The analytical grades of chemicals were purchased from Genetix, Himedia, Fermentas, Titan, SIGMA. The filtered milli-Q (0.22µm) water was used for the preparation of solutions. Different methods were used to sterilized solutions, glass and plastic wares either through autoclaving or by filtration. The appropriate temperatures were used to store all the chemicals. Luria Bertani, the bacterial media was brought form HIMEDIA and antibiotics were brought from SIGMA. Different components like Taq. DNA polymerase enzyme, Restriction enzymes, Glutathione agarose beads were purchased from G Biosciences, New England Biolabs, Genetix and SIGMA, respectively. The vectors used for this study were pTZ57R/T (Thermo Scientific), pGEX2TK (GE healthcare), pCDNA3.1 (Invitrogen) and pEGFP-N1 (Clontech Laboratories, Inc.). DMEM (Dulbecco's Modified Eagle’s Medium and MEM (Minimum Essential Medium). HEK293 and HEK293T cell lines were purchased from NCCS, Pune, INDIA.
Semi-nested PCR amplification of truncated huΔIRF-1 (1-699 bp): The wild-type huIRF-1 DNA sequence was deleted (700–975 bp; 92 amino acid) at the C- terminal end to generate the deleted huΔIRF-1 (1-699 bp, 233 amino acid). pT1.3 clone plasmid DNA (2888 + 975 bp) was used as a template to be amplified by internal primers; Forward Primers: 5’AAGGATCCATGCCCATCACTCGGAT3’ (underlined is BamHI restriction site) Reverse Primers: 5’TTGAATTCCCGGTAACGATCTAGCT3’ (Underlined is EcoRI Restriction site).
Sub-cloning of IRF-1 and ΔIRF-1 in pcDNA3.1 vector
The human IRF-l was transferred from pTZ57R/T into the pcDNA3.1 mammalian expression vector (Invitrogen) to form the pCDNA3.1-IRF-1. This was carried out by EcoRI and BamHI restriction digestion of pTZ57R/T-IRF-1 and pTZ57R/T-ΔIRF-1 recombinant vectors. The smaller fragments of size 991 and 699 bp were gel purified and ligated with double digested pCDNA3.1 vector. The ligation mixture was transformed into E. coli/DH5α cells and the positive clones were selected by colony PCR and plasmid restriction digestion.
Maintenance of HEK-293 and HEK293T cells
Cell lines HEK293 and HEK293T (Human Embryonic Kidney cells) were used in this study. These cell lines (HEK293 and HEK293T) were purchased from National Centre for Cell Science, Pune and maintained in MEM (Minimum Essential Medium) and DMEM (Dulbecco's Modified Eagles Medium with high Glucose) complete medium supplemented with 2 mM L- glutamine, 10% FBS and 1X antibiotic solution. All cells were cultured in a humid atmosphere at 37°C with 5% CO2. When confluence exceeding 80%, sub-culturing of HEK293 and HEK293T cells were done in every 3-4 days. Cells were washed with 1xPBS for sub-culturing further treated with 0.1% trypsin-EDTA solution; the flask was thus gently shake to remove the cells from the plate and produce a single cell-suspension. The cell suspension was centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in 2 ml of fresh MEM or DMEM and maintained at 37°C, 5% CO2 with humidity. Further cells were observed under the inverted microscope.
