Development of a Novel Electroporation Method for the Oyster Parasite Perkinsus marinus – Scientific Reports

Identification of a new buffer operating in the traditional state

So far only the D-023 program of the Amaxa Nucleofector is used to transfect Perkinsus marinus20. There are no reported successes with other devices. We first screened buffers (Table 1) compatible with the D-023 program in the 50×10 Amaxa Nucleofector6 Cells (CB5D4 strain, a strain used in the first transfection report20) to develop a new electroporation method with 10 µg pMGB plasmid23. Transfection efficiency was scored on a 5-point scale using GFP-positive cells with the original buffer, solution V in a transfection kit (Lonza) being 100%. Cytomix24 and 3R buffer16 without CaCl2 did not work while 3R buffer with CaCl2 worked as previously reported16.25 (Table 1). Although the transfection efficiency was low, we found that the Ingenio electroporation solution also worked in the D-023 program (Table 1).

Table 1 Comparison of electroporation efficiency using the D-023 program in Amaxa Nucleofector II among electroporation buffers.

Assessment of the tensions for a possible gene transfer

To estimate the strain conditions in the D-023 program, we used another electroporator, NEPA21, to refine the strain conditions. This NEPA21 generates rectangular electric pulses and can freely control the voltage as well as the frequency and intervals of the pulses and has been successfully transfected into the green alga Chlamydomonas reinhardtii and diatom Phaeodactylum tricornutum26.27. We were able to strain the CB5D4 (50 × 106 cells) with 10 µg of the pMGB plasmid using conventional solution V at 100-200 V, albeit with low efficiency using the NEPA21 (Table 2). This is the first report to break the limitations of electroporation equipment and provides important research motivation to investigate conditions using devices other than the Amaxa Nucleofector.

Table 2 Comparison of electroporation efficiency using Lonza buffer and NEPA21 electroporator.

Next, cell amount and voltage were screened to establish the new electroporation method using the Ingenio buffer (Table 3). In this buffer, an overcurrent error (E01 error) often occurred when the amount of plasmid was 10 µg, so we tested at 5 µg. We found the following two things; (1) 175V × 5 pulses is much better than 175V × 2 pulses at 50 × 106 Cells, (2) 200v × 2 pulse is much better than 200v × 1 pulse at 10 × 106 cells. The results are the first case of highly efficient transfection using a different device and buffer than Lonza products (Table 2 and Fig. 2a).

Table 3 Comparison of electroporation efficiency using the Ingenio buffer and the NEPA21 electroporator.
figure 2
figure 2

Differences in cell damage between cell strains. 10×106 Cells were electroporated with 5 µg of plasmid by 175 V x 5 pulses. Pictures were taken three days after transfection. Merged images of GFP and brightfield are shown. The observed white and spherical objects indicate living cells. Other objects observed as small, black shadows indicate cellular debris. A lot of cell debris can be observed in the CB5D4 strain (Has), while very few were observed in the CRTW-3HE strain (b). The scale bars; 50 µm.

However, the overcurrent error occurred at 20-30% at 50×106 cells. So we reduced the number of cells to 10 × 106 cells; there were no errors and high transfection efficiency with 200 V × 2 pulses (Table 3). However, at 10 × 10, much cell debris was observed6 cells (Fig. 2a).

Strain CRTW-3HE is better suited for electroporation than strain CB5D4

After electroporation at 10 × 106 cells in Ingenio buffer with the NEPA21 electroporator, we observed a lot of cell debris from CB5D4 cells (Fig. 2a), suggesting that the pulse killed many cells. However, the same conditions did not lead to the same observation when applied P.marinus Strain, CRTW-3HE (ATCC50439), a strain used in the development of the drug selection system21.22, with similar transfection efficiency (Fig. 2b). The finding indicates that the CRTW-3HE strain is better suited for transfection experiments than the CB5D4 strain. Therefore, in the following experiments we use the strain CRTW-3HE.

