Preparation of the retroviral vector supernatant (RVS) 
The S3A producer cell line was seeded into tissue culture plates and grown 
to near confluence in DMEM medium containing 10$ fetal bovine serum (D^ q) an( j, 
since the SAX vector contains the Neo R gene. 1 mg/ml G418. Fresh medium 
without selective agent was added and collected 20 to 22 hours later. This 
supernatant, which contained viral particles carrying the retroviral vector, 
was filtered through a 0.22 micron cellulose acetate filter, frozen in 40 ml 
aliquots in conical 50 ml polypropylene tubes, and stored at -80°C until use. 
Other than removal of cells by the filtration process, no other purification of 
the RVS was performed. The S3A RVS used for the primate studies reported below 
was packaged in PA-12 (see Section I.B.l .b.(2)) and contained as a result 
approximately 0.1% contaminant with murine replication-competent virus (see 
Section I.B.2.C.). 
Samples of RVS are tested for contamination by aerobic and anaerobic 
bacteria, mycoplasma, and other viruses. No studies designed to detect VL30 
RNA (an endogenous retroviral -1 ike transcript found in mouse cells), other 
nucleic acids or proteins are contemplated at present. 
(c) If co-cultivation Is employed, what kinds of cells are being 
used for co-cultivation? What steps are being taken (and 
assays used with their sensitivity) to detect and eliminate 
any contaminating materials? Specifically, what tests are 
being done to assess the material to be returned to the 
patient for the presence of live or killed donor cells or 
other non-vector materials (for example. VL30 sequences) 
originating from those cells? 
Not applicable 
(d) If methods other than those covered by (a) -(c) are used to 
Introduce new genetic Information Into target cells, what 
steps are being taken to detect and eliminate any 
contaminating materials? What are possible sources of 
contamination? What is the sensitivity of tests used to 
monitor contamination? 
Recombinant DNA Research, Volume 12 
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