison
Results for cells 3 and CCE are also shown. See I
Table 2 shows the results when C57BL / 6 mouse 4-day embryos were used instead of CR3-day embryos. From Tables 1 and 2, TT2
ES cells do not give rise to offspring in 4-day embryos of C57BL / 6 mice (Table 2), whereas they frequently give rise to offspring with 3-day embryos of ICR (Table 1). In contrast, the other E14, D3 and CCE cells gave rise to progeny in C57BL / 6 mouse 4-day embryos (Table 2, less frequent), but no ICR3-day embryos.

[0014]

[Table 1]

[0015]

[Table 2]

Claims (2)

[Claims] 1. A 4-day embryo derived from a cross between a C57BL / 6 female mouse and a CBA male mouse, wherein the 3-day embryo produces a chimeric mouse and the 4-day embryo does not produce a chimeric mouse. Embryonic undifferentiated cells. 2. Microtechnology Research Institute Microbiology No. 12971 (FERM
The cell according to claim 1, which is a TT2 embryonic undifferentiated cell of P-12971).