Microinjection of dsRNA in tardigrades

Abstract

Classical genetic analysis in the tardigrade Hypsibius exemplaris is a challenge because these animals are parthenogens. The publication of the H. exemplaris genome has facilitated the study of targeted genes by RNA interference (RNAi), a robust mechanism to disrupt gene function. This protocol describes microinjection of double-stranded RNA (dsRNA) in tardigrades using techniques adapted from protocols originally developed in Caenorhabditis elegans. A DNA template (either genomic or cDNA) is used to prepare dsRNA, to which T7 polymerase binding sites are added at the 5′ end of each strand. The dsRNA is injected into adult tardigrades, preferably targeting the gonad or intestine. Injected adults are allowed to recover in spring water and then transferred to culture dishes or individual wells of a 96-well plate.