3.2.1 Linearisation of DNA.
It was important to linearise the plasmid DNA to be transfected, as this increases the efficiency of integration. Ideally, the enzyme site chosen should be at least 200bp from either end of the promoter/poly A of the gene of interest since some bases can be removed from the plasmid during integration. 10 g of linearised DNA were prepared per cell type to be transfected.
The following digest was set up:
3 g DNA, 10 l restriction enzyme buffer (buffer E, buffer D), 5 l appropriate enzyme (10units/ l) (SspI, NotI), ddH20 to 100 l, Incubate for 4 hours at the appropriate temperature
The resulting DNA was then purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences) as per manufacturer‟s instructions. The DNA was then eluted in a final volume of 50 l.
The Pc DNA3 HVEM plasmid and the pGAL CMV Luc Hygo plasmid were used in this study (Figure 3.2, Table 3.2)
Figure 3.2 Pc DNA3 HVEM and the pGAL CMV Luc Hygo plasmids
Table 3.2 Pc DNA3 HVEM and the pGAL CMV Luc Hygo plasmids
Plasmids Linearisation restriction enzyme
Pc DNA3 HVEM Ssp I
pGAL CMV Luc Hygo Not I
3.2.2 Antibiotic killing curves
The aim of the antibiotic killing curve was to determine the minimum concentration of antibiotic (G418 (Gibco), hygromycin (Sigma)) required to give complete cell death level of cell death. It was necessary to change the selecting media every 2 days. After 10 days the cells were fixed and stained as above.
pGL4 C M V luc hygo
3.2.3 Transfection of cells to produce luciferase/HVEM expressing cell lines
AY-27 cells were plate at 6x105 cells per well of 6 well tray (using media without antibiotics) and incubate at 370C o/n. 1 g of linearised plasmid (pcDNA3 HVEM, pGAL CMV Luc Hygo) was diluted in 100 l of RPMI (without FCS) per transfection.
The lipofectin (Invitrogen) was diluted in RPMI (without FCS). Both were let stand at room temperature for 30-45 minutes. The diluted DNA and lipofectin were combined, gently mixed and incubated at room temperature for 15 minutes. The growth media from the AY-27 cells was removed and the cells were washed with 2ml of RPMI without FCS. 800 l of RPMI without FCS was added to DNA/ lipofectin mix and then added to the cells. The cells were incubated at 370C for 7hrs. After this incubation the DNA/ lipofectin mix was removed, 2ml of FGM was added and incubated at 370C o/n.
After 48hrs the cells were split into 90mm culture dishes (x9) and incubated at 370C o/n. After 24 hours FGM was removed and FGM containing the appropriate antibiotic was added.
3.2.4 Selection and scale up of transfected clones
48 hours after transfection cells were split into 90mm culture dishes (x9) culture dishes and the appropriate selection media was added 24 hours later (Table 3.3). The concentrations of antibiotic used to treat the cells were pre-determined by carrying out an antibiotic killing curve (See section 3.2.2).
The cells were observed every day and the selection media was changed every 2-3 days (especially if there is a high level of cell death). Individual cell colonies were visualised between 5-20 days of selection (depending on the cell line and compound). Once the colonies were clearly observable, they could be transferred to individually wells of a 96-well tray containing 100 l of selection media. This was achieved by picking the colonies, with a p20 gilson, directly from the plate whilst observing it under an inverted microscope (Nikon Eclipse TE200). The cells were then transferred to one well of a 96-well tray and pipetted up and down a few times to break up the cells. The individual cell
lines took between 2-14 days to produce a confluent monolayer in a well of a 96-well tray. Once confluent, the cell lines were transferred to one well of a 24-well dish containing 500 l selection media/well, using 50 l trypsin versene to remove the cells from the 96-well dish. The cell lines were grown from 24 well stage to a 6 well stage and finally up to a 25cm2 flask. At all times the cell lines were exposed to selection media. Once confluent, the flask of cells was frozen down into 3 ampoules, one of which was kept as a master stock. Once a suitable cell line was identified, it was scaled up and frozen stocks were produced.
Table 3.3 Concentration of selection agents used in this study
Antibiotics AY-27 cells
G418 (Gibco) dissolve in H2O 300 g/ml Hygromycin (Sigma) dissolve in H2O 100 g/ml
3.2.5 Sub-cloning cell lines
This protocol was carried out after a suitable cell line had been identified from a relevant screening process in order to ensure that the cell line was derived from an individual transfected cell. 1x103 cells per well in 200 l fresh FGM with appropriate concentrations of selection antibiotics were added to every well in row A of 96-well tray. 100 l selection media was added to rows B-H of the dish. Then a 1:2 serial dilution of cells suspension was carried out from row A to row H. The trays were incubated until the wells containing 1 cell become confluent. The new sub-clones were grown up and re-screened to identify positives.
3.2.6 Infection screening assay for AY-27 HVEM
To identified clones of AY-27-HVEM that support replication of HSV-1 test cells were plated at 1x105cells per well of a 24 well tray and incubated at 37oC/5% CO2 o/n. The media was then removed and the cells infected with OncoVexGFP virus at MOI 0.1 (in a volume of 200 l RPMI) and incubated at 37oC/5% CO2 for 1Hr. The virus was removed and 1ml of fresh FGM media was added. The infection was incubated at 37oC/5% CO2
for 72 hrs. Infection of OncoVexGFP virus expressing green fluorescent protein marker
gene were visualised under an inverted fluorescent microscope (Nikon Eclipse TE200) at wavelength 520nm.
3.2.7 Luciferase activation screening assay.
AY-27 HL cells were plated at 5x105 cells per well of 6 well tray and incubated at 370C o/n. After this incubation the FGM was removed and the cells were washed (3x) with 1.5ml ice cold PBS. The PBS was removed and 120 l of cell 1x culture lysis reagent (Sigma 5x cell culture lysis Reagent, C 4707) was added. The lysate was incubated at room temperature for 15 minutes after which the cells were scrape off and spun at 12,000g at 40C for 1 minute. The supernatant was removed and store on ice. The lyophilized luciferase assay substrate (sigma L 0407) was resuspended in 10ml of luciferase assay buffer (Sigma L 0532). The luciferase substrate and the tested cell lysate were allowed to equilibrate to room temperature before use. 20 l of the cell lysate was added to 100 l of the luciferase substrate assay buffer. Light emission was read after 10 seconds incubation at room temperature using Beckman colterDTX 880.
(Luciferase assay kit MB-260)