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LEA29Y transgenic pigs for overcoming cellular rejection in xenotransplantation

Andrea Baehr¹, Lelia van Buerck², Mayuko Kurome¹, Barbara Kessler¹, Hiroshi Nagashima³, Jochen Seissler², Nikolai Klymiuk¹, Eckhard Wolf¹

¹Molecular Animal Breeding, LMU Munich, Munich, Germany; ²Diabetes Center, LMU Munich, Munich, Germany; ³Laboratory of Developmental Engineering, Meiji University, Kawasaki, Japan

Inhibition of the B7/CD28 co-stimulatory pathway of T-cell activation is an approach for overcoming cellular rejection of xenografts.

Pigs transgenic for the coding sequence of the co-stimulation blocking agent LEA29Y under the control of the 1.3kb core promoter from the porcine insulin gene were generated. The gene construct was transfected into porcine fetal fibroblasts and stable, pooled clones were subsequently used for nuclear transfer into enucleated oocytes. Embryos gained after electrofusion and activation were transferred into synchronised gilts. By this, two litters with a total of seven unique transgenic founder piglets could be generated. Genomic analysis by Southern blotting revealed the distinct transgenic properties of each individual. Immunohistochemical analysis of organ spectra of four animals sacrificed at an age of three months illustrated strong pancreas islet specific expression of the transgene product in two piglets which were therefore chosen for re-cloning to generate animals for functional analysis and breeding.

Isolated islet-like clusters of re-cloned neonatal INS-LEA transgenic pigs and wild-type controls were transplanted under the kidney capsule of streptozotocin induced NOD-SCID mice. After mice had re-gaining normoglycaemia, human peripheral blood mononuclear cells were administered to restore their immune system in a humanised way. Mice transplanted with wild-type control islets subsequently became hyperglycaemic, while recipients with LEA29Y transgenic islets retained their normoglycaemia for 30 days but became hyperglycaemic after removal of the transplant. Thus, the functionality of this INS-LEA pig model could be demonstrated.