MILLER AND BUTT! MO RE 
Mol. Cell. Biol. 
pLPL2 
PN2 
spile# 
C (•donor 
pSDHT (uRfl— 
spile# 
accaptor 
i 
1 kb 
L . J 
FIG. 2. VinJ vectors. Selectable marten in these vectors were 
expressed by usin| transcriptional signals present in the viral LTRs. 
The Neo virus pN2 (11) and the DHFR virus pSDHT (16) have been 
described before. The HPRT virus pLPL2 is similar to the pLPL 
virus described previously (14) with two modifications. First, two 
GC-rich regions in the HPRT cDNA (10) that lie between the S' LTR 
and the start codon of HPRT in pLPL were removed. Sae\ was used 
to cut the HPRT cDNA 9 base pain S' of the start codon (10). and 
a Pit I linker was added, cleaved with Pul, and joined to the Pit I site 
present in pLPL at the start of the HPRT cDNA. Second, in addition 
to the normal retroviral packaging signal between the S' LTR and 
HPRT, a second packaging signal lies downstream of the 3’ LTR in 
pLPL. and this signal might allow packaging of RNAs initiating in 
the 3' LTR and extending into pBR322 sequences following trans- 
fection of pLPL into packaging cell lines. To avoid this possibility, 
the second signal was removed by cleavage at a Ball site just 3' of 
the 3' LTR. and an EcoRl to Ball fragment containing the HPRT 
virus was inserted in place of the Tet' gene of pBRJ22 between the 
EcoRl and P*u II sites. HPRT virus production following transfec- 
tion of pLPL2 into packaging cells was about threefold higher than 
that of pLPL. kb. Kilo base. 
present at both ends of pPAM3. A double recombination 
event is presumably quite r are; thus, it might be possible to 
generate helper-free virus by introduction of the N2 vector 
into PA.* 17 cells. 
This prediction was confirmed in the following experi- 
ment. The N2 virus was introduced into PA12 or PA317 
retrovirus packaging cells, and independent clones contain- 
ing the virus were isolated and screened for the production 
of Neo virus and helper virus. All of the PA12-N2 clones 
produced Neo virus, and all but one produced helper virus 
(Table 3). Helper virus production by this clone was de- 
tected after further passage of the cells. The entire experi- 
ment was repeated with a similar result. Similarly, most of 
the PA317-N2 clones produced Neo virus, but in contrast 
none of the clones produced helper virus (Table 3). Passage 
TABLE 1. Ability of clonal cell lines cotransfected with 
packaging constructs to package a retroviral vector (pl_PL2)* 
axumjet 
Highest 
virus titer 
(CFU/mi) 
% at dooes 
producing 
>20% of 
maiimiiBi 
tzter 
%of dooes 
producing 
do virus 
<<10 CFU/ml) 
Best 
dooe 
pPAM 
3 x 10* 
50 
13 
PA12 
pPAM2 
6 x 10* 
40 
40 
PA212 
pPAM3 
6 x 10* 
40 
29 
PA317 
pPAM4 
4 x 10* 
25 
50 
PA 405 
* Qooct were isolated from two separate tranifecboos. At least eight 
clones from each transfectioo were screened. 
TABLE 2. Vims production from packaging cells containing 
SDHT DHFR virus* 
Packaging 
cell line 
SDHT virus- 
infected cell 
done 
DHFR virus 
titer 
(CFU/ml) 
Helper 
titer 
(FFU/mlf* 
DHFR virus 
titer after 
helper virus 
infectioo 
(CFU/ml) 
PA 12 
1 
2 X 10* 
<1 
2 
2 x 10* 
<1 
3 
2 x 10* 
ND* 
4 
1 x 10* 
ND 
3 
4 x 10* 
ND 
6 
4 x 10* 
<1 
PA317 
1 
6 x 10» 
<1 
6 x 10* 
2 
6 x 10 5 
<1 
4 x 10* 
3 
1 x 10’ 
<1 
3 x 10’ 
4 
4 x 10* 
<1 
6 
3 x 10* 
<1 
6 x 10* 
7 
<1 x 10» 
<1 
4 x 10’ 
S 
5 x 10* 
<1 
9 
1 x 10 5 
<1 
10* 
* Packaging ceO Unci containing retroviral vectors were made by transfect- 
ing Pvi-2 cells followed by harvesting of virus 2 days later and infection of 
amphotropsc packaging cell lines as previously described (16). Independent 
vector-infected cell Uses were isolated following drug selection by using 
cloning rings. 
* FFU, Focus-forming units. 
* ND, Not determined. 
of two of the high-titer Neo virus-producing cell lines for 1 
month did not result in helper virus production, as measured 
by the S*L“ assay. In addition, NIH 3T3 cells infected with 
1-ml samples of virus from these lines did not produce helper 
virus or Neo virus when assayed more than 2 weeks after 
infection. Thus, helper virus production from PA317 cell* 
containing N2 virus was not detected, whereas production 
from PA12 cells containing N2 virus was always detected. 
Previous reports indicate that when N2 virus was 
TABLE 3. Vims production from packaging cells containing N2 
Neo vims* 
Neo virus 
Packaging 
ceil line 
N2 virus- 
infected 
ceil done 
Neo virus 
trier 
(CFU/ml) 
Helper 
trier 
(FFU /ml) 
trier after 
helper virus 
mfedjOG 
(CFU/ml) 
PA 12 
1 
5 x 10* 
20 
2 
4 x 10* 
<1 
3 
2 x 10’ 
40 
4 
1 x 10’ 
190 
3 
5 x 10* 
200 
PA317 
1 
8 x 10* 
<1 
2 X 10’ 
2 
1 x 10* 
<1 
3 
2 x 10* 
<1 
3 x 10’ 
4 
4 x 10* 
<1 
3 
2 x 10* 
<1 
6 
1 x 10* 
<1 
4 x 10’ 
7 
4 x HP 
<1 
1 x 10’ 
8 
6 x 10 5 
<1 
10 
2 x 10 s 
<1 
11 
1 x 10’ 
<1 
Population' 
6 x 10* 
<1 
* For experimental coodiootu, see Tabic 2, footnote m. 
* FFU. Focus-forming units. 
' A population of over 100 independent clones was analyzed to check for 
rare events leading to helper virus prod nelson. 
Recombinant DNA Research, Volume 12 
[ 371 ] 