Determination of the fine pulse state

We have optimized the pulse conditions of the NEPA21 electroporator with the CRTW-3HE strain. Because increasing the cell count increases the error rate, we validated at 10 × 106 cells and 5 × 106 cells. The amount of plasmid was 5 µg based on the previous study20, and 10 µg were also tested to see if transfection efficiency is further increased. Pulse conditions were tested under the two conditions identified in Table 3, 175V x 2 pulses and 200V x 2 pulses.

We found that the 175 V x 5 pulses with 5 µg plasmid allow transfection with high efficiency, approximately 2% (Table 4 and Fig. 3a and b); this value is sufficient for the next experiment, drug selection. In the previous report25, transfection efficiency was increased by decreasing cell number and increasing plasmid amount; the plasmid/cell ratio was 10-fold higher than in the other report20 (up to 1.0 pg/cell from 0.1 pg/cell). Therefore, we tested electroporation with a plasmid/cell ratio of 1.0-2.0 pg/cell. However, there was no significant improvement in efficiency. Finally, we determined the optimal pulse state to be 10 × 106 cells, 5 µg plasmid and 175 V x 5 times. We used this condition in the following experiments.

Table 4 Pulse state refinement using the Ingenio buffer and the NEPA21 electroporator.
figure 3
picture 3

Comparison of the GFP signals under the specified pulse conditions and the amount of plasmid in the CRTW-3HE strain. (Has) Percentage of GFP-positive cells after electroporation in each condition. Dots indicate individual data, bars indicate mean values ​​of the data, and error bars indicate standard deviation. (b) GFP images (left side) and merged images (right side) are shown for each condition. The scale bars; 50 µm. E01 error on Has and b; overcurrents.

New expression plasmids for P.marinus

The expression plasmids for P.marinus were constructed based on the pCR4-TOPO vector20. The plasmid has additional sequences that are not involved in gene expression in the parasite cell. Since the molecular mechanism involved in establishing stable expression cells in is unknown P.marinusit is desirable to remove as many sequences as possible that are irrelevant for expression in order to avoid unforeseen events and to establish reproducibly stable cells. Therefore we constructed a new expression plasmid with a simpler sequence based on the pSP72 vector. Exclusion of the kanamycin resistance gene and the LacZa gene in pCR4-TOPO resulted in a plasmid length that was approximately 1.6 kb shorter. The plasmid contains the origin of replication (ori), the ampicillin resistance cassette, the moe Gene promoter and the 5′ and 3′ UTR des moe gene and the puromycin resistance gene (puromycin N-acetyl transferase: pac). We named the plasmid pMGvsp72 (pMOE-GFP-C terminal Pac in pSP72) (Figure 4d). Electroporation of the plasmid using NEPA21 leads to the expression of GFP (Fig. 4b). This showed that the simplified plasmid with different backbone does not affect gene expression P.marinus.

figure 4
figure 4

Requirement of 5′ and 3′ flanking regions of moe Gene for GFP expression in CRTW-3HE strain. (Has) Schematic representations between 5′ and 3′ flanking regions (FR) of the tested plasmids (left side). The length of the FRs and the results of GFP expression are shown on the right. (b and vs) Representative images of GFP expression following promoter activity of 250 bp 5′-FR with and without 3′-FR are shown in () and (e), respectively. The scale bars; 50 µm. Schematic representations of pMGvsP72 () and pM250GcP72-3FR (e) are shown. The images are provided by the SnapGene software.

To further exclude unnecessary sequences, the need for 5′ and 3′ flanking regions was tested. For the 5′ flanking region, even the shortest 250 bp region had promoter activity (Figure 4a, b and c). Furthermore, for the 3′ flanking region, the exclusion of this region did not affect gene expression (Figures 4a and c). Taken together, most of the flanking region sequences of the conventional plasmid are unnecessary for gene expression. Therefore, we designed a new plasmid with minimal flanking regions (FR). We named the plasmid pM250Gvsp72-3FR (pMOE 250-GFP-C terminal Pac without 3′ FR in pSP72) (Figure 4e).

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