VII. APPENDICES: 
APPENDIX A: PRE-CLINICAL STUDIES 
I. VECTOR CONSTRUCTION 
The ADV-tk vector was prepared by inserting HSV-tk into the plasmid pADL.I/RSV 
which contained the Rous sarcoma virus long-terminal-repeat promoter (RSV-LTR) to 
generate pADL.I/RSV-tk (Fig 1, Chen, et al. , 1994). Recombinant adenovirus was produced 
by co-transfecting 293 cells with pADL.I/RSV-tk and a plasmid, pJM17, containing the 
adenovirus genome. The 293 cells are transformed human kidney cells that contain the El 
region of the adenovirus genome. When 293 cells were co-transfected with pADL.I/RSV-tk 
and pJM17 replication-defective adenovirus was produced by homologous recombination 
(Graham and Prevec, 1991). Virus titer was determined by optical absorbance at 260 nm. A 
replication-deficient adenovirus vector carrying the E. coli b-galactosidase gene under 
control of the RSV-LTR (ADV-(3gal) was provided by M. Perricaduet (Institut Gustave 
Roussy, Centre National de la Rechercher Scientfique, Villejuif, France) and was used as a 
control vector (Stratford-Perricaudet et al., 1990). 
II. IN VITRO EXPERIMENTS 
Quantitative Transduction of C 6 Glioma Cells In Vitro by Recombinant Adenovirus 
Adenovirus transduction of C 6 cells in vitro was tested using ADV-pgal. The C 6 cell line 
was originally derived from a rat glial tumor induced by N-nitrosomethylurea (Benda et al., 
1968). The C 6 glioma cells, purchased from the American Type Culture Collection (ATCC), 
were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% 
fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml) in 5% C0 2 at 37°C. 
C 6 cells (5 x 10 5 ) were plated on 1.5 cm diameter wells, transduced with ADV-bgal at 
various viral doses of 0, 125, 500, and 2000 M.O.I. and then stained for bgal activity 48 
hours later (Sanes et al., 1986). As demonstrated by X-gal staining of bgal activity, ADV- 
bgal efficiently transduced the C 6 glioma cells in vitro at a multiplicity of infection ( M.O.I.) 
greater than 125 (Fig. 2). 
Transduction of Human Glial Cells In Vitro by Recombinant Adenovirus 
Several cell lines derived from human neural tumors were tested for their capacity to 
be transformed with adenovirus vectors. Cell lines were purchased from ATCC and cultured 
using the conditions above. The cells lines and their descriptions are: 
A-172 -glioblastoma 
H4 - neuroglioma, brain 
SW-1088 -astrocytoma 
U-373 MG - glioblastoma, astrocytoma, grade III 
Cells were transduced with ADV-bgal at M.O.I.s of 200, 500, 1000, and 4000 and stained for 
bgal activity 48 hours later. The vector transduced all 4 cell lines at different efficiencies 
(Fig. 3). 
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