CHAPTER 2: Materials and Methods 42
2.1 Cell Culture and Virological Methods 42
2.1.1
Cell maintenance
Human cervical (HeLa), human embryonic rhabdomyosarcoma (RD), human embryonic kidney (HEK293T), and murine fibroblast (L929) cells were grown as monolayers in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 100 μg/ml of streptomycin, 100 U/ml of penicillin, 2 mM L-‐glutamine, and 10% heat inactivated (HI)-‐foetal bovine serum (FBS). Baby hamster kidney cells stably transfected with a T7 RNA polymerase expression plasmid (BsrT7) were maintained in Glasgow Minimum Essential Medium (GMEM) supplemented with 100 μg/ml of streptomycin, 100 U/ml of penicillin, 2 mM L-‐glutamine, and 10% HI-‐FBS. Antibiotic G418 was also added to BsrT7 cell medium to select cells containing the T7 RNA polymerase gene under control of the cytomegalovirus (CMV) promoter and the neomycin resistance gene (Buchholz et al., 1999). All cells were passaged using trypsin-‐ ethylenediaminetetracetic acid (EDTA).
2.1.2
RNA transfection of mammalian cell lines
Purified RNA (amounts as specified elsewhere) in H2O was made up to 100 μl in serum and
antibiotic free medium and mixed with an equal volume solution of Lipofectamine 2000 (3:1 Lipofectamine 2000 to μg RNA), (Invitrogen) in similar medium. The mixture was incubated according to the manufacturer’s instructions and added to cell monolayers in either 6 or 12-‐well tissue culture plates.
2.1.3
Luciferase assay
Supernatant was removed from transfected cell monolayers, cells were briefly washed with PBS and lysed using 200 μl 1 x Glo Lysis Buffer (Promega®) per well in a 12-‐ well plate. The
oxidation reaction wascatalysed by the addition of 50 μl cell lysate (luciferase enzyme) to 50 μl room temperature Bright-‐GloTM Luciferase Assay System (Promega®) substrate and shielded
Biosystems) with values normalised to mock transfection controls.
2.1.4
Isolation of media from transfected cells
Media from individually or co-‐transfected cells was isolated at 48-‐72 hrs post transfection. Any cellular debris was pelleted by centrifugation, with media being placed into new 1.5 ml
microfuge tubes. Plaque assays were then carried out to detect virus.
2.1.5
Tissue culture infectious dose50 (TCID50)
Recombinant virus from co-‐transfections was titred by TCID50 which measures the quantity of
virus required to infect 50% of inoculated microplate wells per sample (Minor, 1985). Each well of 96-‐well plates was seeded with 2.5 x 104 HeLa cells, allowing for four replicates of titration
per sample. In a separate 96-‐well round-‐bottomed plate, a serial 10-‐fold dilution of sample with DMEM/10% FBS/penicillin-‐ streptomycin (PS) from 10-‐1 to 10-‐12 was performed. Media was
removed from confluent cells, 100 μl of the appropriate virus dilution was added to each well, (including 100 μl DMEM/10% FBS/PS to control wells) and incubated for 1 hour at 37°C/5% CO2. An extra 100 μl DMEM/10% FBS/PS was added per well and plates were incubated for four
days. Plates were stained with crystal violet for one hour and rinsed with tap water before drying upside down on paper towel. Virus titre was expressed as log10 TCID50/ml (Reed &
Muench, 1938).
2.1.6
Plaque assay
RD or HeLa cells were seeded in 6 or 12-‐well plates and grown to 95% confluency. Where appropriate ten-‐fold dilutions of virus stock were made in DMEM. Once medium was removed from seeded wells, cells were washed with sterile PBS then inoculated with 500 μl of prepared virus and incubated for 30 mins at 37°C in the presence of 5% CO2/air to allow absorption to
occur. Plaque overlay media (refer to section 2.6 for contents) was added to each well and plates were incubated inverted for two to three days at 37°C in the presence of 5% CO2/air.
Cells were stained with crystal violet solution and re-‐stained post-‐removal of the plaque overlay media. Plaques were counted and the virus titre expressed as plaque forming units per millilitre (PFU/ml).
2.1.7
Plaque purification by limiting dilution
Recombinant virus from RNA transfections was biologically cloned by limiting dilution in 96-‐ well microtitre plates. Virus dilutions were performed using a multichannel pipette in a round bottomed 96-‐well microtitre plate (Sterilin) prior to transfer onto fresh cell monolayers. Fifty-‐ five microlitres of serum-‐free media was added to each well prior to addition of diluted virus. Approximately 55 μl diluted virus supernatant was added to each well in the top row. After mixing by pipetting, 55 μl of solution was transferred to the next row (already containing 55 μl serum-‐ free media). Pipette tips were discarded after the mixture was mixed in each row and these two-‐fold dilutions continued down the plate. Fifty microlitres was transferred from each well onto a fresh cell monolayer in flat bottomed 96-‐well plates seeded with 2.6 x 104 HeLa
cells/well. The plates were left to allow for virus absorption for 30 mins at 37°C in the presence of 5% CO2/air. An extra 200 μl serum-‐free media was added to each well and plates were
incubated at 37°C/5% CO2/air. After 4 days, the supernatant was transferred to a new
microtitre plate and cell monolayers were stained by crystal violet for the presence of complete CPE. RNA was extracted from the highest dilution of virus supernatant per column and RT-‐PCR was carried out to amplify the crossover region of the recombinant virus.
2.1.8
Virus infections
Cell monolayers (RD or HeLa) were inoculated with virus at the stated MOI. Virus was absorbed onto monolayers for 30 mins at 37°C/5% CO2/air. Virus supernatant was removed and replaced
with medium supplemented with or without 10% HI-‐FBS. For passaging, virus supernatant was removed 24 hours post-‐infection, upon completion of full cytopathic effect (CPE; appearance of cell rounding or detached cells).
2.1.9
Disruption of cellular microtubules and nocodazole treatment
Following the protocol taken from Egger & Bienz, 2005. To disrupt microtubules, cells were kept for 10 min at 0°C. Nocodazole at concentrations of 5 mM (Sigma) was added to prevent re-‐ polymerization of microtubules upon rewarming to 37°C.