Three minutes at 93 °C were programmed as the initial step, followed by 35 cycles of 1 min at 93 °C, 1 min at 52 °C and 5 min at 74 °C. A final polymerization step of 5 min at 74 °C was added. The amplified gene was inserted in the plasmid pLW previously digested with the enzyme EcoR V. The recombinant plasmid was named pLW-hah5. In vitro culture of HEK-293 cell line (ATCC CRL-1573) was carried out in flasks of BLZ945 concentration 25 cm2 (Greiner Bio-One, Germany) using the culture medium Dulbecco’s

modified Eagle’s medium (DMEM) (Sigma, USA) supplemented with 10% of fetal calf serum (FCS) (PAA, Canada), 0,3 mg/mL of l-glutamine (Sigma, USA), 1 mM of sodium pyruvate (Sigma, USA) and an antibiotic–antimycotic solution 100× (GibcoBRL, USA) at a final concentration of 1×. Cells were incubated at 37 °C, 5% of CO2 and 95% of relative humidity. One hour before the transfection, the medium of the cell culture at 80% of confluence was changed for fresh medium without FCS. Transfection was performed using the polycation polyethylenimine 25 000 (PEI) (Sigma, USA) at 0,81 mg/mL, pH 7 and the plasmids pAEC-hah5 and pEGFP. The last plasmid was generated by including a transcription unit with the enhanced green fluorescent protein (EGFP) gene under the control of the CMV enhanced promoter into selleck compound the

pMOS-Blue backbone. DNA was used at 0,72 μg/cm2. The ratio pAEC-hah5/pEGFP was 36:1 and the ratio PEI/DNA was 1 μL/1 μg. DNA and PEI were diluted in separate tubes using 5% of glucose until reaching 50 μL each. After samples were vigorously mixed during 10 s and allowed to stand for 5 min, PEI was added to DNA, which were vigorously mixed during 1 min and allowed to stand for 20 min. Subsequently, 900 μL of fresh DMEM was added to the PEI/DNA complex and the mixture of 1 mL was carefully added to the cell culture. Six hours later, FCS was added at a final concentration of 10%. Negative control was performed as above but with the plasmid pAEC-Spt. Transfection was verified after 72 h by observing the production

of the EGFP Parvulin protein in transfected cells at the fluorescence microscope using a magnification of 400×. Transfection of the HEK-293FT cell line (Invitrogen, USA) with the plasmids pLP1, pLP2, pLP/VSVG (Invitrogen, USA), pEGFP and pLW-hah5 was carried out in 6 flasks of 175 cm2 (Greiner Bio-One, Germany) using PEI as explained above. In each flask, DNA was used at 0,411 μg/cm2. The ratio DNA/pEGFP was 36:1 and the ratio pLW-hah5/each helping plasmid (pLP1, pLP2 and pLP/VSVG) was 2:1. Negative control was performed in the same way but with the plasmid pLW. Six hours after adding the mixture of PEI/DNA to the cell culture, FCS was added until reaching 10%. After 48 h, the supernatant was centrifuged at 1000 × g, filtered using a pore size of 0,45 μm and ultracentrifuged at 25 000 × g for 1:30 h. The supernatant was removed and lentiviral particles were resuspended in fresh DMEM.