Buie Cullen R.

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Buie
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Cullen R.
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  • Preprint
    Toward establishing model organisms for marine protists : Successful transfection protocols for Parabodo caudatus (Kinetoplastida: Excavata)
    ( 2017-06) Gomaa, Fatma ; Garcia, Paulo A. ; Delaney, Jennifer ; Girguis, Peter R. ; Buie, Cullen R. ; Edgcomb, Virginia P.
    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 with the EF1 alpha promoter, pUB-GFP with Ubiquitin C promoter, and pEYFP37 Mitotrap with CMV promoter). We evaluated three electroporation approaches: 1) a square-wave electroporator designed 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 electroporation of 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 gene was 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.