Cell culture

All cell lines were stored long term in liquid nitrogen and during culture were maintained at 37°C in either a 5% CO^ incubator in a humidified atmosphere (for flasks) or on a rotary roller apparatus (for roller bottles). All manipulations of cells were carried out under sterile conditions using standard aseptic techniques.

2.3.1 Freezing and recovery of cell stocks.

Cell stocks for long term storage were prepared by suspending the cells from one

175cm flask in 1.8 ml of 8% (v/v) dimethylsulphoxide (DMSO), 30% PCS, 6 8%

appropriate medium (see section 2.1.4). The freezing vials were slowly cooled to -70°C, then immersed in liquid nitrogen. Cells were recovered by rapidly thawing the contents of one vial and transferring the cells to a 25cm^ flask o f pre-warmed medium containing the appropriate selection if required. The medium was changed or the cells passaged the following day.

2.3.2 Routine cell passage

2 2 2

Cells were grown in 175cm flasks, 500cm plates or 800cm roller bottles in the appropriate growth media (see section 2.1.4). When they were 80-90% confluent the cells were passaged as follows. For BHK, BHK-based cell lines, HEK293, HEK293- based cell lines and vero cells, the monolayers were rinsed with Hank’s Balanced Salt Solution (HBSS), detached with trypsin/versene (1:10) and disaggregated. Fresh flasks were seeded at a ratio of 1:10 (BHK), 1:6 (vero) or 1:3 (HEK293). Roller bottles were gassed with CO^ to give a final concentration o f 5%.

2.3.3 Transfection of plasmids for transient expression

BHK or BHK-based cells were transiently transfected with 5-lOp.g supercoiled plasmid

DNA per well o f a 6-well plate. The protocol was based on the standard calcium

HEBES Transfection Buffer: 140mM NaCl 5mMKCl 0.7mMNa2HPO4 5.5mMD-glucose 20mM Hepes pH 7.05 with NaOH

Filter sterilised with a 0.2pm filter. Stored at 4°C BHK or BHK-based cells were grown until they were at 70% confluency.

Two tubes were set up:

Tube A: 3 Ipl 2M CaCE Tube B: 400pl HEBES transfection buffer

lOpg plasmid DNA

20pg herring sperm DNA (phenol/chloroform extracted)

The contents of tube A were thoroughly mixed and then added slowly to tube B whilst it was being continually vortexed. The mixture was then left for 20-40 minutes at room temperature to allow the DNA to precipitate. Media was removed from the cells and the precipitated DNA mixture was slowly added. The cells were then incubated for 20- 40minutes at 37°C. 1ml o f growth media without selection was then added to the cells

and the plate incubated for 4 hours at 2>TC. The media was then removed and the cells

washed twice with 2mls of growth media. 1ml o f 25% (v/v) DMSO in HEBES transfection buffer was then added and left on the cells for no longer than 2.5 minutes. The DMSO solution was removed and the cells immediately and quickly washed twice with 2mls o f growth media. A final 2mls of growth media without selection was added to the cells which were then incubated at 37°C/5% 00% for 24 or 48 hours.

2.3.4 Detection of P-galactosidase expression by X-Gal staining

Media was removed and the cells were washed twice with 2mls of Ix PBS. The cells were then fixed in 1ml o f Ix PBS containing 0.05% glutaraldehyde for 10 minutes at room temperature. The cells were then washed twice with 2mls o f Ix PBS and

incubated at 3T C for 1-16 hours in 3mls/well o f X-Gal stain (5mM K3Fe(CN)ô, 5mM

K4Fe(CN)6.6H2 0, ImM MgClz, 150)Lig/ml X-Gal in DMSO in Ix PBS). The X-Gal

stain was then removed and replaced with 70% (v/v) glycerol for storage.

2.3.5 Detection of GFP expression

Cells expressing GFP required no pre-treatment and were visualised directly under an inverted fluorescent microscope at a wavelength of 500nm.

2.3.6 Determination of antibiotic resistance threshold (“killing curve”)

Cells were plated in 6-well plates and grown to approximately 70% confluency (except

if zeocin was the antibiotic to be used, when cells were grown to 25% confluency). The appropriate antibiotic was applied at a range o f concentrations around that recommended by the manufacturer. The selective media was changed every 2-3 days and the minimum concentration o f antibiotic required to cause complete cell death in 5 days was determined.

2.3.7 Construction of stable cell lines

Stable cell lines were constructed by an adaptation of the transfection protocol detailed in section 2.3.3. lOpg of plasmid DNA was linearised, phenol/chloroform extracted and resuspended in lOpl ddHiO, all of which was used in one transfection. The protocol was then carried out exactly as described in section 2.3.3, with the exception that the cells to

be transfected were grown in 1 0 0mm wells and the volumes of other reaction products

were scaled up accordingly (89pl 2M CaCb, lOpl HS DNA, llSOpl HEBES transfection buffer). After the DNA precipitate has bound to the cells, 3mls of growth media was added and after the DMSO shock, lOmls of growth media was added. The day after transfection, the cells were washed and the selective media was applied at the concentration determined by the killing curve (section 2.3.6). If transfected cells were to be selected by zeocin resistance, the 100mm wells were split 1:15 24 hours after transfection and the selective media applied to all wells 24 hours after the split. This extra step was necessary as zeocin is able to kill unresistant cells most efficiently when the monolayer is not more than 25% confluent.

The selective media was changed every 2-3 days until colonies were clearly visible but still well isolated. Colonies were picked under an inverted microscope and transferred to 96-well plates. When 80-90% confluent, the cells were split and grown

through 24 and 6-well plates with changing o f the selective media every 2 days. Cells

were always moved before they became 100% confluent. Cell lines were then transferred to 25cm^ flasks and 2 ampoules o f each were frozen down by the method described in section 2.3.1. At this stage, 24-well plates of all cell lines were set up to enable screening of the cells for the ability to grow disabled HSVl.

2.3.8 Screening of cell lines

Each cell line was split into 2 wells o f a 24-well plate and grown to 80% confluency. The wells were infected at a multiplicity of infection o f 0.01 with an appropriately disabled virus. The virus was harvested and titred 48 hours post infection (see section 2.4.2). A small number of the best cell lines were selected for further analysis by growth curves (see section 2.4.8). The optimal cell line was then selected and cloned out.

2.3.9 Obtaining a pure population of cells

Once the optimal cell line had been identified, this was cloned out by limiting dilution in a 96-well plate and slowly amplified under continual selection. A large number of 175cm^ flasks of the pure cell line were grown and frozen down. These frozen ampoules were stored in liquid nitrogen and a new one was defrosted every 4-6 weeks (see section 2.3.1)