Miller et aL 
lines, which avoids the phenomenon of viral 
interference by using retrovirus packaging 
lines with different host ranges. 
We next tested several vector-infected 
packaging cell clones derived using the 
method outlined in Figure 1 for the titer of 
viral vector produced and for the presence of 
helper virus. Since the amphotropic packag- 
ing line PA1 2 produced virus at a titer similar 
to Psi-2 cells (Table 1) (2, 4) but has a wider 
host range (29, 32), we have chosen to test 
infected PA 12 cell clones generated as shown 
in Figure 1A. DHFR-virus and Neo-virus 
production was assayed by using NIH 3T3 
(TK - ) cells. Of the six DHFR-virus-infected 
PA 12 cell clones (Table 2A), three produced 
DHFR-virus at titers of over 10* per ml of 
medium exposed to the cells. None of these 
clones produced detectable helper virus using 
a modified S + L“ assay (4), a sensitive assay 
for viruses capable of rescuing other viruses, 
i.e., “helper” viruses. We also tested for helper 
virus production by infecting NIH 3T3 cells 
with 1 ml of virus supernatant from DHFR- 
virus producer clone 6 followed by assay of the 
infected cells for DHFR and helper virus 
production after three weeks of culture. Virus 
production from the infected cells was not 
detected, indicating that no helper virus was 
present. This pattern is typical of experiments 
we have performed with many viral vectors 
(data not shown). Although all clones are 
DHFR* and secrete DHFR-virus, some of the 
clones only produce low-titer virus. This find- 
ing could be explained by heterogeneity in 
production of proteins required for virus repli- 
cation among cells in the PA 12 population, as 
was hypothesized above to explain the incom- 
plete block in these cells to infection by 
amphotropic pseudotype virus. Alternatively, 
there may be mutations in the integrated 
DHFR-viruses which inhibit virus replication 
but do not inhibit DHFR expression. 
In contrast, five Neo-virus-infected 
PA 12 cell clones (Table 2B) all produced 
Neo-virus at high titer, and all but one pro- 
duced helper virus. The level of helper virus 
production was very low in comparison to cells 
Tabic 2. Virus Production from Vector-Infected 
PA 12 Cells' 
A. 
DHFR-vinis- 
DHFR-virus 
Helper 
infected PA 12 
titer 
titer 
cell clone 
(CFU/ml) 
(FFU/ml) 
1 
2 x 10* 
<1 
2 
2 x 10* 
<1 
3 
2 x 10* 
N.D.* 
4 
1 x 10* 
N.D.* 
5 
4 x 10* 
N.D.* 
6 
4 x 10* 
<1 
B. 
Neo-virus (N2) 
Neo- virus 
Helper 
-infected PA 1 2 
titer 
titer 
cell clone 
(CFU/ml) 
(FFU/ml) 
1 
5 x 10* 
20 
2 
4 x 10* 
<1 
3 
2 x 10’ 
40 
4 
I x 10’ 
190 
5 
3 x 10* 
200 
C 
Neo-virus (LNL2) 
Neo- virus 
Helper 
-infected PA 12 
titer 
titer 
cell clone 
(CFU/ml) 
(FFU/ml) 
10 
5 x 10* 
<1 
11 
<10* 
<1 
12 
10* 
<1 
13 
10* 
<1 
14 
10* 
<1 
13 
<10* 
<1 
16 
2 x 10* 
<1 
*Virus titer was measured in medium exposed to cells for 
24 h. 
*N.D., not determined. 
productively infected with amphotropic 
helper virus (10*— 10 7 FFU/ml by using the 
S*L” assay), but is clearly detectable. The 
clone which initially did not produce helper 
virus eventually produced helper virus after 
three weeks of culture. In addition, both 
helper virus and Neo-virus production from. 
NIH 3T3 cells was detected 2 weeks after 
infection with virus harvested from the Neo- 
virus producer cells. We repeated the entire 
procedure for generating Neo-virus producer 
cell lines and screened these clones for Neo- 
virus and helper virus production. High-titer 
Neo-virus (2-5 x 10‘ CFU/ml) was secreted 
by four of five clones analyzed, while one of 
the five did not produce Neo-virus (<100/ 
ml). Three of the four Neo-virus-secreting 
clones also secreted helper virus (data not 
[364] 
Recombinant DNA Research, Volume 12 
