TITLE: Transfection of Crypthecodinium cohnii using labelled DNA
AUTHORS: José A. Fernández Robledo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">José A. Fernández Robledo </div><div class = "text-block">Nastasia J. Freyria</div><div class = "text-block"> Bigelow Laboratory for Ocean Sciences</div></div>

[STEPS]
?. [Reagents]
Liquid mediumL1 + 10 gr/L glucose NPN (https://ncma.bigelow.org/ccmp316#.XLOpDZNKi2w)L1 medium with nitrogen limited (without yeast extract and glucose) to obtain cyst from C. cohnii swimming cellsPlatesPrepare Petri dishes containing L1 medium bacteriological agar (1.5%)Prepare  PEG 8000 solution (0.4%) with L1 medium, sterilized by using a filter (0.22μm)Pour 1 ml of L1-PEG8000 solution onto dry agar plates; alternatively, pour 12.5 ml of L1-PEG8000 solution onto dry agar platesLet the PEG800 solution infuse for 24 hStore at 4°Cuntil usePlasmid vectorsPropage  pPmMOE[MOE]:GFP-11 (Cold et al., 2016; Fernández Robledo et al., 2008) in JM109 for miniprep    Linearize 20 μg with NotI (e.g., New England Biolabs® inc., Ipswich, MA, USA), clean the restriction digestion (e.g., GenElute® PCR Clean-up kit)Store at -20°C until useDNA probe construction and DNA labeling*Use forward (5’ -CCGCACATGTATGGTGAGCAAGGGCGAGGAGC- 3’) the reverse (5’ –CGTAGGACATGTCTTGTACAGCTCGTCCATGCCG- 3’ primers targeting pPmMOE[MOE]:GFP-11 (Fernández Robledo et al., 2008) to amplify a 739 bp DNAClean the amplicon as above and label 1 µg using UlysisTM Alexa Fluor® 488 Nucleic Acid Labeling Kit (ThermoFisher Scientific) following the manufacturer’s instructionsStore labelled DNA were stored at -20°C until usePlatesPrepare Petri dishes containing L1 medium bacteriological agar (1.5%)Prepare  PEG 8000 solution (0.4%) with L1 medium, sterilized by using a filter (0.22μm)Pour 1 ml of L1-PEG8000 solution onto dry agar plates; alternatively, pour 12.5 ml of L1-PEG8000 solution onto dry agar platesLet the PEG800 solution infuse for 24 hStore at 4°Cuntil usePlasmid vectorsPropage  pPmMOE[MOE]:GFP-11 (Cold et al., 2016; Fernández Robledo et al., 2008) in JM109 for miniprep    Linearize 20 μg with NotI (e.g., New England Biolabs® inc., Ipswich, MA, USA), clean the restriction digestion (e.g., GenElute® PCR Clean-up kit)Store at -20°C until useDNA probe construction and DNA labeling*Use forward (5’ -CCGCACATGTATGGTGAGCAAGGGCGAGGAGC- 3’) the reverse (5’ –CGTAGGACATGTCTTGTACAGCTCGTCCATGCCG- 3’ primers targeting pPmMOE[MOE]:GFP-11 (Fernández Robledo et al., 2008) to amplify a 739 bp DNAClean the amplicon as above and label 1 µg using UlysisTM Alexa Fluor® 488 Nucleic Acid Labeling Kit (ThermoFisher Scientific) following the manufacturer’s instructionsStore labelled DNA were stored at -20°C until usePlasmid vectorsPropage  pPmMOE[MOE]:GFP-11 (Cold et al., 2016; Fernández Robledo et al., 2008) in JM109 for miniprepLinearize 20 μg with NotI (e.g., New England Biolabs® inc., Ipswich, MA, USA), clean the restriction digestion (e.g., GenElute® PCR Clean-up kit)Store at -20°C until useDNA probe construction and DNA labeling*Use forward (5’ -CCGCACATGTATGGTGAGCAAGGGCGAGGAGC- 3’) the reverse (5’ –CGTAGGACATGTCTTGTACAGCTCGTCCATGCCG- 3’ primers targeting pPmMOE[MOE]:GFP-11 (Fernández Robledo et al., 2008) to amplify a 739 bp DNAClean the amplicon as above and label 1 µg using UlysisTM Alexa Fluor® 488 Nucleic Acid Labeling Kit (ThermoFisher Scientific) following the manufacturer’s instructionsStore labelled DNA were stored at -20°C until useDNA probe construction and DNA labeling*Use forward (5’ -CCGCACATGTATGGTGAGCAAGGGCGAGGAGC- 3’) the reverse (5’ –CGTAGGACATGTCTTGTACAGCTCGTCCATGCCG- 3’ primers targeting pPmMOE[MOE]:GFP-11 (Fernández Robledo et al., 2008) to amplify a 739 bp DNAClean the amplicon as above and label 1 µg using UlysisTM Alexa Fluor® 488 Nucleic Acid Labeling Kit (ThermoFisher Scientific) following the manufacturer’s instructionsStore labelled DNA were stored at -20°C until use*Alternatively use any other set of primers/target DNA to generate the amplicon for labelingReferencesCold, E.R., Freyria, N.J., Martínez Martínez, J., Fernández Robledo, J.A., 2016. An agar-based method for plating marine protozoan parasites of the genus Perkinsus. PLoS One 11, e0155015.Fernández Robledo, J.A., Lin, Z., Vasta, G.R., 2008. Transfection of the protozoan parasite Perkinsus marinus. Mol Biochem Parasitol 157, 44-53.
