Microinjection System for Single-Microalgal Cells Based on Electroosmotic Flow
Authors: Akasaka Kaoruko , Yasui Rein , Yoshino Tomoko , Nojima Daisuke , Mochiduki Makoto , Tanaka Tsuyoshi 
Published: December 2024

Introduction

Microalgae are attracting attention as promising candidates for CO2 fixation and biomaterial production, due to no competition with food and feed, and high growth rates and photosynthetic ability. To expand the use of microalgae as a host for biomaterial production, it is crucial to develop efficient and versatile gene introduction technologies for microalgae, enabling diverse metabolic modifications. Several methods, such as microparticle bombardment, electroporation, and bacterial conjugation, have been developed to deliver foreign DNA to microalgae. However, these methods resulted in low transformation efficiency and limited applicable microalgae species. To overcome these problems, we have developed a novel microinjection system for single-microalgal cells based on electroosmotic flow using nano-sized pipette (nanopipette). The nanopipette enables automatic and precise control of solution delivery into cells at the femtoliter level. In this study, we investigated optimum conditions for substance introduction using the electrochemically controlled nanopipette into a green alga, Haematococcus sp..

Materials & Methods

A pipette with tip outer diameter of several tens of nanometers, was used for the injection. Fluorescein isothiocyanate (FITC)-labeled dextran (MW: 70,000) was used as an injection material. Initially, FITC-dextran dissolved in PBS was filled inside the nanopipette and an electrode was set. The cell sample was also filled with PBS and a reference electrode was placed inside. Voltage was then applied to the electrode during injection and the ion current generated was monitored. The nanopipette automatically detects the cell surface by identifying changes in ionic current. Once the cell surface is detected, the nanopipette penetrates the cell to a predetermined depth and injects the reagent using electroosmotic flow caused by the application of voltage. A green microalga, Haematococcus sp. strain NKG7001, which is an astaxanthin-producer, was used to optimize the conditions for microinjection using the nanopipette. Four types of algal cells, i.e. flagellated, palmelloid, intermediates or cyst cells based on their cell cycle were investigated. Algal cells were applied and fixed on the surface of agarose gels. Subsequently, these cells on the agarose gels were immersed in PBS, and then, dextran was injected into each cell using the nanopipette under different applied voltages and injection time. The injection efficiency was calculated as the number of successfully injected cells divided by the number of trials. Furthermore, the proposed method was applied to other green algae, a polysaccharide producer, Tetraselmis sp. strain NKG400013, and a green alga, Clamydomonas reinhardtii.

Results & Discussion

The injection of dextran into Haematococcus cells was succeeded in flagellated and palmelloid cells, but not intermediate and cyst cells. In general, cyst cells possess thick cell wall, so the thick cell wall might prevent the nanopipette from penetrating the cells. Therefore, flagellated and palmelloid cells were used in the following experiments. Subsequently, the optimum injection voltage and injection time for Haematococcus cells were determined. The injection efficiency of this method reached 44%. Based on the above conditions, the microinjection using nanopipette was successfully carried out in other green microalgae, Tetraselmis sp. and C. reinhardtii. These results suggest that microinjection using the nanopipette can be widely utilized in green microalgae. The proposed method will be applied to efficient genome editing and subsequent metabolic engineering of various microalgae.