Nepa21 electroporator

Electroporation using an Amaxa Nucleofector II (Lonza, Basel, Switzerland) was performed as described in the previous study^{21 (link)}. The other machine NEPA21 electroporator (Nepa Gene, Chiba, Japan) can control two types of pulses: poring and transfer. We determined the pulse conditions for P. marinus as follows; Voltage (V): 175, msec: 1, Pulse number: 5, Interval (msec): 50, Decay (%): 10, Polarity: + /− for poring pulse, and Voltage (V): 20, msec: 50, Pulse number: 10, Interval (msec): 50, Decay (%): 40, Polarity: + /− for transfer pulse. For the experiments in Table 2, we did not change the transfer pulse condition. 5 or 10 µg plasmid was ethanol-precipitated, and the pellet was resuspended in 100 µl of the Ingenio electroporation solution (Mirus Bio, Madison, WI). 5 × 10⁶ cells were collected by centrifugation at 1000 × g for 3 min at room temperature. The cell pellet was resuspended in the plasmid-containing Ingenio solution and transferred all into a 2-mm cuvette (Nepa Gene), and electroporated by NEPA21 electroporator using the pulses described above. The treated cells were transferred into a well in a 12-well plate with 2 ml of fresh ATCC medium 1886. Then, it was divided into 1 ml × 2 wells and incubated at 26 °C. The percentages of the GFP-positive cells were calculated by counting at least 3000 cells by 3 individuals 1 week after the electroporation.

The BFF cells derived from cloned fetuses from the first round of SCNT carried the AcGFP cassette for use in positive selection; to remove this cassette, piggyBac transposase mRNA was introduced. BFF cells that had undergone less than three passages were trypsinized and suspended (1 × 10⁶ cells) in 100 µl of Opti-MEM (Thermo Fisher Scientific) with 5 µg of Excision-only piggyBac transposase mRNA (System Biosciences). Cells were placed in a 10-mm gap cuvette and subjected to electroporation using a NEPA21 electroporator (NEPA GENE). The electroporation conditions were as follows: 1) poring pulse: input voltage, 150 V; pulse width/interval, 5/50 ms; pulse number, 2; 2) transfer pulse: input voltage, 20 V; pulse width/interval, 50/50 ms; pulse number, 5. After electroporation, cells were diluted immediately with culture medium and plated onto 60-mm dishes. Five days after electroporation, cells with low AcGFP expression were enriched using a MoFlo Astrios cell sorter and, after several passages, cells that were negative for AcGFP were used for SCNT to generate a second round of clones.