Nucleofection of Parasite Forms: An Electroporation-Based Transfection Method to Deliver Fluorescent Protein Encoding-Plasmids Into Sporozoite Nucleus

Protokol

1. Nucleofection of merozoites or sporozoites

1. Preparation before nucleofection of parasites
   1. Prepare about 10⁷ merozoites or sporozoites in one tube. If transfecting merozoites, prepare 3-4 tubes.
   2. 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).
   3. 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.
   4. 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.
2. Nucleofection
   1. Centrifuge the sporozoite or merozoite suspension at 600 x g for 10 min. Then discard the supernatant.
   2. 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.
   3. Transfer the suspension to a nuclear transfection cup. Put the cup into a nuclear transfer groove.
   4. 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.
   5. When the program finishes, press the X button of the nucleofection device, and the screen should display OK, indicating that the nucleofection is successful.
   6. 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
*water: distilled water

Table 1: Composition of Buffers.

Malzemeler

Name	Company	Catalog Number	Comments
DMEM	MACGENE	CM15019
Nucleofection device	LONZA/amaxa	90900012 (Nucleofector II)
Glucose	Sigma	No. V900116
ATP-disodium	Sigma	A26209
Cellulose DE-52	Solarbio	C8350
KH2PO4	Sigma	No. V900041
Low Speed Centrifuge	BEIJING ERA BEILI CENTRIFUGE CO., LTD.	DT5-2
MgCl2	Sigma	449164
NaHCO3	Sigma	144-55-8
PBS	Solarbio	P1010
Sorvall Legend Micro 17 Microcentrifuge	ThermoFisher Scientific	75002430
Trypsin	Solarbio	T8150
Percoll (DG gradient stock solution)	GE Healthcare	17-0891-09