Nucleofection of Parasite Forms: An Electroporation-Based Transfection Method to Deliver Fluorescent Protein Encoding-Plasmids Into Sporozoite Nucleus
Published: April 30, 2023
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
- 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 |
| *water: distilled water |
Table 1: Composition of Buffers.
Declarações
The authors have nothing to disclose.
Materials
| 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 |
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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).