polyadenyl ation. the deletion of the 3'LTR removed the site of initiation of 
second-strand DNA synthesis and the 3' R region required for translation of 
reverse transcriptase during first strand DNA synthesis (Varmus and Swanstrom. 
1985 ). Sequences required for viral integration (Panganiban and Temin. 1983) 
upstream from the 5 ' LTR viral enhancer were removed, making pPAM3. A pBR322 
clone of pPAM3 was used to make the helper-free packaging line PA-317. 
The constructs were introduced into NIH 3T3 TK- cells by cotransfection 
with the herpes simplex thymidine kinase gene (Colbere-Garapin et al.. 1979) a 
a selectable marker. TK + colonies were screened for production of replication 
competent virus using the S + L’ assay and for their ability to package a 
standard retroviral vector containing the HPRT gene previously described. No 
helper virus was detected from any of more than 70 clones analyzed. Roughly 
equivalent titers were obtained from the cell lines containing pPAM. pPAM2 and 
pPAM3. The best pPAM3 transfected clone was named PA-317 and was henceforth 
used In all of the transfection experiments. 
Assay for helper virus with N2 as previously done yielded no detectable 
Neo PA-317 clones which produced helper virus on S L assay (Table 3). 
Neither did NIH 3T3 cells Infected with virus from these lines assayed more 
p 
than two weeks after Infection. A population of over 100 independent Neo 
clones was also negative for helper virus when assayed by S + L . 
Recombinant DNA Research, Volume 12 
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