?. [Cell Preparation]
Liquid cultureIncubate at 24°C in the dark with 125 rpm in a rotatory shaking incubator; the doubling time of C. cohnii cells under replete nitrogen conditions is 24-48 hoursHarvest cells at log phaseCentrifuge cells at 2,500 g for 10 min at room temperatureDiscard supernatant and maintain cells on ice until useSpheroplasts inductionCrypthecodinium cohnii spheroplasts are derived from both swimming cells and cyst cells (Kwok et al., 2007; Pozdnyakov et al., 2014). Prepare PEG8000 (20%) (wt/vol) in L1 mediumResuspend C. cohnii swimming cells with 1 ml of 20% L1-PEG8000 solution and vortex for 10 minSpread 1 ml of C. cohnii swimming cells or cysts on Petri dishes containing L1 medium bacteriological agar and L1-PEG8000 solution (0.4%)Incubate for 2 days at 28°CCover the colonies on the plate with L1 medium, pour off the mediumPour new L1 medium and retain the second elutant containing the spheroplastsCount spheroplasts using a haemocytometer (x3)Spheroplasts inductionCrypthecodinium cohnii spheroplasts are derived from both swimming cells and cyst cells (Kwok et al., 2007; Pozdnyakov et al., 2014). Prepare PEG8000 (20%) (wt/vol) in L1 mediumResuspend C. cohnii swimming cells with 1 ml of 20% L1-PEG8000 solution and vortex for 10 minSpread 1 ml of C. cohnii swimming cells or cysts on Petri dishes containing L1 medium bacteriological agar and L1-PEG8000 solution (0.4%)Incubate for 2 days at 28°CCover the colonies on the plate with L1 medium, pour off the mediumPour new L1 medium and retain the second elutant containing the spheroplastsCount spheroplasts using a haemocytometer (x3)ReferencesKwok, A.C., Mak, C.C., Wong, F.T., Wong, J.T., 2007. Novel method for preparing spheroplasts from cells with an internal cellulosic cell wall. Eukaryot Cell 6, 563-567.Pozdnyakov, I., Matantseva, O., Negulyaev, Y., Skarlato, S., 2014. Obtaining spheroplasts of armored dinoflagellates and first single-channel recordings of their ion channels using patch-clamping. Marine drugs 12, 4743-4755.
?. [Electroporation]
Collect Crypthecodinium cohnii cells in log phase or spheroplasts (1.0 x106 cells/mL) Electroporated with 5 μg (2.5 μg supercoiled, 2.5μg NotI linearized) of pPmMOE[MOE]GFP-11 using Amaxa®Cell Line Optimization Nucleofector™ solution V and program X-001 in a Nucleofector™  (Lonza)Recover cells from the cuvette and incubate in L1 medium As control for plasmid and electroporation, electroporate P. marinus PRA240 with pPmMOE[MOE]:GFP-11 as reported elsewhere (Fernández Robledo et al., 2008)Monitor cells for green fluorescence using standard FITC excitation/emission filters (488/507nm) under a transmitted-light fluorescence and/or confocal  microscope
?. [Lipofection]
Lipofectamine® 3000 Reagent kit (ThermoFisher Scientific) in tissue culture plate 24-wells and 6-wells plates containing respectively 2- 3 mL of  L1 mediumUse 15 µl of Lipofectamine and 1 µg of labeled DNA in 1.0 x 106 cellsMonitor cells for green fluorescence using standard FITC excitation/emission filters (488/507nm) under a transmitted-light fluorescence and/or confocal  microscope
?. Crypthecodinium cohnii viability after electroporation