METHOD OF TRANSFERRING GENE INTO ALGAL CELL INVOLVING UTILIZING MULTIPLE SQUARE-WAVE PULSES IN THREE STEPS

摘要

To provide a technology that solves the above-mentioned problems, and is directly applicable to eukaryotic algal cells with cell-wall, the technology enabling gene transfer and transformation to be performed with high efficiency and good reproducibility irrespective of species of algae. Also provided is a method of transferring an exogenous gene into a eukaryotic algal cell, the method comprising performing electroporation using multiple square-wave pulses to the solution containing a cell of a green alga with cell-wall, and a nucleic acid molecule by the following three steps: applying a square-wave electric pulse (first electric pulse) with a high voltage for a short period of time under the condition that its total electric energy falls within a predetermined range; then applying a square-wave electric pulse (second electric pulse) with a low voltage for a long period of time two or more times; and then applying a square-wave electric pulse (third electric pulse) that is opposite in polarity to the second electric pulse, with a low voltage for a long period of time, two or more times.

主权项

1. A method of transferring an exogenous gene into a eukaryotic algal cell, the method comprising performing electroporation using multiple square-wave pulses by the following three steps:
applying a square-wave electric pulse that satisfies a condition as defined in the following item (A) to a solution one time, two times or more so that the square-wave electric pulse has a total electric energy of 1.3 to 4.9 J/100 μL, the solution comprising a cell of a green alga with cell-wall, and a nucleic acid molecule; applying a square-wave electric pulse that satisfies conditions as defined in the following items (B1) and (B2) to the solution at least two times; and applying a square-wave electric pulse that satisfies conditions as defined in the following items (C1) to (C3) to the solution at least two times: (A) a square-wave electric pulse having a voltage per pulse of 750 V/cm or more; (B1) a square-wave electric pulse having a voltage per pulse of 150 V/cm or less; (B2) a square-wave electric pulse having an electric energy per pulse of 0.02 to 0.6 J/100 μL; (C1) a square-wave electric pulse opposite in polarity to the electric pulse that satisfies the conditions as defined in the items (B1) and (B2); (C2) a square-wave electric pulse having a voltage per pulse of 150 V/cm or less; and (C3) a square-wave electric pulse having an electric energy per pulse of 0.02 to 0.6 J/100 μL.