OBJETIVOS

2.9.1 Transfection of plasmid DNA into mammalian cells

Transfection of plasmid DNA into HEK-293 cells using Calcium Phos- phate method

In this approach precipitate containing calcium phosphate and DNA is formed by mixing HEPES buffered saline solution (HBS) with a solution containing calcium chloride and DNA. The precipitate adheres to the sur- face of the cells and is taken up by the cells via yet unknown mechanism. Solutions HBS buffer 50mM Hepes 280mM NaCl 1.5mM NaP* filter sterilized

pH is adjusted with NaOH to 7.2

*NaP is 1:1 mixture of equal molar amounts of Na2HPO4 and NaH2PO4.

pH range of HBS solution optimal for transfection is very narrow. There- fore, 5 aliquots of HBS solution with pH ranging from 6.6 to 7.8 with the step of 0.3 were prepared and tested using pCDNA3 GFP for the highest transfection efficiency.

CaCl2 Solution for transfection

250 mM CaCl2 in H2O

filter sterilized

250 mM CaCl2 Solution was prepared shortly before transfection by dilut-

ing 2.5 M CaCl2 stock solution using sterile endotoxin-tested water (Sigma,

USA).

HEK-293 cells were sub-cultured as described in 2.7. The day before transfection cells were seeded at 1.7 X 106 cells per 6 cm tissue culture plate. 10 µg of DNA was diluted in 119 µl of 250 mM CaCl2 solution.

solution was then added dropwise to DNA/ CaCl2 mixture and tubes were

vortexed immediately for 3-4 seconds at low speed. Culture medium was changed to 4 ml of fresh medium and DNA/ CaCl2/ HBS mixture was added to the cells dropwise within two minutes after addition of HBS buffer. 12-24 hours after the transfection, culture medium was changed to 4 ml of fresh medium. 36-48 hours after transfections cells were either lysed or supernatant containing recombinant retrovirus was harvested.

Transfection of COS-7 cells using Effectene Transfection reagent kit In this method, DNA is condensed by interaction with Enchancer reagent of the kit in a defined buffer system. Effectene reagent, which is based on a proprietary non-liposomal lipid, is then used to coat the condensed DNA. DNA-Effectene complexes fuse with the plasma membrane of tissue culture cells resulting in both uptake and expression of the DNA.

COS-7 cells were sub-cultured as described in 2.7. The day before trans- fection cells were split 1:3, which resulted in about 50% confluent cellular monolayer on the day of the transfection. Right before transfection, medium was changed to 2 ml of DMEM containing 1.5 g/L of glucose, supplemented with 10% FBS, 100 Units/ml of penicillin and 0.1 mg/ml of streptamycin. 1-2µg of DNA was mixed with 100µl of EC buffer from the kit and 12µl of Enchancer. The mixture was vortexed and incubated at room temperature for 5 minutes. 14 µl of Effectene Reagent was added to the mixture. The components were mixed by vortexing for 10 seconds. The mixture was then incubated at room temperature for 10 minutes. 900µl of DMEM containing 4.0 g/L of glucose, supplemented with 10% FBS, 100 Units/ml of penicillin and 0.1mg/ml of streptamycin, was added to the mixture. Samples were mixed carefully and the mixture was added to the cells dropwise. Culture medium was changed 8-16 hours after the transfection. Cells were lysed 48 hours after the transfection.

2.9.2 Production of recombinant retrovirus using transient trans- fection of HEK-293 cells

Recombinant replication-incompetent retroviruses have been generated by co-transfecting HEK-293 cells with the packaging vector (ecopac) and the retroviral vector (pMSCV neo, pMSCV IRES EGFP, MIY). Packaging vec- tor carries retroviralgag, pol andenv genes that are required for replication of retrovirus. Retroviral vector carries the gene of interest, Ψ+ packaging signal and LTRs, which encode most of the viral control elements including promoter, enchancer, the polyadenylation signal and the integration signal. HEK-293 cells were co-transfected with ecopac and appropriated retroviral vector using calcium phosphate method. 10 µg of retroviral vector and 5

µg of ecopac were usually used per transfection of 80-90% confluent cells in 6 cm dishes. 12 hours after the transfection, medium was changed to 3

ml of fresh medium. Supernatant, containing recombinant retrovirus was harvested 36-48 hours after the transfection, aliquoted and stored at - 80oC. 2.9.3 Titering retroviral stocks

Following infection and reverse transcription of the viral genome, integration of the viral DNA into the host cell DNA takes place. The number of proviral integrants into the cellular genome depends on the infection efficiency. Ac- cording to Poisson statistics, the uninfected fraction is equal to e−l, wherel

is the mean proviral copy number per cell. Therefore, when 10% of cells are infected, the mean proviral copy number per cell is -ln(0.9)=0.1. So, most infected cells have single proviruses. To generate cell lines, in which most cells have single proviruses, infections efficiency was kept at 10% or lower.

