Poemas de la memoria

Transfections were performed using cationic lipid, which relies on the binding of the positively charged lipid headgroup to the negatively charged

phosphate groups of the DNA to form lipid DNA complexes (Feigner et al., 1987). Prior to transfection of eukaryotic cells with plasmid DNA, fresh plasmid DNA was prepared using the Qiagen QIAprep plasmid miniprep column as described in section 2.2.3 and cleaned as described in section 2.2.3.4. The concentration of the DNA was calculated by measuring the absorbance at 260nm (A2 6 0) using a UV visible spectrophotometer (Cam Spec M302).

Target cells used were 293 cells (ECAGG No:85120602), which are derived from human embryonal kidney cells transformed with sheared human adenovirus type 5 DNA. The 293 cells were seeded at a concentration of 1x10^ cells/ml in a Nunclon 6 well plate, with a total of 2 mis in each well, in Dulbecco’s Modified Essential Medium (DMEM) (Gibco), supplemented with 10% Foetal Galf Serum (FGS) (GibcoBRL), penicillin (GibcoBRL) at a concentration of 50IU/ml and streptomycin (GibcoBRL) at a concentration of 50|Lig/ml. The cells were cultured at 37°G and 5% carbon dioxide (GO2) until

they reach 40-60% confluency. The desired amount of plasmid DNA to be transfected was placed in a 17 x 120mm conical polystyrene sterile tube (Falcon) and to this lOOpI of serum free medium was added (Opti-MEM GibcoBRL). In separate 12x75 mm sterile tubes was aliquoted the cationic lipid (Lipofectin (1:1 ratio N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoyl phosphotidylethanolamine (DOPE) GibcoBRL), which was at a concentration of 1 mg/ml, to which was added 1 0 0|il of serum

free medium. The plasmid DNA and lipofectin solutions were mixed gently and incubated at room temperature for 15 minutes, after which 800pl of serum free medium was added to each tube, making the total volume 1ml. Meanwhile the cells to be transfected were washed by adding once to each well 2 mls of serum

free medium. This was aspirated from each well and to each well was added the DNA/lipofectin mixture ensuring the cell monolayer was completely covered. The cells were incubated for 5 hours at 37°C and 5% carbon dioxide (CO2),

after which the DNA:lipofectin mixture was carefully aspirated and replaced with DMEM supplemented as before. Incubation was then continued for 48 hours before harvesting of the cells to assess expression of the gene of interest.

2.1.6.1 Detection of p-Galactosidase Activity in Transfected Cells

To assay the cells for p-galactosidase activity the medium was carefully aspirated from the cell monolayer from each transfected well. The cells were then washed twice with PBS buffer (137mM NaCI, 2.7mM KCI, 4.3mM Na2HP0 4 , 1.47mM KH2PO4, pH adjusted to 7.1) (Mg^"^ and Ca^"" free) taking

care not to dislodge any of the cells. The residue of the PBS was aspirated using a pipette tip. 400pl of 1x Reporter Lysis Buffer (Promega) was added to the cells so that the cell monolayer was completely covered. This was incubated at room temperature for 15 mins, gently rocking the cells once midway through this period. The cells were then carefully scraped off from each well and transferred to separate 1.5ml microcentrifuge tubes (Eppendorf). Each tube was vortexed for 15 seconds and centrifuged at 10,000 x g in a benchtop microcentrifuge for 2 minutes. After this the supernatants containing p-galactosidase activity were transferred to fresh microcentrifuge tubes.

25pl of each cell extract was added to a flat bottomed 96 well plate (Nunc), including the negative control and was diluted with 25pl of 1x Reporter Lysis Buffer (Promega). In a separate 96 well plate 50pl of Assay 2x Buffer (120mM Na2HP0 4 , 80mM NaH2P0 4 , 2mM MgCl2, lOOmM p-mercaptoethanol

and 1.33mg/ml o-nitrophenyl-p-D-galactopyranoside (ONPG)) was added to the appropriate number of wells. To each of these wells was added 50pl of each cell extract, the samples mixed by pipetting up and down and the plate was covered and incubated at 37°C for 30 minutes. The reaction was then stopped by the addition of 150pl of 1M Sodium carbonate. The absorbance of the samples was read at 405nm using a plate reader (Anthos reader 2001).

2.1.6.2 Generation of Stable Transfectants

293 cells were cultured in a 96mm sterile petri dish (Nunc) in supplemented DMEM as described in 2.1.6 until they had reached 60% confluency. The plasmid was transfected into the 293 cells as described in 2.1.6, using lipofectin. 5 hours post transfection, the lipofectin:DNA complexes were removed and replaced with fresh medium. After incubation overnight at 37°C with 5%C02, the medium was replaced with fresh supplemented DMEM containing G418 sulphate at an active concentration of 500pg/ml (Canaani and Berg, 1982). Culture was continued, with death of non resistant cells between 3 and 7 days post exposure to G418. During selection, the medium was replaced every day and dead cells removed. When the cells had reached confluency, they were harvested by incubation with 5mls of trypsin-EDTA (0.5g trypsin and 0.2g EDTA in lOOOmIs PBS), after aspiration of the medium and washing the cell monolayer once with HBSS. The cells were then analysed by flow cytometry for expression of the gene of interest, as described in section 2.1.3.

2.1.6.3 Sorting of 293 B7-1 and B7-2 293 Cells into Uniformly Expressing Populations

Stable transfectants expressing either B7-1 or B7-2 were harvested by trypsinisation. 1x10® cells from each population were washed and labelled with either anti-B7-1 FITC (Pharmingen - clone BB1) or anti-B7-2 FITC (Pharmingen - clone FUN-1) respectively. After labelling and washing the cells were resupended in 1ml of FACSFlow. The cells were sorted on a FACS Vantage, the cells separated into two populations, the highest surface expressors of B7-1 and B7-2 and the low or negative expressors of B7-1 or B7-2. The high expressors of B7-1 or B7-2 were washed once in 1ml of HBSS, centrifuged at 200 X g, supernatant discarded and the cell pellet resupended in 2mls of DMEM, supplemented with 10% FCS, penicillin and streptomycin and G418 sulphate at 500pg/ml. The B7-1 and B7-2 high expressors were each inoculated into one well of a 6 well plate and cultured at 37°C and 5%C02 until the cells had reached confluency. An aliquot of each of the two samples was harvested and labelled with either lOpI anti-B7-1 PE or lOpI anti-B7-2 FITC accordingly, as described in section 2.1.3, prior to flow cytometric analysis.