Cells were diluted with trypan blue stain at 1:1 dilution for cell counting. Then 10 µl of stained and mixed cells were counted on a haemocytometer. Cell count in a four corner square is divided by four to find out an average number of cells in one area of haemocytometer. The number of cells was counted by the following formula: No. of cells/ml = Total No. of cells in 4 square/4 X 104 X Dilution factor
Transfection of HEK-293 and HEK-293T cells with plasmid DNA
For high cell density, high efficiency, high expression levels, and to minimize cytotoxicity, transfection of cells by using different DNA (μg) to Lipofectamine 2000 (μl) ratio i.e., of 1:2 to 1:3. One day before transfection 0.5-2 x 105 HEK-293 and HEK293T cells in 500 μl of the growth medium was grown in DMEM supplemented with 10% FBS with 1x antibiotic mix solution so that cells will be 70-90% confluent at the time of transfection. Preparations of complexes for transfection of plasmid DNA are as follows: Different sets of plasmid DNA contained a gene of interest (pCDNA-IRF-1, pCDNA-IRF-1, pCDNA3.1) were diluted and mix gently in 50 μl of Opti-MEM Reduced Serum Medium (Solution A) in three different MCT. Lipofectamine 2000 was also diluted in the three different 1.5 ml MCT with 50 μl of Opti-MEM Medium (Solution B). These different diluted solutions were incubated for 5 minutes at RT. DNA (Solution A) was diluted with Lipofectamine 2000 solutions (Solution B). The samples were briefly mixed and kept at room temperature for 20 minutes and prepare three different sets of plasmid DNA: Lipofectamine diluted solutions. Solution A: Solution B mixture (100 μl) was then added directly to the cells with medium and mixed the plate gently by rocking back and forth. For observation of transgene, expression cells were incubated at 37°C in a CO2 incubator for 24-48 hours.
Measurement of Cytotoxicity by MTT assay
MTT assay was used to determine qualitative and quantitative evaluations of the viability of cells. Cells were seeded in 96 well plates contain 100 μL of the complete medium at the density of approximately 1x105 cells per well. After an incubation of 48 hours, culture medium was removed and replaced with aliquots of 100 μL of medium containing the appropriate concentration of extracts particles. PCDNA3.1 empty vector transfected the cells were treated as control. A total of three wells were assigned for each transfected cell. The cells were transfected with different sets of plasmid DNA (pCDNA-IRF-1, pCDNA-IRF-1, pCDNA3.1) for 24 hours. After the incubation period, these cells were observed microscopically to assess the transfection positive cells; this was the indication of qualitative evaluation of gene of interest. Added 80-100 μL of MTT reagent to each well and gently mixed at least for 60 seconds and the plates were incubated at 37°C in the incubator for approximately 4 hours; cells were lysed by adding 100 μL of Dimethyl Sulfoxide (DMSO) to each well. At 560 nm wavelength absorbance was measured to determine the cytotoxicity effect and quantitative evaluation of gene of interest using the ELISA plate reader linked to a computer using ChromaxPro software.
Preparation of Protein extracts from cell lines
Protein extracts from cultured cells that were used for immunoblot were made by using Radio immunoprecipitation assay (RIPA) buffer. 100 µl of an appropriate lysis buffer was used per 1x106 cells. The cultured monolayer cells were rinsed with cold PBS 3 times. Through trypsinization or by using a cell scraper, transfer the cell suspension in 2 ml of MCT tube with a final rinse. Cells were centrifuged at 1,500 rpm for 5 minutes at 4°C and discarded the supernatant as much as possible. For preparation ice-cold lysis buffer, 5 µl Protease Inhibitor Mixes were added to 1 ml of extraction buffer. 100µl of ice-cold cell lysis buffer was added and resuspended the pellets followed by incubation on ice for 10 minutes. Vortexes tubes briefly and proceed to sonication with the following settings 40% amplitude with 30 seconds on and 30 seconds off cycles, repeated 5-8 cycles by using Ultrasonic Homogenizer Sonicator in ice. Briefly vortexed the sample and visually checked the samples after 5 cycles, the viscosity should be reduced after perfect sonication. To prevent protein damaged the shortest sonication time should be chosen. The supernatant was transferred to a new MCT tube and samples were centrifuged at 14,000 rpm for 15 minutes at 4°C to remove any remaining insoluble material. Take an aliquot for the quantification and the further analysis if needed. Stored the extracted proteins at -80°C.
The unbound antibody was removed by washing with PBST washing buffer. The blot was incubated for 1 hour at RT with horseradish peroxidase-conjugated anti-mouse secondary antibody (1:10,000 in 3% BSA; Santa Cruz, USA) after three washing with PBST. The unbound antibody was removed with PBST washing buffer and the blot was developed using 0.6 mg/ml DAB substrate solution (0.1 mM NaCl pH 7.6, 50 mM Tris-HCl, 0.01% H2O2) at RT for 0.5-2 minutes of incubation. The bands of expressed protein were visualized in the ChemiDoc TM MP imaging system for their analysis.