Gene pulser 2

For the segregation assay, Jurkat cells were grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum (FCS). Transfections were performed via electroporation (210 V, 1000 μF) using a Bio-Rad Gene Pulser II with a capacitance extender (Bio-Rad Laboratories). U2OS cells were obtained from American Type Culture Collection (ATCC no: HTB-96), and grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FCS. U2OS cells were transfected with the indicated amounts of HPV-18 miniplasmid by electroporation (220 V, 975 μF) using a Bio-Rad Gene Pulser II with a capacitance extender (Bio-Rad Laboratories).

Parasites were cultivated using established methods with some changes (38 (link), 39 (link)). Cultures of Plasmodium falciparum clone Dd2 were grown in vitro at 37°C in solutions of 2% hematocrit (serotype A positive human erythrocyte) in RPMI 1640 medium (Invitrogen) containing l-glutamine and 25 mM HEPES and supplemented with 0.5% Albumax I and 0.1 g/liter hypoxanthine (ThermoFisher). Cultures were maintained in sterile, sealed flasks and flushed with a blood gas mixture (5% CO₂, 5% O₂, and 90% N₂). Transfections were done with the direct electroporation method on sorbitol-synchronized ring-stage parasites as described previously (31 (link), 32 (link)). Briefly, Dd2 clone was electroporated with 100 μg of each plasmid using Bio-Rad Gene Pulser II set at 0.31 kV and 960 μF. Selection pressure (2.5 nM WR99210 and 2 μg/μl blasticidin) and 200 μM IPP (Isoprenoids, LC and home supplies) were applied 48 h posttransfection. Media supplemented with IPP and selection pressure were renewed every day for 6 days. On day 8 posttransfection, selection pressure was removed and medium was supplemented with IPP and changed every other day. Parasite proliferation was monitored by Giemsa-stained thin-smeared blood samples taken at each medium change three times a week.

To construct the subgenomic replicon, JM-PnL (Figure 1), the puromycin N-acetyl-transferase (PAC), and the deep sea shrimp-derived nano-luciferase (nLuc) genes were individually amplified by PCR and the obtained DNA fragments were inserted into the previously reported JEV Muar strain-based replicon clone [23 (link)]. A similar strategy was applied to construct the DENV NGC-derived replicon, DN-PnL (Figure 1), bearing the PAC and nLuc genes. The obtained clones of plasmid DNA were linearized by Swa I digestion and then subjected to in vitro RNA transcription using a MEGAscript T7 Kit (Thermo Fisher Scientific). The prepared replicon RNAs were electroporated into BHK cells by using the GENE PULSER II (Bio Rad, Hercules, CA, USA) at 500 V and 25 µF [22 (link)]. To select the cells successfully harboring the replicon RNA, puromycin was added at 10 µg/mL from the next day and the surviving cells were designated as BHK/JM-PnL and BHK/DN-PnL, respectively.