308 Development of Transgenic Livestock With Reduced Myostatin Expression Using Rna Interference

Abstract

RNA interference (RNAi) is a means of regulating gene expression by targeting mRNA in a sequence-specific manner for degradation or translational inhibition. Short hairpin RNAs (shRNAs) and siRNAs have been extensively employed for manipulating gene expression in a wide range of species. However, the great majority of this work has involved in vitro studies with cells grown in culture. Our goal for this project is to produce transgenic livestock in which myostatin, a negative regulator of muscle growth, has been targeted for silencing by RNAi. In theory, livestock in which myostatin has been silenced should exhibit increased muscle growth and development. To that end, we designed shRNAs to target the bovine myostatin mRNA sequence. The shRNAs were cloned into a lentiviral vector that contains a cytomegalovirus promoter controlling green fluorescent protein and shRNA expression as well as neomycin resistance. Infective lentivirus was made in HEK293T cells through co-transfection of the lentiviral vector, a packaging plasmid, and a plasmid expressing the VSVG pseudotype. Bovine fetal fibroblasts were transduced, selected using Geneticin®, and nuclear transfer was utilized to produce cloned transgenic embryos. There were 186 fusion attempts resulting in 160 fused embryos (fusion rate = 86%). Of these, 54 reached the blastocyst stage (34%) and 10 embryos were transferred into 5 recipient females (2 embryos per recipient). At 40 days, ultrasound revealed 1 confirmed pregnancy. Current plans are to harvest this fetus at 90 days and analyze it for evidence of myostatin knockdown. The production of transgenic animals exhibiting myostatin knockdown through lentiviral-mediated RNAi will demonstrate the utility of RNAi in the study of gene function in large animal models without the need for homologous recombination techniques, which are currently inefficient in species other than mice.