APPENDIX A 
S+/L- Assay: The C3t fibroblast: line PG-4 (Haapala et. al . 1985) is used as 
the indicator cell line. Cells are maintained in McCoy's 5A medium with 5% fetal 
calf serum and 2mM L-glutamine and grown at 37*C, in 5% CO 2 . Twenty four hours 
prior to virus exposure, cells are plated on 6 well dishes, 1x10^ cells per well 
in 1 ml of medium. On the day of infection, 0.5 ml of DEAE Dextran (20 ug/ml in 
medium) is added to each well and incubated with cells for 30 minutes. Dextran is 
removed and 0.5 ml of test sample is added and incubated for 2 hours at 37*C. 
After 2 hours the test material is replaced with 2.5 ml of medium and incubated 
for 3 days. Foci of transformed cells are determined microscopically with a 10X 
objective and the focus - forming units per ml (ffu/ml) calculated. 
3T3 Amplification: 3T3 cells are maintained in Dulbecoo's modified Eagle's 
medium with 10% fetal calf serum and 2 mM L-glutamine; the cells are grown at 
37"C, in 5% C02- Cells are plated at 1x10^ cells per well In 6 well dishes twenty 
four hours prior to sample exposure. Medium is removed and 0.5 ml of supemate 
samples are added per well; polybrene (8ug/ml) is used to enhance viral 
infection. Coculivation with cells is performed overnight in a volume of 1 ml 
with polybrene (8 ug/ml). After infection the samples are removed and 2.5 ml 
fresh medium is added. Cells are split 1:10 when confluent and carried for 3 
weeks. At the end of the first and third week, when plates are confluent, fresh 
medium is added to the well and collected 20 hours later for analysis in the 
S+/L- assay. 
Polymerase Chain Reaction: DNA is isolated from both uninfected and 
transduced TIL cells using standard techniques (Maniatis et. al . ) and subjected 
to polymerase chain reaction. In this assay 1-5 ug of DNA is added to a 0.5 ml 
microcentrifuge tube containing 100 ul of 50 mM KCL, 10 mM Tris-HCl (pH-8.3), 1.5 
mM MgCl 2 , 0.01% gelatin, 200 uM each deoxynucleotide triphosphate, 1.0 uM 
envelope primer 1, 1.0 uM envelope primer 2 and 2.5 U of Taq DNA polymerase. All 
reagents, with the exception of TIL cell DNA and the envelope primers, are 
supplied in kits purchased from Perkin Elmer Cetus (GeneAmp kit) . The samples are 
then placed in a DNA Thermal Cycler instrument (Perkin Elmer Cetus) and subjected 
to 30 cycles of 3 minutes each of 94*C denaturation , 53“C annealing, and 72*C 
polymerization conditions. At the completion of the cycling, the amplified DNA 
sample is removed and analyzed by agarose gel electrophoresis followed by 
Southern blot analysis (Maniatis et al . ) using a radiolabeled DNA probe isolated 
from the envelope gene of the amphotropic virus 4070A. Final analysis for the 
presence of amphotropic envelope sequences is obtained by autoradiography of the 
Southern blot. 
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