Embryonic or stem-like cell lines produced by cross species nuclear transplantation and methods for enhancing embryonic development by genetic alteration of donor cells or by tissue culture conditions
An improved method of nuclear transfer involving the transplantation of differentiated donor cell nuclei into enucleated oocytes of a species different from the donor cell is provided. The resultant nuclear transfer units are useful for the production of isogenic embryonic stem cells, in particular human isogenic embryonic or stem cells. These embryonic or stem-like cells are useful for producing desired differentiated cells and for introduction, removal or modification, of desired genes, e.g., at specific sites of the genome of such cells by homologous recombination. These cells, which may contain a heterologous gene, are especially useful in cell transplantation therapies and for in vitro study of cell differentiation. Also, methods for improving nuclear transfer efficiency by genetically altering donor cells to inhibit apoptosis, select for a specific cell cycle and/or enhance embryonic growth and development are provided.
1. A method for producing a nuclear transfer human/rabbit blastocyst by cross-species nuclear transfer to give rise to embryonic stem-like cells comprising:
(i) inserting a human cell or human cell nucleus into an enucleated recipient rabbit oocyte under conditions suitable for the formation of a nuclear transfer (NT) unit;
(ii) activating the NT unit formed in step (i); and
(iii) culturing the NT unit until the blastocyst stage to give rise to embryonic stem-like cells, wherein the embryonic stem-like cells are pluripotent.
2. The method of claim 1 which further comprises transferring the nucleus of the NT unit from step (i) into another rabbit oocyte which is enucleated before transfer of the nucleus.
3. The method of claim 1 wherein the nuclear transfer blastocyst produced by step (iii) is cultured on a feeder layer.
4. The method of claim 3 wherein the blastocyst is cultured on a fibroblast feeder layer.