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2.20.1 Maintenance of cell lines

M urine cell lines were m aintained in DMEM Com plete m edia and stable transfected cell lines w ere m aintained in DMEM Com plete supplem ented w ith SOOpgml^ G418. AU ceU lines w ere split every 3-4 days to prevent the ceUs from becom ing over-confluent. CeU lines w ere split by rem oving the media, washing the cells once w ith 1.5ml trypsin (Gibco BRL) and trypsinising at 37°C in a further 1ml trypsin for 3 minutes. Following trypsinisation the ceUs w ere hom ogenised in 3.5ml DMEM Com plete m edia by pipetting up and dow n several times against the bottom of the plate. Between 4 and 10 drops of this ceU suspension w ere then used to seed a fresh 10cm plate of media, depending on the grow th rate of the particular ceU line.

2.20.2 Transfection of M us dunni cells and selection for clonal cell lines

Transfection protocol: Stable transfection of M us dunni cells was carried out

using Superfect (Qiagen) according to a modification of the m anufacturer's standard protocol. Cells w ere grow n in 6cm tissue culture plates to 20-40% confluency in 5ml DMEM Complete. The sam e m iniprepped DNA that w as used

for sequencing was used to transfect m ouse cells. Approximately Ip g of

construct (of a m inim um concentration of O.lpgpD) was mixed w ith ISOpl DMEM containing no serum or antibiotics. After mixing and spinning this dow n briefly, 7.5pl of Superfect was added to the DNA solution and mixed by pipetting up and dow n 5 times. This was then incubated at room tem perature for 5-10 m inutes to allow complex formation. While this was happening, the g ro w th m edium was rem oved and the cells washed once w ith PBS. After adding 1ml of DMEM Com plete to the reaction tube containing the transfection complexes, this DNA complex solution was mixed by pipetting up and dow n twice and transferred to the cells. A fter incubation for 2-3 hours at 37°C and 5% COg, this m edium was

rem oved and the cells washed 4 times w ith PBS. Fresh DMEM Com plete

m edium was then added and the cells incubated for 3 days to ensure sufficient expression of the m arker gene.

Selection fo r stable cell lines: Following transfection and recovery, the

m edium was rem oved from the plates. The cells w ere then w ashed once w ith

1ml trypsin and incubated w ith 1ml trypsin at 37°C and 5% CO2 for 3 minutes.

After ensuring the cells had completely detached following trypsinisation, they w ere then resuspended in 3.5ml DMEM Com plete supplem ented w ith 40pgml'^ G418 and pipetted vigorously to reduce cell clumping. In order to isolate single transfected clones, the ceU suspension was diluted and aHquoted: approxim ately 150pl (3 drops) and 750jil (15 drops) of this ceU suspension was added to separate 150ml volum es of DMEM Com plete supplem ented w ith G418 and mixed thoroughly. Both of these dilutions w ere then transferred to 6 24-well plates,

approxim ately 1m l/well. The m edia was replaced every 3 days to ensure

m aintenance of selection. After about 10 days, the wells w ere regularly

exam ined for grow th. Transfected clones from plates with less than 6 wells positive for g ro w th in total w ere transferred to 6cm plates w hen confluent; plates w ith m ore than 6 positive wells w ere discarded. Transfected ceU lines w ere m aintained in G418-supplemented media.

2.20.3 Storage and reviving of stable cell lines

Cell lines w ere grow n to near-confluency on 10cm tissue culture plates p rio r to freezing. The m edia w as rem oved and the cells w ashed twice in 1.5ml trypsin

before incubating at 37°C and 5% CO2 for 3 minutes. After ensuring the cells had

completely detached following trypsinisation, they w ere then resuspended in 5ml tissue culture freeze m edium and pipetted vigorously to reduce cell clumping. This suspension was then aliquoted into 3 cryo tubes (Nunc) and transferred in an insulated box to a -80°C freezer overnight. The tubes w ere transferred to a liquid nitrogen store the following day. This 2-step freezing procedure reduces loss of ceU viability caused by rapid freezing.

2.20.4 Virus infection of cell cultures

Supernatant from virus-producing cell lines was aliquoted into 1.5ml eppendorf

tubes and stored at -80°C. These stocks w ere assayed by infecting the F v f M us

dunni ceU line at various dilutions. In the case of the MTV stocks used in the RT assay, the lowest dilution that gave a productive infection was used. In the case of the pseudotyped virus used in the lacZ assay, the dilution that gave -500 blue cells per plate was used.

Cell lines to be assayed were plated out in a 6-weU tissue culture plate to a density of -20-40% confluency. The m edia was rem oved from the cells and 1ml of DMEM Com plete containing the virus and 15pg polybrene was then added. For the control well, 1ml DMEM Com plete contcdning only polybrene was

added. The cells were then incubated at 37°C and 5% CO2 for 1 hour. This m edia

was replaced w ith 3ml fresh DMEM Com plete and the cells incubated for 3 days

at 37°C and 5% CO2 before being assayed.