Chimeric rat produced using rat embryonic stem cells in the presence of an ES cell differentiation suppressant
The present invention provides a preparation method of a chimeric embryo and a chimeric rat, which is characterized by contacting a rat pluripotent stem cell and a host embryo in the presence of an ES cell differentiation inhibitor. The method includes (a) a step for contacting a fertilized host embryo collected from a female rat and a rat pluripotent stem cell in the presence of an ES cell differentiation suppressant, and (b) a step for culturing the host embryo in contact with the rat pluripotent stem cell to form a chimeric embryo.
1. A method of preparing a chimeric embryo, comprising
(a) a step for introducing a rat pluripotent stem cell into a fertilized host embryo collected from a female rat, wherein the introducing is in a medium comprising an ES cell differentiation suppressant comprising an MEK inhibitor, a GSK3 inhibitor, and a TGFβ receptor inhibitor, to produce a chimeric rat embryo.
2. The method according to claim 1 , wherein the introducing is achieved by injecting the rat pluripotent stem cell into the host embryo.
3. The method according to claim 1 , wherein the rat pluripotent stem cell is introduced into the host embryo in a medium comprising the ES cell differentiation suppressant of step (a) and a ROCK inhibitor.
4. The method according to claim 1 , wherein the host embryo is precultured in a medium comprising the ES cell differentiation suppressant before introduction of the rat pluripotent stem cell.
5. The method according to claim 4 , wherein the preculture is performed in a medium comprising the ES cell differentiation suppressant and a ROCK inhibitor.
6. The method according to claim 2 , wherein the medium comprises the ES cell differentiation suppressant and a ROCK inhibitor.
7. The method according to claim 1 , wherein the pluripotent stem cell is an ES cell.
8. The method according to claim 1 , wherein the rat pluripotent stem cell is a pluripotent stem cell prepared from a rat of a strain that does not produce a germline-transmitted chimeric rat when introduced into a host embryo in the absence of the ES cell differentiation suppressant in the step (a).
9. The method according to claim 1 , wherein the host embryo is derived from a rat of a strain that does not produce a germline-transmitted chimeric rat when introduced with a rat pluripotent stem cell in the absence of the ES cell differentiation suppressant in the step (a).
10. The method according to claim 1 , further comprising (b) a step for culturing the chimeric embryo in a medium comprising the ES cell differentiation suppressant to form a chimeric blastocyst.
11. The method according to claim 10 , wherein the medium of step (b) comprises the ES cell differentiation suppressant and a ROCK inhibitor.
12. A method of preparing a chimeric rat, comprising transplanting a chimeric blastocyst prepared by the method according to claim 10 to the uterus or oviduct of a psuedopregnant female rat to allow an offspring rat to be born.
13. A method of preparing a rat with a contribution of a rat pluripotent stem cell to the whole body, comprising mating a chimeric rat obtained by the method according to claim 12 with a rat of the opposite sex.