Helper-Free Rrtrt t lnd Vector* 
SD 
SA 
pSDHT 
K|TPj 1 
1 1 Hhfr trI 
L. 
P u H 
so 
9 
m 
pN2 
r- — > ^ 
|S LTRff— 
, / 
< 
1 nao 
pPAM 
• / 
so •/ 
i 
1 
po* 
SA 
1 . 
p Lm i 
pLNL2 
• \ 
nao | 
|5Ttr 
Rf.2. Viral vector*. The DHFR-virus plaimid pSDHT *11 constructed as followt. A HindHI to Nool fragment from 
the plasmid pFR400 (28) that contains the coding region for a metbotreaate-resistant DHFR gene was inserted in place 
of the gp52 envelope gene of the SFFV A -L clone of spleen focus-forming virus (37) between the Xmalll and Clal sites. 
The mRNA encoding DHFR is presumably a spliced message, as is the SFFV envelope mRNA. The Neo-virus plasmid 
pN2 was constructed by inserting the coding region for neomycin resistance (30) between the long terminal repeats 
(LTRs) and viral replication signals from Mo-MLV (13). .The plasmid pPAM has been described (4) and was used to 
make the PA 1 2 retrovirus packaging cell line. pPAM lacks the signal required for RNA packaging into virions, or psi 
site, but encodes all of the proteins required for viral replication. pLNL2 was made by inserting a Bglll to Sail fragment 
from the plasmid Tn5, that contains the coding sequences for neomycin phosphotransferase, into the BamHI site of a 
Mo-MLV-bascd retrovirus vector, after addition of a BamHI linker to the Sail site of the Neo insert. The overlap in the 
gag region of pN2 and pPAM, and the area of the psi site deletion in pPAM as compared with pN2 and pLNL2 are 
shown by dashed lines. SD, splice donor; SA, splice acceptor. 
the existence of ceils in the population which 
no longer express the amphotropic envelope 
glycoprotein, or alternatively, that the cells in 
general do not express enough envelope glyco- 
protein to block all of the amphotropic virus 
receptors. Most important in relation to our 
scheme for producing helper-free vectors 
(Figure 1), PA 12 cells were efficiently 
infected with virus generated from Psi-2 cells, 
and Psi-2 cells were efficiently infected with 
virus generated from PA 1 2 cells. Thus expres- 
sion of proteins required for retrovirus encap- 
sidation in the packaging cell lines does not 
affect entry of virions generated in the alter- 
nate packaging line. This is important because 
if expression of the packaging proteins did 
adversely affect virus entry, we might inad- 
vertently select for vector-infected packaging 
cells expressing lower amounts of the proteins 
required for retrovirus encapsulation, and 
which would presumably produce lower titer 
virus. We conclude that the strategy outlined 
in Figure 1 is a viable method for rapid 
production of retrovirus vector producing cell 
Table 1. Apparent Titer (CFU/ml) of Replication-Defective Viruses Rescued Following Transfection of Packaging 
Cell Lines, as Measured on Various Cell Lines 
Recipient cells 
Transfected 
DNA 
Transfected 
cells 
Uninfected 
NIH3T3 
Mo-MLV- 
infected 
NIH JT3 
AM-virus- 
infected 
NIH 3T3 
Psi-2 
(NIH 3T3) 
PA12 
(NIH 3T3) 
Neo- virus 
PA 12 
10* 
10* 
<10 
10* 
2 x 10* 
Psi-2 
3 x 10* 
<10 
4 x 10* 
<10 
« x 10* 
DH FR- virus 
PAI2 
6 x 10* 
5 x 10* 
20 
7 x 10* 
2 x 10* 
Psi-2 
6 x 10* 
<10 
4 x 10* 
<10 
8 x 10* 
Recombinant DNA Research, Volume 12 
[363] 
