In this video, we describe nucleofection, an electroporation-based transfection procedure to deliver fluorescent reporter protein-encoding plasmid DNA into sporozoite form of the chicken Eimeria parasite.
Protocol
1. Nucleofection of merozoites or sporozoites
Preparation before nucleofection of parasites
Prepare about 107 merozoites or sporozoites in one tube. If transfecting merozoites, prepare 3-4 tubes.
Prepare an amount of plasmid DNA or purified PCR fragment that is greater than or equal to 10 µg. NOTE: The plasmid used in this study contains 2 genes: enhanced yellow fluorescent protein (EYFP) and dihydrofolate reductase thymidylate synthase derived from Toxoplasma gondii (TgDHFR-TS).
Prepare 25 U of restriction enzyme. If plasmids are linearized, the restriction enzyme can improve the transfection efficiency. If the plasmids are circular, omit the restriction enzyme.
Prepare 85 µL of nucleofection buffer (Table 1): mix 20 µL of nucleofection buffer I and 1 mL of nucleofection buffer II and use a part of the solution. The volume of the total buffer is 100 µL.
Nucleofection
Centrifuge the sporozoite or merozoite suspension at 600 x g for 10 min. Then discard the supernatant.
In the following order, add 85 µL of nuclear transfection buffer, 10 µg of plasmid (PCR fragment), and 25 U of restriction enzyme (usually 5 µL) into the 1.5 mL tube containing sporozoites or merozoites.
Transfer the suspension to a nuclear transfection cup. Put the cup into a nuclear transfer groove.
Turn on the nucleofection device by using the power button and select the transfection procedure U-033. If the nucleofection device starts in the Free Program Choice mode, exit this mode by pressing the X button.
When the program finishes, press the X button of the nucleofection device, and the screen should display OK, indicating that the nucleofection is successful.
Add 0.5-1 mL of Dulbecco's Modified Eagle's Medium (DMEM) to the nucleofection cup to stop the reaction and transfer the suspension to 1.5 mL tube after mixing gently.
Buffer
Composition
Nucleofection buffer I
ATP-disodium 2g, MgCl2-6H2O 1.2g, 10 ml water
Nucleofection buffer II
KH2PO4 6 g, NaHCO3 0.6 g, glucose 0.2 g, 500 ml water
Nucleofection of Parasite Forms: An Electroporation-Based Transfection Method to Deliver Fluorescent Protein Encoding-Plasmids Into Sporozoite Nucleus. J. Vis. Exp. (Pending Publication), e20993, doi: (2023).