Genetically modified rat derived from rat embryonic stem cell
View Patent ↗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.
1. A heterozygous knockout or knockin rat obtained by
(I) producing a chimeric rat by a process comprising the following steps (X)-(Z):
(X) a step for introducing a targeting vector into a target gene in the genome of a rat embryonic stem cell by homologous recombination to produce a homologously recombined rat embryonic stem cell,
(Y) a step for preparing a chimeric rat embryo for transplantation by (i) injecting the rat embryonic stem cell, into which the targeting vector was introduced, into a blastocoele of a rat blastocyst or a morula stage-, 16-cell stage- or 8-cell stage-rat embryo or (ii) co-culturing the rat embryonic stem cell, into which the targeting vector was introduced, with an 8-cell stage- or 16-cell stage-rat embryo without zona pellucidas,
(Z) a step for transferring the chimeric rat embryo for transplantation into the uterus of a pseudopregnant female rat to produce the chimeric rat, wherein the rat embryonic stem cell has the following properties (a)-(k):
(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,
(j) having an ability to produce a chimeric rat,
(k) being germline-competent,
wherein the rat embryonic stem cell is obtained by the method consisting 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,
(E) culturing the dissociated primary embryonic stem cells on inactivated mouse embryonic fibroblasts to establish an embryonic stem cell,
wherein an rLIF-containing culture medium is used in steps (C)-(E); and
(II) crossing the chimeric rat with a wildtype rat, thereby producing a heterozygous knockout or knockin rat.
2. A homologous knockout or knockin rat obtained by mating the heterozygous knockout or knockin rats according to claim 1 .