(b) If a virus, how Is It prepared from the DNA construct? In 
what cell Is the virus grown (any special features)? What 
medium and serun are used? How Is the virus purified? What 
is Its structure and purity? What steps are being taken 
(and assays used with their sensitivity) to detect and 
eliminate any contaminating materials (for example. VL30 RNA. 
other nucleic acids, or proteins) or contaminating viruses 
or other organisms In the cells or serum used for preparation 
of the virus stock? 
The protocol would be essentially Identical to the protocol developed for 
non-human primates described in Kantoff et al.. J Exp. Med ., in press (copy 
supplied in Appendix B). Specifications for equipment, chemicals, and 
biological agents in direct or indirect contact with patient tissues are 
provided In Appendix A. 
Preparation of ADA vector-containing producer cell line, S3A 
The SAX vector, in a pBR322 backbone, was transfected into the helper 
virus free 3T3 packaging cell line 1^2 (see Section I.B.l.b(2)) using the 
standard calcium phosphate mediated DNA transfer technique (Wigler et al.. 
1977) and then trans-infected into the helper-free amphotropic packaging cell li 
PA-12. Stably transformed clones were isolated by their resistance to 1 mg/ml 
G418. a neomycin-like antibiotic that is lethal for mammalian cells. These 
clones were then analyzed for viral particle production by serially diluting 
supernatant medium (from a 20 hour incubation on near-confluent plates) on 3T3 
cells. One ml of viral supernatant to be tested was added together with 8 
j^g/ml polybrene to 3T3 cells (5 x 10 4 inoculated 24 hours previously) in a 60 
mm petri dish. After a two hour incubation at 37°C, 4 ml DMEM plus 10% fetal 
calf serum (Djq) was added. Two days later, the medium was changed to D 10 plus 
1 mg/ml G418. Colonies were read 10-12 days later. The producer cell line 
with the highest titer was S3A: 2-10 x 10 6 Neo R colony forming units (cfu) per 
ml. This producer cell line was frozen in individual aliquots (Master Cell 
Bank) and stored in liquid nitrogen. 
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Recombinant DNA Research, Volume 12 
