Gene pulser 2 electroporator

To generate DT40 cells expressing GFP-CENP-A or GFP-CENP-A^QD, ggCENP-A_pEGFP-C2 or ggCENP-A^QD_pEGFP-C2 plasmids containing a Blasticidin resistance gene were transfected into CENP-A conditional knockout cells [CENP-A (–/Flox), Mer-Cre-Mer]^{22 (link)} with a Gene Pulser II electroporator (Bio-Rad). Cells transfected with the ggCENP-A_pEGFP-C2 plasmid were selected in medium containing 2 mg/ml G418 (Santa Cruz Biotechnology), and cells transfected with the ggCENP-A^QD_pEGFP-C2 plasmid were selected in medium containing 25 μg/ml Blasticidin S hydrochloride (Wako). After 10 days of selection, the drug resistant and GFP-positive clones were isolated. To knockout the endogenous CENP-A gene in the isolated clones, 100 nM 4-hydroxytamoxifen (OHT, Sigma) was added to the culture medium to activate the Mer-fused Cre-recombinase (Mer-Cre-Mer), and then the 5′ portion of the CENP-A gene [from exon 1 to 3 (CENP-A 1–87)] flanked by the LoxP sequences was excised from the genome. After the OHT treatment, we further isolated the monoclonal lines by limited dilution in 96-well plates, and verified the depletion of endogenous CENP-A in the isolated clones by a southern blot analysis. CENP-A disruption was also confirmed by an immunoblot analysis.

Infectious clones were individually transformed into Agrobacterium tumefaciens C58 by using a GenePulser II electroporator (Bio‐Rad). Agroinoculation was conducted according to the procedure described by Llave et al. (2000) with some modifications. Briefly, 0.5 ml of an overnight bacterial culture was recultured in 10 ml of Luria Bertani (LB) medium (pH 5.6) containing kanamycin (50 μg/ml), streptomycin (50 μg/ml), 2‐N‐morpholino‐ethanesulfonic acid (MES, 10 mM), and acetosyringone (AS, 0.04 mM) at 28 °C for 16 hr on a shaker set at 200 rpm. The bacterial cells were pelleted by centrifugation at 5,000 × g for 10 min and resuspended in 10 ml of an infiltration solution (10 mM MgCl₂, 0.15 mM AS, pH 5.6). Agrobacteria carrying the infectious DNA‐A or DNA‐B constructs were co‐injected in equal amounts into the leaves of N. benthamiana, oriental melon (C. melo “Silver Light”) or cucumber (C. sativus “Vantage”). After agroinoculation, N. benthamiana leaves with symptoms (c.1 g) were collected and ground in 0.01 M potassium phosphate buffer (pH 7) (1:20, wt/vol). The resultant sap was inoculated onto N. benthamiana leaves or cotyledon explants of oriental melon and cucumber plants by rubbing with carborundum powder. All inoculated plants were kept in a greenhouse at 25–28 °C to observe symptom development.

Transfections were performed as described previously (46 (link)). Briefly, 150 μg of plasmid DNA was precipitated and resuspended in Cytomix (25 mM HEPES [pH 7.6], 120 mM KCl, 0.15 mM CaCl₂, 2 mM EGTA, 5 mM MgCl₂, 10 mM K₂HPO₄). A ring-stage P. falciparum culture was washed with Cytomix and resuspended in the DNA/Cytomix solution. Cells were electroporated using a Bio-Rad Gene Pulser II electroporator at 950 μF and 0.31 kV. Electroporated cells were washed with media and returned to normal culture conditions. Parasites expressing the construct were selected by continuous treatment with 2 μg/ml blasticidin S HCl (Thermo Fisher Scientific). Transfectants were cloned by limiting dilution, and diagnostic PCRs were performed using genomic DNA from resultant transfectants using primer sets specific for episomal plasmids or genome integrants. Primer A and D sequences are as follows: (X) 5′-TAAGAACATATTTATTAAACTGCAG-3′; (Y) 5′-GAAAAACGAACATTAAGCTGCCATA-3′.

After trypsinization, 5 × 10⁶ cells of each HaloTag stain or fluorescent protein were mixed, pelleted at 250g, and resuspended in 180 μl medium before being transferred to an electroporation cuvette (Bio-Rad, Hercules, CA, USA). Cells were repelleted, then electroporated using 220 V/900 μF in a Gene Pulser II electroporator (Bio-Rad). The cuvette was left to incubate at 37°C for 30 min; the cells were then resuspended and plated. Plates were fixed at appropriate time points using 4% v/v formaldehyde.