Method and system for fusion and activation following nuclear transfer in reconstructed embryos

A method for producing cultured inner cell mass cells, comprising:

• (i) obtaining desired differentiated mammalian cells to be used as a source of donor nuclei;
• (ii) obtaining at least one oocyte from a mammal of the same species as the cells which are the source of donor nuclei;
• (iii) enucleating said at least one oocyte;
• (iv) transferring the desired differentiated cell or cell nucleus into the enucleated oocyte;
• (v) simultaneously fusing and activating the cell couplet to form a first transgenic embryo;
• (vi) activating a cell-couplet that does not fuse to create a first transgenic embryo but that is activated after an initial electrical shock by providing at least one additional activation protocol including an additional electrical shock to form a second transgenic embryo;
• and
• (vi) culturing cells obtained from said cultured activated embryo to obtain cultured inner cell mass cells.

A method for cloning a non-human mammal through a nuclear transfer process comprising:

• (i) obtaining desired differentiated mammalian cells to be used as a source of donor nuclei;
• (ii) obtaining at least one oocyte from a mammal of the same species as the cells which are the source of donor nuclei;
• (iii) enucleating said oocytes;
• (iv) transferring the desired differentiated cell or cell nucleus into the enucleated oocyte;
• employing at least two electrical shocks to a cell-couplet to initiate fusion and activation of said cell-couplet into an activated and fused embryo.
• (vii) culturing said activated and fused embryo until greater than the 2-cell developmental stage;
• (viii) transferring said first and/or second transgenic embryo into a host mammal such that the embryo develops into a fetus;
• wherein the second of said at least two electrical shocks is administered at least 15 minutes after an initial electrical shock;
• and
• wherein a desired gene is inserted, removed or modified in said differentiated mammalian cell or cell nucleus prior to insertion of said differentiated mammalian cell or cell nucleus into said enucleated oocyte.