activity, Western blot and Southern blot. The titer of 1 x 10 6 cfu/ml for the Psi-cre 
producer of N2-SV-GC was based on the number of G418 resistant colonies of infected NIH 3T3 
cells. The titer of the MFG-GC vector was estimated by Southern blots of non-selected 3T3 
cells infected by the N2-SV-GC vector. The MFG-GC viral producer line clone (GC-Psi-cre 
#4) with a titer of 1 x 10 6 was chosen for use in the experiments described in Sections IV: 
C,D and E. 
The amphotropic producer line of MFG-GC was developed by cross infection of the Psi- 
crip amphotropic packing cell line with viral supernatants from the ecotropic GC-Psi-cre 
#4 producer. Multiply infected (4x) Psi-crip cells were plated at limiting dilution in 96 
well plates. Of twenty-two clones, twelve were expanded and were assayed for enzyme 
activity. Supernatants were collected and assayed for transduction efficiency based upon 
enzyme activity in 3T3 targets. A single clone, cc-2, yielded superior supernatants and 
was chosen for quantity production (Figure 25). This producer exhibited slow cell growth 
and contact inhibition, at the same time yielding consistently high titer supernatant. From 
one batch of confluent cells, supernatants were collected every day for almost three weeks 
without trypsinization and without measurable decline in viral production (Figure 26). 
The cc-2 producer was titered as follows. Target 3T3 cells were transduced with 
varying volumes of a representative supernatant. The infected cells were harvested, and 
DNA was extracted and analyzed by Southern blot hybridization using a probe for GC. A Beta 
scanner was used to quantitate the amount of the vector fragment for each target cell DNA 
sample. These radioactivities were compared to the radioactivity from DNA prepared from 
a cloned cell line containing a known number of copies (3 copies) of the vector sequence. 
(The copy number of this infected clone was determined on a separate blot using EcoRI 
restriction to produce a unique band for each integration site. An identical 3-band pattern 1 
was observed in 5/5 subclones.) A linear relationship was observed between copy number in 
the target 3T3 cells and amount of supernatant used for infection (Figure 27). The average j 
copy number of the target cells multiplied times the number of target cells present during 
infection therefore provides a minimum estimate of the number of virus particles present 
in the volume of supernatant used. The linearity of the relationship between volume of 
supernatant and average copy number supports the validity of this approach for estimation 
of titer. Since an average copy number of 35 was observed for infection of 3 x 10 5 cells i 
with 1 ml of supernatant, the titer of the supernatant was at least 35 x 3 x 10 5 /ml = 10 7 | 
infectious virus particles/ml. | 
The absence of helper virus was verified by coculture of the producer cc-2 cells with 
BAG infected 3T3 indicator cells. Cocultures were split 4 times in the presence of 8ug/ml 
polybrene over a period of three weeks. Supernatants prepared from these cocultures were 
used at full strength to infect 3T3 target cells. The supernatants from BAG/cc-2 cocultures 
were negative for producing Xgal staining cells or for G418 R cells among infected targets. 
Control cultures of BAG/3T3 cells with pA3 1 7 producer cells, from which helper activity had 
been induced by previous coculture with an ecotropic packaging line, were positive in this 
series of assays. This assay is extremely sensitive for detection of helper virus, 
particularly when multiple passages over time in the presence of polybrene are incorporated. 
The viral producer line of R-GC will be developed by transfection of the psi-crip 
packaging line and selection of high producers as outlined above. Neither ping-pong 
infection nor cross-infection will be used in developing the R-GC vector. The VPL of R-GC 
will be characterized as to its ability to reproducibly produce a high titer viral 
supernatant. The titering methods have been described in earlier sections of this 
application. The selected VPL will be placed in cultures and expanded to provide at least 
25 cryovials. The VPL will be brought into culture and a minimum of 100ml of viral 
supernatant produced and tested for titer. This test will be repeated on separate mini- 
production runs. Our experience with different producers is that the cell line remains 
stable for many months. Tests will also be conducted to assess the stability of the frozen 
viral supernatant. Again, our experience is that the supernatant is stable for many months. 
JC 
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Recombinant DNA Research, Volume 17 
