enzyme called neophosphotransferase (NPT) vhich Inactivates G418, a neomycin- 
analogue that kills eukaryotic cells. Therefore, cells that express the NeoR 
gene can be detected by their continuing growth when incubated in toxic levels of 
G418. 
2 . Retroviral infection trotocol used for successful eene insertion 
into TIL 
Human TIL were cultured at 1 x 10^ cells/ml in T-75 flasks in their standard 
growth medium (AIM-V (Gibco) with 1000 U IL-2/ml, 0.5% penicillin/streptomycin, 
0.1% Fungizone and 1% glutamine). Cell culture supernatant containing the N2 
retroviral vector, amphotropically packaged in the PA317 cell line at a titer of 
5 X 10^, was added directly to the medium of the culture flask at a 1:1 (vol:vol) 
ratio. Protamine sulfate, a polycation known to facilitate the binding of 
retroviruses to cell surface receptors, was added at 5 This infection 
procedure was repeated a second time 18 hours later. The following day, G418 was 
added at a concentration of 0.3 mg/ml .and the cells were cultured under this 
selection pressure for 7 days. 
3. Efficiencv of retroviral infection and exnression of the vector in 
The N2- infected G418- selected human TIL were evaluated for the expression of 
the NeoR gene using two different methodologies; 1) their ability to proliferate - 
in G418 using ^ [H] -thymidine incorporation and 2) the presence of intracellular 
NPT using an enzymatic assay. 
To assess proliferation in G418, 1 x 10^ TIL/well in a volume of 200 /xl of 
medium were cultured in 96 -well flat bottom plates with different concentrations 
of G418 (0, 0.1, 0.3, 0.6 and 1.0 mg/ml). After 72 hours, ^ [H] -thymidine 
incorporation was measured. TIL with no vector did not proliferate in 0.3 mg/ml 
(or higher) G418, while N2-containing cells continued to proliferate even in 1.0 
mg/ml G418 (Figure 2) . 
To further document the expression of the NeoR gene, TIL were assayed for 
the presence of the neomycin phosphotransferase (NPT) enzyme (9). Figure 3 
presents the findings in three patients comparing uninfected TIL, N2- infected 
G418- selected TIL, and N2 producer cells. Infected TIL show a high level of NPT 
activity while no activity is detected in uninfected cells. 
Therefore, we have clear evidence that TIL can be infected with the 
retroviral vector N2 with essentially 100% of the cells expressing the NeoR gene 
after selection. 
Additionally, we have assayed TIL for the presence of murine ampho tropic 
helper virus 7 days after infection with N2 supemate . .Control and infected cells 
from the three patients studied were negative by S+/L- assay (the sensitivity of 
the S+/L- assay is 1 viral particle/ml) . 
Recombinant DNA Research, Volume 14 
[7] 
