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 cellmediated 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 + CTLmediated 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.

Immunogenetics of xenotransplantation

“…Similarly, researchers developed transgenic pigs overexpressing Fas Ligands (CD95L) or TNF‐α‐related apoptosis‐inducing ligand (TRAIL) protecting their cells from xeno T‐cell attack. Indeed, cells from transgenic pigs expressing a membrane‐bound form of CD95L are described to be protected against human T‐cell cytotoxicity (Seol et al., 2010). Likewise, expression of human TRAIL on porcine cells notably decreased xenogeneic response (Kemter et al., 2012; Klose et al., 2005).…”

“…Similarly, researchers developed transgenic pigs overexpressing are described to be protected against human T-cell cytotoxicity (Seol et al, 2010). Likewise, expression of human TRAIL on porcine cells notably decreased xenogeneic response (Kemter et al, 2012;Klose et al, 2005).…”

Recent advances in critical nodes of embryo engineering technology

“…in reprogramming, which makes somatic cell reprogramming incomplete, there is an extremely low potential for nuclear transfer embryo development. For example, the success rate of somatic cell cloning technology ranges from 5% for cattle 12 to 2% for pigs 13 and monkeys 11 , and its success rate is less than 1%. This greatly limits the application prospects of this technology.…”