Cell Lines and Viruses

  1. CSL503 fetal ovine lung cells (CSL Ltd, Parkville, VIC) were frozen as a cell bank at doubling 41. Cells were grown in minimum essential medium (MEM) supplemented with nonessential amino acids (0.1 mM), glutamine (2 mM), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) (28 mM) (all from Gibco; Invitrogen Corp, Carlsbad, CA), penicillin/streptomycin (50 IU/50 pg/L) (Gibco/BRL), and fetal bovine serum (FBS) (10%) (Thermo Electron Corp, Melbourne, Victoria, Australia).
  2. PC3 human prostate cancer cells (ATCC CRL-1435) for testing virus potency were frozen down as a working cell bank (1-2.5 x 106 cells/vial) at doubling 18 and used within five passages. For cell growth, RPMI with glutamine and without NaHCO3 (Gibco/BRL), made according to manufacturer's instructions, was supplemented with 10% FBS and penicillin/streptomycin (50 IU/50 pg/L). Typically, cells were passaged twice per week at a split ratio of 1:5 by treating them lightly with trypsin.
  3. Recombinant virus OAdV623 contains the wild-type genome with a gene cassette inserted in a nonessential site (III) between the E4 and RH transcription units (2). The gene cassette comprises the PSMA enhancer and rat probasin promoter (the combination provides prostate cell-specific expression) linked to the E. coli purine nucleoside phosphorylase coding sequence and bovine growth hormone polyadenylation signal (8). OAdV220 contains a similar cassette in which the RSV promoter is used. In this case the cassette is inserted in site I between the pVIII and fiber genes (5).
  4. OAdV623 formulated in Tris sucrose PEG buffer containing 10 ||M cationic lipid CS087 (not commercially available) is known as FP253. CS087 is a Tris-conju-gated cationic lipid (trilysine-capryloyl-tris-trilaurate ditrifluoroacetate [T-shape]; empirical formula: C66H130N8O10-2C2F3O2; molecular weight: 1421.81) (12). Lipid formulation may enhance virus infectivity in cell types that lack the viral receptor (13). FP253 is stored at -80°C in a minimum volume of 200 |L in 2-mL borosilicate vials with snap-sealed rubber stoppers.
  5. A reference batch of OAdV623 was set aside in 25 |L-aliquots and used as a standard in gel analysis and assays of potency and infectivity. Compiling results for the reference batch in these assays made it possible to monitor interassay variation and the long-term performance of individual assays.
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