Production of Transgenic Cloned Miniature Pigs with Membrane-bound Human Fas Ligand (FasL) by Somatic Cell Nuclear Transfer

Abstract

Cell-mediated xenograft rejection, including NK cells and CD8+ CTL, is a major obstacle in successful pig-to-human xenotransplantation. Human CD8+ CTL and NK cells display high cytotoxicity for pig cells, mediated at least in part by the Fas/FasL pathway. To prevent cell-mediated xenocytotoxicity, a membrane-bound form of human FasL (mFasL) was generated as an inhibitor for CTL and NK cell cytotoxicity that could not be cleaved by metalloproteinase to produce putative soluble FasL. We produced two healthy transgenic pigs harboring the mFasL gene via somatic cell nuclear transfer (SCNT). In a cytotoxicity assay using transgenic clonal cell lines and transgenic pig ear cells, the rate of CD8+ CTL-mediated cytotoxicity was significantly reduced in transgenic pig's ear cells compared with that in normal minipig fetal fibroblasts. Our data indicate that grafts of transgenic pigs expressing membrane-bound human FasL control the cellular immune response to xenografts, creating a window of opportunity to facilitate xenograft survival.