Successful Suppression of Endogenous <i>&alpha;-1,3-Galactosyltransferase</i> Expression by RNA Interference in Pig Embryos Generated <i>In Vitro</i>

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 doublestranded 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. Key words: Blastocyst, α-1,3-Galactosyltransferase, Pig, RNAi, Somatic cell nuclear transfer (J. Reprod. Dev. 58: [69][70][71][72][73][74][75][76] 2012) S uppression of endogenous target gene expression by gene targeting has been proven as a powerful tool for exploration of the biological function of a gene of interest. Recently, attempts to use antisense technology, now termed RNA interference (RNAi), as a convenient and valuable tool for understanding the in vivo function of a gene of interest have been made [1][2][3][4][5][6][7][8]. RNAi is a multiple-step process that involves the generation of 21-25 nt small interfering (si) RNA and results in degradation of the homologous RNA [2]. Since siRNA can be easily synthesized chemically, it is easy to study the role of endogenous genes by injecting double-stranded (ds) RNA or siRNA into early embryos. However, in the former cases, the effectiveness of dsRNA introduced into cultured cells is known to be retained for a relatively short time (less than one week) [5][6][7][8][9]. In contrast, use of siRNA should be more beneficial for conferring long-term expression of siRNA in transfected cells. In fact, this technology (which is termed "transgenic RNAi") was found to be effective in knocking down ta...

Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations

“…Therefore, it may be required to establish a single embryo-based assay for detecting such mutations at the protein and gene levels, which will enable researchers to test whether the CRISPR/Cas9-related components constructed are effective for inducing mutations in the target locus. Blastocysts, stages just prior to implantation, are appropriate materials to test this issue since they can successfully be used for PCR-based amplification for molecular biological analysis [ 38 , 39 ] and express many types of mRNAs, including α-1,3-galactosyltransferase (α-GalT) gene ( GGTA1 ) [ 40 ]. It takes seven days to obtain blastocysts after fertilization in pigs.…”

Effects of Downregulating Oct-4 Transcript by RNA Interference on Early Development of Porcine Embryos

“…Most recently, RNA interference (RNAi) has been proven to be functional in oocytes and preimplantation embryos of the pig [23, 24]. Knockdown of Oct-4 expression in porcine embryos may be achieved using siRNA.…”

Xenotransplantation literature update, May to June 2012

“…Chi et al [14] examined whether RNA interference (RNAi) technology is useful for the suppression of the GT enzyme in early pig embryos. GT synthesis was monitored by cytochemical staining with Bandeiraea simplicifolia isolectin-B 4 and by RT-PCR analysis.…”