Toward establishing model organisms for marine protists: successful transfection protocols for Parabodo caudatus (Kinetoplastida: Excavata)

Summary We developed protocols for, and demonstrated successful transfection of, the free-living kinetoplastid flagellateParabodo caudatus with three plasmids carrying a fluorescence reporter gene(pEF-GFP with the EF1 alpha promoter, pUB-GFP with Ubiquitin Cpromoter, and pEYFP-Mitotrap with CMV promoter). We evaluated three electroporationapproaches: 1) a square-wave electroporatordesigned for eukaryotes, 2) a novel microfluidic transfection system employing hydrodynamically-controlled electric field waveforms, and 3) a traditional exponential decay electroporator. We found the microfluidic device provides a simple and efficient platform to quickly test a wide range of electric field parameters to find the optimal set of conditions for electroporationof target species. It also allows for processing large sample volumes (> 10 ml) within minutes, increasing throughput 100 times over cuvettes. Fluorescence signal from the reporter genewas detected a few hours after transfection and persisted for 3 days in cells transformed by pEF-GFP and pUB-GFP plasmids and for at least 5 days post-transfection for cells transformed with pEYFP-Mitotrap. Expression of the reporter genes(GFP and YFP) was also confirmed using reverse transcription-PCR (RT-PCR). This work opens the door for further efforts with this taxon and close relatives toward establishing model systems for genome editing. This article is protected by copyright. All rights reserved.