[Show abstract][Hide abstract] ABSTRACT: α-1,3-Galactosyltransferase (α-GalT), an enzyme creating Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species such as pigs, is known to be a key factor that causes hyperacute rejection upon transplantation from pigs to humans. To establish the RNA interference-based suppression of endogenous α-GalT messenger RNA (mRNA) synthesis in porcine preimplantation embryos, we determined the suitable embryonic stage at which stage such approach is possible by using the semi-quantitative RT-PCR (qRT-PCR) and the cytochemical method using a fluorescence-labeled Bandeiraea simplicifolia Isolectin B(4) (BS-I-B(4) ). Staining with BS-I-B(4) demonstrated that α-Gal epitope expression was first recognized at the 8-cell stage, and increased up to the hatched blastocyst stage. Single embryo-based qRT-PCR also confirmed this pattern. These results indicate that creation of α-Gal epitope is proceeded by de novo synthesis of α-GalT mRNA in porcine preimplantation embryos with peaking at the blastocyst stage.
[Show abstract][Hide abstract] ABSTRACT: RNA interference (RNAi) technology using small interfering RNAs (siRNA) has been widely used as a powerful tool to knock down gene expression in various organisms. In pig preimplantation embryos, no attempt to suppress the target gene expression with such technology has been made. The purpose of this study is to demonstrate that the RNAi technology is useful for suppression of endogenous target gene expression at an early stage of development in pigs. Alpha-1,3-Galactosyltransferase (α-GalT) is an enzyme that creates the Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species, and removal of the epitope is considered to be a prerequisite for pig-to-human xenotransplantation. We decided to suppress the endogenous α-GalT mRNA expression in pig early embryos, since reduction of α-GalT synthesis is easily monitored by cytochemical staining with Bandeiraea simplicifolia isolectin-B(4), a lectin that specifically binds to the α-Gal epitope, and by RT-PCR analysis. Cytoplasmic microinjection of double-stranded RNA and pronuclear injection of an siRNA expression vector into the embryos generated in vitro resulted in a significant reduction in expression of the α-GalT gene and α-Gal epitope in blastocysts, at which stage the α-Gal epitope is abundantly expressed. Somatic cell nuclear transfer of embryonic fibroblasts stably transfected with an siRNA expression vector also led to a significant reduction in the level of α-GalT mRNA synthesis together with decreased amounts of the α-Gal epitope at the blastocyst stage. These results indicate that the RNAi technology is useful for efficient suppression of a target gene expression during embryogenesis in pigs and suggest the possibility of production of siRNA-expressing pigs for use in xenotransplantation.
Journal of Reproduction and Development 10/2011; 58(1):69-76. DOI:10.1262/jrd.10-165M · 1.52 Impact Factor