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2.1.1. Cell lines

The continuous African green monkey kidney cell line, BSC-1 and chick embryo fibroblasts (CE), prepared by previous members of the PVL laboratory were used to investigate the tdCE mutant phenotype. The BSR-T7 cell line which was derived from the BHK-21 cell line and constitutively expresses the bacteriophage T7 RNA polymerase (Buchholz et al., 1999) was used in all minigenome and full-length virus rescue transfections. BSC-1 cells were routinely maintained in Glasgow Modified Eagle’s Medium (GMEM) supplemented with 10% foetal bovine serum (FBS), 0.2 mM L-glutamine and 100 µg/ml penicillin/streptomycin. BSR-T7 cells were also grown in complete GMEM (as described above) and every second pass in GMEM supplemented with 1 mg/ml G418, an aminoglycoside antibiotic that prevents polypeptide synthesis by irreversibly attaching to the 80S ribosomal subunit. G418 is required for the selection of cells stably transfected with a plasmid encoding the T7 polymerase and the neomycin-phosphotransferase gene which confers resistance to G418. Primary cultures of CE cells were maintained in medium 199

supplemented with 10% FBS, 0.2 mM L-glutamine and 100 µg/ml penicillin/streptomycin.

The mosquito (Aedes albopictus) cell line, C6/36 (Igarashi, 1978), was used to investigate the difference in cytopathic effect of CHPV in mammalian and insect cells. C6/36 cells were grown in Leibovitz’s L-15 medium with 10% FBS, 0.2 mM L-glutamine and 100 µg/ml penicillin/streptomycin. The packaging cell line HEK

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293T was used to generate lentivirus for pseudotyping experiments as they constitutively express the large T antigen of simian virus 40 (SV40). HEK 293T cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% FBS, 0.2 mM L-glutamine and 100 µg/ml penicillin/streptomycin. The astrocytoma U373, human embryonic oligodendrocyte Oligo/TL, human rhabdomyosarcoma (RD) and hepatocellular carcinoma Hep-2 cell lines were also used in this investigation. U373 cells were cultivated in Eagle’s Minimal Essential Medium (EMEM), while

Oligo/TL, RD and Hep-2 cells were grown in DMEM, all supplemented with 10% FBS, 0.2 mM L-glutamine and 100 µg/ml penicillin/streptomycin.

2.1.2. Maintenance of cells in culture

Cultures of each of the cell lines were grown in 75 cm2 tissue culture flasks with 20 ml of the appropriate medium and incubated at 37°C in 5% CO2, except C6/36 cells which were incubated at 28°C without CO2. Cells were passaged every 3-4 days in 1 in 4 (C6/36, U373) 1 in 6 (BSC-1, Hep-2 and CE) or 1 in 8 (BSR-T7, 293T,

Oligo/TL and RD) dilutions. Confluent monolayers of cells were washed in 1:6 trypsin (0.25% (w/v) trypsin in PBS, pH 7.4 (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) /versene (0.02% (w/v) EDTA and 0.002% (w/v) phenol red in PBS, pH 7.4) before incubation at 37°C (28°C for C6/36 cells) with trypsin until all the cells had detached from the flask surface, approximately 5-10 minutes. Growth medium was then added to inhibit trypsin activity and cells were

resuspended to a homogenous suspension and an aliquot transferred to seed a new flask or roller bottle containing fresh medium or a tissue culture plate.

57 2.1.3. CHPV minigenome rescue

BSR-T7 cells were seeded into 12-well tissue culture plates 24 hours before transfection so that the cells were approximately 70% confluent when transfected. 0.6 µg pT7N, 0.1 µg pT7P, 0.1 µg pT7L and 0.2 µg mgT7NGFPL (a minigenome containing an eGFP reporter gene cloned between the N and L gene) were

transfected using TransIT®-LT1 transfection reagent (Mirus, USA). DNA was added to Opti-MEM® Reduced Serum Medium (Invitrogen Life Technologies, UK) and gently mixed before 3 µl of TransIT®-LT1 transfection reagent was added to the diluted DNA. The TransIT®-LT1 reagent: DNA complexes were gently mixed and incubated at room temperature for 15-30 minutes, after which the complexes were added dropwise to the cells and incubated at 37°C for 24 hours. The level of GFP expression was determined by fluorescence microscopy. The plasmids used in the minigenome rescue were all obtained from Dr A. C. Marriott (Marriott & Hornsey, 2011).

2.1.4. Rescue of infectious recombinant viruses

BSR-T7 cells grown to approximately 70% confluence were transfected with 0.8 µg pT7CV (the whole genome clone) and the support plasmids in the following

proportions: 0.2 µg pT7N, 0.15 µg pT7P, 0.15 µg pT7L using 6 µl TransIT®-LT1 transfection reagent, as described in section 2.1.3. 48 hours after transfection the supernatant (containing virus) was inoculated onto confluent monolayers of BSC-1 cells growing in complete GMEM with 2% FBS. The supernatant was harvested approximately 24 hours post infection.

58 2.1.5. Generation of CHPV-G glycoprotein pseudotyped lentivirus

The established HIV-1-based self-inactivating lentiviral vector system (Coleman et al., 2003) was used to generated lentivirus pseudotyped with the CHPV G

glycoprotein. The CHPV G gene was cloned into the pHEF-VSVG envelope plasmid (Addgene plasmid 22501) to give pHEF-CHPVG. HEK 293T cells grown to approximately 90% confluence in T75 cm2 tissue culture flasks were transfected with 7.1 µg pNHP (Addgene plasmid 22500), 3.5 µg pTYF-1xSYN-EGFP (Addgene plasmid 19973) and 2.8 µg of either pHEF-VSVG or pHEF-CHPVG. Virus was harvested 24 hours and 48 hours post-transfection and pooled.

2.1.6. Cryopreservation of cell lines

Cells were treated with trypsin/versene as described in section 2.1.2. Following addition of complete growth medium, the cells were transferred into falcon tubes and centrifuged at 1,300 rpm in a benchtop centrifuge (Sorvall Legend) at 4°C for 5 minutes. The supernatant was then discarded and the cells resuspended in an appropriate volume (1 ml per T75 cm2 tissue culture flask) of freezing medium, comprising of 90% FBS and 10% dimethyl sulfoxide (DMSO) (Sigma, USA). Aliquots were transferred into cryovials and placed in a Mr Frosty freezing container (Thermo Scientific, USA) which was immediately placed at -70°C. For long-term storage (>1 month), vials of cells were transferred to liquid nitrogen after 24 hours at -70°C.

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