To normalize between different viral stocks, 32D cl.3 cells were infected with serial dilutions of retrovirus. Infection efficiency was measured 36-48 hours after the infection by FACS analysis of GFP or YFP expression. Dilu- tions, at which infection efficiency was 10% or less were used for generation of 32D cl.3-based cell lines.

2.9.4 Infection of mammalian cells with recombinant retroviruses Infection of 32D cl.3 cells with retorovirus was achieved by incubation of retrovirus with cells in the presence of polycations (Polybrene).

Polybrene Solution 800 µg/ml of Polybrene in H2O

filter sterilized

Fresh aliquot of the HEK-293 cells’ supernatant, containing appropriate recombinant retrovirus was thawed in 370C water bath shortly before the infection. Exponentially or sub-confluently growing cultures of 32D cl.3 cells were used for infection. 5 X 104 cells per infection were resuspended in 250

µl of RPMI, supplemented with 10%FBS, 10% WEHI-3B supernatant, 100 Units/ml of penicillin and 0.1mg/ml of streptamycin, in 24-well plate. 16

µg/ml (2X final concentration) of polybrene was added to the cells. When necessary (see 2.9.3), viral stocks were diluted in RPMI supplemented with 10% FBS, 10% WEHI-3B supernatant, 100 Units/ml of penicillin and 0.1 mg/ml of streptamycin. 250µl of diluted or undiluted viral stock was added to the cells. Cells were mixed gently and returned to the incubator for 3-5 hours. 1.5 ml of fresh culture medium was then added to the cells. Cells were incubated for 48 hours at 370C before subsequent analysis or fluorescence- activated cell sorting.

For infection of higher numbers of cells, the volumes of all the components were multiplied accordingly.

2.9.5 Fluorescence-activated cell sorting

Modular Flow cytometer (MoFlo), Cytomation, USA, was used for cell sort- ing. In flow cytometer equipped for cell sorting, fluorescent light emitted by each cell is measured and cell suspension is passed through a nozzle, which forms droplets containing at most single cells. At the time of formation, each droplet is given an electric charge proportional to the amount of fluo- rescence in the cell. Droplets containing electric charge which corresponds to the fluorescence of desired cells are separated by an electric field and col- lected.

Cells to be sorted were pelleted by centrifugation at 1000g for 5 minutes, resuspended in the small volume of culture medium to the final concentra- tion of 5x107 cells/ml. Cell suspension was passed through sterile 40 µl cell strainers to obtain single cell suspension right before sorting. Immediately after sorting cells were resuspended in the appropriated culture medium and returned to the cell culture incubators.

2.9.6 Generation of 32D cl.3 cells expressing Flt3 and Flt3 F692T using antibiotic selection

Retroviruses encoding Flt3 constructs and neomycin resistance gene were generated by transient transfection of HEK-293 cells. 32D cl.3 cells were infected with retroviruses as described. 48 hours after the transfection, cells were transfered to the medium containing 1 mg/ml of G418. Infected cells were selected in the presence of G-418 for 1 week until all cells in the uninfected control have died. Resulted cell lines were sub-cultured in the presence of 0.5 mg/ml of G-418.

2.9.7 Generation of 32D cl.3-based cell lines expressing Flt3 and Hck constructs using FACS

To generate 32D cl.3 cell lines expressing Flt3 and Hck constructs, cells were first infected with retrovirus containing Flt3-IRES-EYFP and YFP- positive cells were sorted as described in 2.9.5. Within a few days after sorting, sorted cells were infected the second time with retroviruses contain- ing Hck-IRES-EGFP, and GFP/YFP-positive cells were sorted. Infection with retrovirusese were performed as described in 2.9.4. Infectiion efficiency was always kept at 10% or lower. Aliquots of sorted cells were frozen within a few days after sorting. Cells, which were in culture for no longer than 3 weeks were used for subsequent analysis.

2.10

Analysis of mammalian cells