the flask (39). The flasks will be incubated at 37°C for six hours at which 
time the medium will be changed. This procedure will be repeated up to three 
times. After 24 to 48 hours medium containing 300 ug/ml G418 will be added 
directly to the flask and the cells will be grown and subcultured for 7 to 14 
days in G418 containing medium. The G418 concentration may be raised to 1 
mg/ml depending on the health of the culture. 
4. Tests on the transduced tumor population. Following transduction, 
growth and selection of the tumor populations the following tests will be 
performed on the tumor prior to injection into patients. 
1) Cell viability will be greater than 70% as tested by trypan 
dye exclusion. 
2) Sterility will be assured by testing for aerobic and 
anaerobic bacteria, fungus and mycoplasma. 
3) S+/L- assay must be negative. 
4) Southern blot or PCR analysis will be run on the transduced 
tumor to assure that proviral sequences are present. 
5) TNF protein assay to assure the production of TNF. Cells 
must be producing at least 100 pg TNF/10 6 cells/24 hours. 
The S+/L- assay, the 3T3 amplification assay and the polymerase chain 
reaction assay are detailed in Appendix A. 
5. Injection of tumor cells . Gene-modified tumor cells will be 
harvested from the culture flasks by exposure to 0.25% versene (EDTA) for 10 
minutes. The cells will be washed three times by suspension in 50 mis normal 
saline and centrifugation. The final cell pellet will be suspended in normal 
saline and counted. 2 x 10 8 viable cells in 1 ml normal saline will be 
injected subcutaneously just beneath the skin in the anterior mid thigh and 
the overlying skin marked with a tatoo dot. If 2 x 10 8 cells are not 
available, fewer may be given but not less than 2 x 10 7 cells will be 
injected. About 3 cm lateral or vertical to this injection the patient will 
receive two intradermal injections (separated by 1 cm) of 2 x 10 7 gene 
modified tumor cells in 0.1 ml normal saline and these sites also marked by a 
Recombinant DNA Research, Volume 15 [71] 
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