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 derive…
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 |
Tags
Citar este artigo
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).