title: A modified single-cell electroporation method for molecule delivery into a motile protist, Euglena gracilis.
abstract: 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.
authors: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan.
authors: Masashi
authors: M
authors: Ohmachi
authors: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan.
authors: Yoshie
authors: Y
authors: Fujiwara
authors: Research & Development Department, euglena Co., Ltd., Shiba, Minato-ku, Tokyo 108-0014, Japan.
authors: Shuki
authors: S
authors: Muramatsu
authors: Research & Development Department, euglena Co., Ltd., Shiba, Minato-ku, Tokyo 108-0014, Japan.
authors: Koji
authors: K
authors: Yamada
authors: Research & Development Department, euglena Co., Ltd., Shiba, Minato-ku, Tokyo 108-0014, Japan.
authors: Osamu
authors: O
authors: Iwata
authors: Research & Development Department, euglena Co., Ltd., Shiba, Minato-ku, Tokyo 108-0014, Japan.
authors: Kengo
authors: K
authors: Suzuki
authors: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan. Electronic address: dwang@icems.kyoto-u.ac.jp.
authors: Dan Ohtan
authors: DO
authors: Wang
mesh_terms: D001483:Base Sequence; D002470:Cell Survival; D018274:Electroporation / Q000379:methods*; D005056:Euglena gracilis / Q000166:cytology* / Q000235:genetics* / Q000502:physiology; D017403:In Situ Hybridization; D008954:Models, Biological; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics; D059010:Single-Cell Analysis / Q000379:methods*; D013997:Time Factors; D014162:Transfection / Q000379:methods*
chemical_list: D009841:Oligonucleotides; D015347:RNA Probes; D012333:RNA, Messenger
keywords: ECHO probe; Euglena gracilis; Motile cells; RNA imaging; Single-cell electroporation