Methods of Reprogramming Animal Somatic Cells

A method for reprogramming an animal differentiated somatic cell to an undifferentiated stem cell, comprising the steps of:

• (a) isolating the nucleus from one or more animal differentiated somatic cells and injecting said nucleus into an oocyte, wherein said oocyte is from a species that is different from the species of said somatic cell, and allowing the remodeling of the nucleus of said somatic cell, wherein said remodeling results in
• (i) the remodeling of the nuclear envelope of said nucleus, wherein one or more components of the nuclear envelope of said nucleus are removed and wherein one or more components from said oocyte are added to the nuclear envelope of said nucleus of said somatic cell, and
• (ii) the reprogramming of the chromatin of said somatic cell resulting in the expression of genes of an undifferentiated cell, wherein said reprogramming of said chromatin comprises the removal of regulatory factors from said chromatin and the addition of regulatory factors from said oocyte to said chromatin;
• and
• (b) transferring or fusing the resulting remodeled nucleus from step (a) into the enucleated cytoplasm of an undifferentiated embryonic cell to form an undifferentiated stem cell, and allowing the resulting undifferentiated stem cell to grow.

A method for creating an undifferentiated animal cell of specific genetic identity, the method comprising the steps of:

• (a) providing compositions of:
• (i) a differentiated animal somatic cell, or nucleus or chromatin isolated therefrom, said differentiated animal somatic cell bearing a genetic identity desired for the undifferentiated animal cell to be created;
• and
• (ii) an undifferentiated embryonic cell;
• (b) performing chromatin or nuclear transfer with the compositions of step (a), wherein the composition of (a) (i) comprises the donor material and the composition of (a) (ii) comprises the recipient cell;
• (c) isolating the nucleus resulting from the chromatin or nuclear transfer of step (b) from within the undifferentiated embryonic cell;
• and
• (d) fusing the nucleus of step (c) with an enucleated cytoplasm of an embryonic stem cell to yield an undifferentiated animal cell of specific genetic identity.

A method for reprogramming multiple animal somatic cells, the method comprising the steps of:

• (a) providing:
• (i) at least two differentiated animal somatic cells that have their nuclear envelope remodeled to that of an undifferentiated cell;
• and
• (ii) at least two undifferentiated enucleated embryonic cell cytoplasts;
• (b) performing nuclear transfer with the cells of step (a), wherein the remodeled animal somatic cell nuclei are transferred to the cytoplasts in (ii);
• (c) isolating the nuclei resulting from the nuclear transfer of step (b) from within the Undifferentiated embryonic cell;
• and
• (d) repeating steps (a)-(c) to yield multiple reprogrammed animal pluripotent stem cells.

An assay to identify molecules useful to include or remove from a nucleus in the process of reprogramming to an undifferentiated state comprising the steps of:

• (a) providing:
• (i) an extract prepared from undifferentiated or germ-line cells, wherein said extract is capable of remodeling the nucleus of a differentiated cell and wherein said extract is modified by the addition or deletion a particular molecular component;
• (ii) at least one differentiated animal somatic cell;
• and
• (iii) at least one undifferentiated enucleated embryonic cell cytoplast;
• (b) remodeling the nucleus of said somatic cell of (ii) with the extract of (i);
• (c) performing nuclear transfer with the remodeled somatic cell nucleus of step (b), wherein the remodeled animal somatic cell nucleus is transferred to a recipient cytoplast;
• (d) culturing cells resulting from step (c);
• and
• (e) comparing the gene expression of said resulting cells of step (c) with normal embryonic cells of the same type as the cytoplast to determine whether the added or deleted molecular component plays a role in cellular reprogramming.