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

Abstract

Source: Duan, C., et al. Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites. J. Vis. Exp. (2020)

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

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 en....

Materials

List of materials used in this article

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