Double knockout (GT/CMAH-KO) pigs, organs and tissues
The invention provides double knockout transgenic pigs (GT/CMAH-KO pigs) lacking expression of any functional aGAL and CMAH. Double knockout GT/CMAH-KO transgenic organs, tissues and cells are also provided. Methods of making and using the GT/CMAH-KO pigs and tissue are also provided.
1. A knockout pig comprising disrupted α(1,3)-galactosyltransferase and CMAH genes, wherein the disruption of said α(1,3)-galactosyltransferase gene is selected from the group of disruptions to the sequence described in SEQ ID NO:18 comprising a 3 base pair deletion adjacent to a G to A substitution, and the group of disruptions to the sequence described in SEQ ID NO:24 comprising a single base pair deletion, a single base pair insertion, a six base pair deletion, a two base pair insertion, a ten base pair deletion, a seven base pair deletion, and an eight base pair substitution for a five base pair sequence; wherein the disruption of said CMAH gene is selected from the group of disruptions to the sequence described in SEQ ID NO:16 comprising a four base pair insertion, and the group of disruptions to the sequence described in SEQ ID NO:25 comprising a two base pair deletion, a single base pair insertion, an eight base pair deletion, a five base pair deletion, a three base pair deletion, a two base pair substitution for a single base pair, and a twenty base pair deletion; and wherein expression of functional α(1,3)-galactosyltransferase and CMAH in said knockout pig is decreased as compared to a wild-type pig and when tissue from said knockout pig is transplanted into a human, a hyperacute rejection related symptom is improved as compared to when tissue from a wild-type pig is transplanted into a human.
2. Porcine organs, tissues and cells obtained from the knockout pig of claim 1 .
3. A cell culture reagent obtained from the knockout pig of claim 1 , wherein the amount of Neu5Gc or alphaGal epitopes in said cell culture reagent is lower than the amount of Neu5Gc or alphaGal epitopes in a cell culture reagent obtained from a wild-type pig, and wherein said cell culture reagent is selected from the group consisting of cell culture media, cell culture serum, and an isolated cell capable of proliferation.