Method to enrich for α(1,3)-galactosyltransferase null pig cells
The invention relates to the genetic manipulation of non-human animals. More particularly, the invention relates to genetic manipulation of non-human animals to be used for xenotransplantation. The invention provides a method of selecting GGTA 1 null cells, a viable GGTA 1 null swine, methods for making such swine, and methods of using cells, tissues and organs of such swine for xenotransplantation.
1. A method of selecting GGTA1 null cells comprising the steps of:
(a) obtaining a line of cells obtained from a GGTA1 heterozygous swine or swine fetus;
(b) enriching the cells for GGTA1 null cells by treating the cells with agents that specifically bind an α(1,3)-galactose epitope and that deplete cells that express the epitope; and
(c) scanning the line for viable GGTA1 null cells.
2. The method of claim 1 wherein in step (b), the cells are enriched by at least one treatment selected from the group consisting of: (a) treating the said cells with anti-galactose-α(1,3)-galactose antibodies, in the presence of complement; (b) depleting the said cells with magnetic micro-beads bound with anti-gal reagents; (c) treating the said cells with anti-galactose-α(1,3)-galactose antibodies and depleting the said cells with magnetic micro-beads bound with anti-antibodies; and (d) treating the said line with gal epitope ligands and depleting the said line with magnetic micro-beads bound with anti ligand antibodies.
3. The method of claim 1 wherein in step (b), the cells are enriched by multiple treatments selected from the group consisting of: (a) treating the said cells with anti-galactose-α(1,3)-galactose antibodies, in the presence of complement; (b) depleting the said cells with magnetic micro-beads bound with anti-gal reagents; (c) treating the said cells with anti-galactose-α(1,3)-galactose antibodies and depleting the said cells with magnetic micro-beads bound with anti-antibodies; and (d) treating the said cells with gal epitope ligands and depleting the said line with magnetic micro-beads bound with anti ligand antibodies.
4. The method of claim 1 wherein in step (b), the cells are enriched by three treatments of each of the following: (a) treating the said cells with anti-galactose-α(1,3)-galactose antibodies, in the presence of complement; (b) treating the said cells with gal epitope ligands and depleting the said line with magnetic micro-beads bound with anti ligand antibodies.
5. The method according to any of claims 1 - 4 wherein the line of cells is a line of swine fetal fibroblast cells.
6. The method according to any of claims 1 - 4 wherein the line of cells is a clonal population of swine fetal fibroblast cells.
7. The method of claim 5 wherein the swine fetal fibroblast cells originate from miniature swine.
8. The method according to claim any of claims 1 - 4 wherein the line of cells is a line of stem cells.
9. The method of claim 8 wherein the stem cells are primordial stem cells.
10. The method according to any of claims 2 - 4 wherein the anti-galactose-α(1,3)-galactose antibodies are primate antibodies.
11. The method according to any of claims 2 - 4 wherein the anti-galactose-α(1,3)-galactose antibodies are monoclonal antibodies or fragments thereof.
12. The method according to any of claims 2 - 3 , wherein the anti-gal reagents are selected from a group consisting of anti-galactose-α(1,3)-galactose antibodies and lectin.
13. The method according to any of claims 2 - 4 , wherein the gal epitope ligands are IB4 conjugates and the anti-epitope ligands are anti-IB4 conjugates.
14. The method according to claim 13 wherein the IB4 conjugates are selected from a group consisting of IB4 biotin and IB4-FITC and the anti-IB4 conjugates are selected from a group consisting of anti-biotin and anti-FITC.
15. The method of claim 6 wherein the swine fetal fibroblast cells originate from miniature swine.