Gene pulser electroporation system

Jurkat-control cells and PBMCs were transiently transfected with an Easyjet Plus Electroporator (Equibio). In brief, cells were collected in 350 μl of RPMI without supplement and mixed with 1 μg of plasmid DNA per million of cells. Cells were transfected in a cuvette with 4 mm electrode gap, at 280 V or 320 V respectively for Jurkat or PBMCs, 1500 μF and maximum resistance by using a Gene Pulser electroporation system (Bio-Rad). Transfections with luciferase expression vector were incubated for 18 hours and luciferase activity was measured by using Luciferase Assay System (Promega Biotech) in a Sirius luminometer (Berthold Detection Systems), according to manufacturer’s instructions. pEGFP vector was co-transfected as control of transfection efficiency and measured by flow cytometry on a FACScalibur Flow Cytometer (BD Biosciences) using CellQuest software or by immunofluorescence using Leica DMI 4000B Inverted Microscope. The Relative Light Units (RLUs) were normalized with protein concentration in each sample and with the percentage of efficiently transfected cells. The percentage of Jurkat cells expressing Tat101 or Tat72 within the whole population was calculated by transient transfection of pEGFP under the control of the HIV-1 LTR promoter (pLTR-EGFP) as previously shown.[8 (link)] Transfection of HIV-1 expressing vectors are described below.

For transfection of Blastocystis, cells were cultured to log-phase and harvested by centrifugation at 1,000 g for 10 min at room temperature. Cells were then washed once with pre-reduced incomplete cytomix buffer (10 mM K₂HPO₄/KH₂PO₄, pH 7.6, 120 mM KCl, 0.15 mM CaCl₂, 25 mM HEPES, 2 mM EGTA, and 5 mM MgCl₂), and then resuspended in pre-reduced complete cytomix (incomplete cytomix supplemented with 2 mM ATP and 5 mM glutathione). These cells were ready for transfection.
Blastocystis cells were mixed with an appropriate amount of plasmid DNA in a 0.4-cm transfection cuvette (Bio-Rad) and subjected to 1 pulse using the Bio-Rad Gene Pulser electroporation system under the protocol and programs shown in Fig. 1B. After electroporation, cells were transferred into fresh pre-reduced culture medium and maintained at 37 °C anaerobically for 12–16 hrs.
For stable transfection in Trypanosoma brucei, the constructs were linearized with MluI and transfected into YTat1.1 cells using Bio-Rad Gene Pulser. The stable transformants were selected with 10 μg/ml blasticidin (Invitrogen, R210-01).

Desktop microcentrifuge, shaking incubator at 37°C, 1.5 ml collection tubes, filtered sterile pipette tips, thermoblocks at 90°C and 50°C (e.g., Thermo Fisher, 88870004), an ultracentrifuge capable of spinning 50 ml falcon tubes at 10,000 rpm (Beckman Coulter Avanti J-30 I ultracentifuge and a Beckman JA-12 fixed angle rotor), falcon tubes (polypropylene, 50 ml (Corning 352070)), a Bio-Rad Gene Pulser electroporation system (BioRad 164–2076), electroporation cuvettes Plus (2 mm, Model no. 620 (BTX)), a gel electrophoresis chamber, and erlenmeyer flasks (glass, 100 ml).

K562 cells were permanently transfected with pLTR2-C3G [25 (link)], pSuper-C3Gi [26 (link)] and CRISPR-C3G (Santa Cruz Biotechnology, Inc.) and the corresponding empty vectors (pLTR2-CT, pSuper-CT and CRISPR-CT), for up-regulation, down-regulation or abrogation of C3G expression, respectively, using the Gene Pulser Electroporation System (Bio-Rad). Additionally, the pLTR2 clones were co-transfected with pSuper.gfp/neo vector to express GFP fluorescence (pLTR2-CT/GFP and pLTR2-C3G/GFP clones). These clones were maintained with complete medium supplemented with 1:20 Killer Hat solution [25 (link)], 250 μg/ml neomycin or 2 μg/ml puromycin, as appropriate.

2 × 10⁶ JM8A1.N3 ES cells were cultured on inactivated feeder cells plated on the day before. After 5 days of culture cells were trypsinized and electroporated with 50 μg of the DNA construct using a gene pulser electroporation system (BioRad, Hercules, CA, USA). After another 24 h of cell culture ES cells selection started by addition of gentamycin (G-418, Sigma-Aldrich, Seelze, Germany). After 7 days of culture under selection conditions, appr. 350 clones were picked and further cultured in 48 well plates for two more days under selection. Then cells were trypsinized and 2/3 were frozen for later blastocyst injection while 1/3 was cultured on gelatinized 24-well plates for another 6–10 days for southern blot analysis.

Regarding the presence of the HPV-16 genome within the CaSki cells and the expression of E5 oncogene in these cells, this type of cervical cancer cell line was chosen for the following tests. E5-siRNA (Bioneer, South Korea) (Table 1) and FITC-labeled siRNA (scrambled siRNA) (GenePharma Co., Shanghai, China) were transfected within the CaSki cells with the serial doses using the Gene Pulser electroporation system (Bio-Rad). The scrambled siRNA was checked by nucleotide BLAST to find if there is no target sequence for this siRNA in the human transcriptome. Also, utilizing Nucleotide BLAST, the specificity of the designated siRNA was evaluated. The results showed that this siRNA could effectively target all the strains of HPV-16 and all transcript variants of E5. Following the application of the electroporation protocol (TC = 12.5 ms and Voltage = 160 v), the proper number of cells were seeded in different cell culture plates regarding the ongoing test. Also, the scrambled siRNA was transfected to the CaSki cells to assess the efficacy of transfection.

Regarding the higher expression of B7-H7 in MKN-45 cells in comparison with KATO III and AGS cells, MKN-45 cells were selected. Using the Gene Pulser electroporation system (Bio-Rad), the MKN-45 cells were transfected with B7-H7 siRNA (20-80 pmol) (Table 1). After using the electroporation protocol (TC = 12.5 ms and voltage = 130 V), the appropriate amount of cells was seeded in 6, 24, and 96 well plates according to the ongoing test.