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Toward establishing model organisms for marine protists: Successful transfection protocols for Parabodo caudatus (Kinetoplastida: Excavata)
Author(s) -
Gomaa Fatma,
Garcia Paulo A.,
Delaney Jennifer,
Girguis Peter R.,
Buie Cullen R.,
Edgcomb Virginia P.
Publication year - 2017
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1111/1462-2920.13830
Subject(s) - electroporation , biology , transfection , green fluorescent protein , reporter gene , plasmid , microbiology and biotechnology , kinetoplastida , gene , gene expression , genetics , protozoal disease , malaria , immunology
Summary 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 pEYFP‐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 transfected by pEF‐GFP and pUB‐GFP plasmids and for at least 5 days post‐transfection for cells transfected 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.