Toward establishing model organisms for marine protists: Successful transfection protocols for Parabodo caudatus (Kinetoplastida: Excavata).
Environ Microbiol
; 19(9): 3487-3499, 2017 09.
Article
em En
| MEDLINE
| ID: mdl-28631386
ABSTRACT
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Plasmídeos
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Transfecção
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Kinetoplastida
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Genes Reporter
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Eletroporação
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Proteínas de Fluorescência Verde
Limite:
Animals
Idioma:
En
Revista:
Environ Microbiol
Assunto da revista:
MICROBIOLOGIA
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SAUDE AMBIENTAL
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Estados Unidos