TITLE: Electroporation transformation of FITC-dextran into Oxyrrhis marina - an early branching dinoflagellate.
AUTHORS: Nick Irwin,  Elisabeth Hehenberger, Liz Cooney, Yoshihisa Hirakawa,  Patrick Keeling

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">INTRODUCTION</span></div><div class = "text-block">Here we describe a protocol for transforming Oxyrrhis marina with FITC-labelled dextran (a DNA analog) by electroporation. </div><div class = "text-block">This protocol is reproducible, and results in a transformation efficiency of around 1%.</div><div class = "text-block"><span>We have confirmed the viability of transformed cells by allowing them to recover in the dark (and thus maintain fluorescence intensity) and then by observing fluorescent cells swimming normally, days after transformation (see the video </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">here</span></a><span>). </span></div><div class = "text-block"><span style = "font-weight:bold;">NOTE ON AUTOFLUORESCENCE</span></div><div class = "text-block"><span>Although </span><span style = "font-style:italic;">Oxyrrhis</span><span> is a non-photosynthetic dinoflagellate and therefore lacks plastid autofluorescence, we have observed a small amount of green autofluorescence that is typically isolated in the anterior of the cell (See top panel of Figure 1).</span></div><div class = "text-block"><span>The autofluorescence was observed in wildtype cells, cells electroporated without FITC-dextran, and cells that were incubated with FITC-dextran but not electroporated. The autofluorescence was observed in cultures with both </span><span style = "font-style:italic;">Dunaliella tertiolecta </span><span>or heat-killed </span><span style = "font-style:italic;">E. coli</span><span>. The brightness and frequency of the autofluorescence also seemed to be dependent on culture conditions. Not all cells had the autofluorescence.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bh3yeh3w.png" /></div><div class = "text-block"><span style = "font-weight:bold;">ADDITIONAL INFORMATION</span></div><div class = "text-block">As of yet we have been unable to demonstrate the actual uptake of plasmid DNA. We have tried using rhodamine labelled plasmids to demonstrate this but the results were inconclusive due to issues with cellular autofluorescence. </div><div class = "text-block"><span>We have also tried this method with a variety of expression plasmids encoding different promoters. For example, we tried the MOE promoter from </span><span style = "font-style:italic;">Perkinsus</span><span style = "font-style:italic;font-style:italic;"> </span><span style = "font-style:italic;">marinus </span><span>(a closely related species) and the generic eukaryotic CMV promoter. However no GFP fluorescence was detected in these samples. </span></div><div class = "text-block"><span>We have also tried transforming </span><span style = "font-style:italic;">in vitro</span><span> synthesized GFP encoding mRNA using this protocol. As of yet, no GFP production has been detected, but this may reflect non-cannonical translational systems in dinoflagellates as the same mRNA could be translated in starfish embryos.</span></div><div class = "text-block"><span>Although the development of expression systems in dinoflagellates remains a challenge, our results suggest that </span><span style = "font-style:italic;">O. marina</span><span> may be amenable to genetic transformation.</span></div></div>

[STEPS]
?. [Culturing and cell collection]
Cultures were fed heat-killed E. coli and grown on a 16h/8h light/dark cycle at 20 degrees, cultures were grown in f/2 media.The O. marina strain we are using is CCMP604.Cultures were grown to a minimum density of 20,000 cells/mL
?. [Resuspend cells in electroporation buffer]
Resuspend the O. marina pellet in 100 uL Biorad Gene Pulser Electroporation buffer. Prior to resuspension we add FITC-dextran (Sigma FD70S) to 2 mg/mL in the electroporation buffer.
?. [Electroporate the cells]
Transfer the resuspended cells to a 0.2 cm cuvette.Electroporate using a Biorad Gene Pulser Xcell using the following conditions:

Square wave pulse - 100 V, 3 x 5 ms pulses, 0.1 ms pause.or for lower voltage conditions:

Square wave pulse - 75 V, 5 x 5 ms pulses, 0.1 ms pause.We tried using the exponential protocol with short high voltage pulses (1000 V, 10 uF) and long low voltage pulses (100 V, 1000 uF) but in all instances the exponential protocol resulted in cell lysis.We also tried raising the voltage with the square wave protocol (200V, 3 x 5 ms, 0.1 ms pause) but this also resulted in lysis.After electroporation, add 1 mL of f/2 media to the cuvette and transfer the cells to a microcentrifuge tube.
?. [Wash the cells and allow them to recover]
After electroporation, spin down the cells for 3 minutes at 7000 xg and wash once with 1mL f/2 media.We allow the cells to recover in 24-well plates for 24 hours before observing.If you want to avoid photobleaching (ie. for FITC-dextran), you can allow the cells to recover in the dark. A video demonstrating the results can be seen here: https://www.youtube.com/watch?v=8eHpX-GGwW0 (sorry for the low quality, the cells also seemed to be slightly squished beneath the slide)
?. [Culturing and cell collection]
Perform cell counts and spin down 1,000,000 cells per sample in a 50 mL falcon tube at 3000xg for 10 minutes.
?. [Background information]
BEFORE STARTING: Read the abstract for more information and expected outcomes (ex. efficiency, autofluorescence, etc...).