Blastomere culture to produce mammalian embryonic stem cells
View Patent ↗The present invention relates generally to the field of somatic cell nuclear transfer (SCNT) and to the creation of cloned animals and cells. The disclosure relates to a method of cloning a mammal, obtaining pluripotent cells such as embryonic stem cells, or for reprogramming a mammalian cell using an oocyte and a fertilized embryo.
1. A method of producing an mammalian embryonic stem (ES) cell, comprising
(a) culturing a mammalian blastomere for 12 to 18 hours;
(b) transferring the blastomere to blastocyst medium further comprising laminin and seeded with fibroblasts; and
(c) culturing the blastomere of (b) until ES cells are produced.
2. The method of claim 1 , wherein the fibroblasts are mitotically inactivated, wherein step (c) comprises culturing in conditions that reduce embryonic vesicle formation, wherein the blastocyst medium comprises 2.5 μg/ml of laminin, and wherein step (c) comprises culturing in blastocyst medium seeded with fibroblasts for 5 days.
3. The method of claim 1 , wherein step (c) further comprises culturing until the blastomeres form cell clumps of about 20 cells and transferring the cell clumps to medium seeded with ES cells that express a marker or are labeled.
4. The method of claim 1 , wherein step (a) comprises culturing said blastomere with a mammalian parental embryo from which said blastomere was obtained.
5. The method of claim 1 , wherein said blastomere is human.
6. The method of claim 1 , wherein step (b) comprises culturing said blastomere in indirect contact with pluripotent cells.
7. The method of claim 1 , wherein said fibroblasts are mouse embryonic fibroblasts.