First Efficient Transfection in Choanoflagellates using Cell-Penetrating Peptides

Abstract

ABSTRACTOnly recently, based on phylogenetic studies choanoflagellates have been confirmed to form the sister group to metazoan. The mechanisms and genes behind the step from single to multicellular organisation and as a consequence the evolution of metazoan multicellularity could not be verified yet, as no reliable and efficient method for transfection of choanoflagellates was available. Here we present cell-penetrating peptides (CPPs) as an alternative to conventional transfection methods. In a series of experiments with the choanoflagellate Diaphanoeca grandis we proof for the first time that the use of CPPs is a reliable and highly efficient method for the transfection of choanoflagellates. We were able to silence the silicon transporter gene (SIT) by siRNA, and hence, to suppress the lorica (characteristic siliceous basket) formation. High gene silencing efficiency was determined and measured by light microscope and RT-qPCR. In addition, only low cytotoxic effects of CPP were detected. Our new method allows the reliable and efficient transfection of choanoflagellates, finally enabling us to verify the function of genes, thought to be involved in cell adhesion or cell signaling by silencing them via siRNA. This is a step stone for the research on the origin of multicellularity in metazoans.