A modified single-cell electroporation method for molecule delivery into a motile protist, Euglena gracilis
| Autor: | Masashi Ohmachi, Yoshie Fujiwara, Koji Yamada, Shuki Muramatsu, Kengo Suzuki, Dan Ohtan Wang, Osamu Iwata |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Microbiology (medical) Time Factors Euglena gracilis Cell Survival ved/biology.organism_classification_rank.species Cell Oligonucleotides Biology Transfection Models Biological Microbiology Green fluorescent protein 03 medical and health sciences medicine RNA Messenger Molecular Biology In Situ Hybridization Alexa Fluor Base Sequence ved/biology Oligonucleotide Electroporation Nucleic Acid Hybridization RNA Probes Fluorescence Cell biology 030104 developmental biology medicine.anatomical_structure Single-Cell Analysis |
| Zdroj: | Journal of Microbiological Methods. 130:106-111 |
| ISSN: | 0167-7012 |
| Popis: | Single-cell transfection is a powerful technique for delivering chemicals, drugs, or probes into arbitrary, specific single cells. This technique is especially important when the analysis of molecular function and cellular behavior in individual microscopic organisms such as protists requires the precise identification of the target cell, as fluorescence labeling of bulk populations makes tracking of individual motile protists virtually impossible. Herein, we have modified current single-cell electroporation techniques for delivering fluorescent markers into single Euglena gracilis, a motile photosynthetic microalga. Single-cell electroporation introduced molecules into individual living E. gracilis cells after a negative pressure was applied through a syringe connected to the micropipette to the target cell. The new method achieves high transfection efficiency and viability after electroporation. With the new technique, we successfully introduced a variety of molecules such as GFP, Alexa Fluor 488, and exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) RNA probes into individual motile E. gracilis cells. We demonstrate imaging of endogenous mRNA in living E. gracilis without interfering with their physiological functions, such as swimming or division, over an extended period of time. Thus the modified single-cell electroporation technique is suitable for delivering versatile functional molecules into individual motile protists. |
| Databáze: | OpenAIRE |
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