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

• DOI: 10.1111/1462-2920.13830
• Publication Date: 2017-06-20T04:08:02Z
• Authors: Fatma Gomaa; Paulo A. Garcia; Jennifer Delaney; Peter R. Girguis; Cullen R. Buie; Virginia P. Edgcomb
• Keywords: Kinetoplastida

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