HHp*r-Fre* Rrtwin* Vector* 
shown). Again, the Neo-virus-sccrcting clone 
which initially did not produce helper virus 
eventually produced helper virus after one 
month in culture, as measured by using the 
S*L" assay. Thus production of helper virus 
after infection of PA 12 cells with the N2 
Neo-virus is very frequent, if not unavoid- 
able. 
The 5' end of the N2 Neo-virus is identi- 
cal to that of Mo-MLV, and this identity 
extends into the gag region of Mo-MLV. Thus 
about 500 base pairs of N2 downstream of the 
virus packaging signal are also present in the 
packaging system DNA (Figure 2). A recom- 
bination event in this area that combined the 
5' end of N2 with the packaging system DNA 
would result in production of amphotropic 
helper virus. The virus that is secreted from 
the Neo-virus producer clones has the charac- 
teristics of an amphotropic virus, as deter- 
mined by its ability to infect both mouse and 
cat cells (data not shown). We tested the 
possibility that the common gag region of N2 
and the packaging system DNA were involved 
in helper virus production by constructing a 
plasmid called pLNL2 (Figure 2) that con- 
tains a Neo-virus which does not contain the 
gag region, but is otherwise similar to pN2. 
We used our technique for generating PA 12- 
derived cell lines that secrete the LNL2 virus, 
and screened these lines for secretion of Neo- 
virus and helper virus (Table 2C). Three of 
seven clones secreted more than 10 5 Neo-virus 
per mililiter of medium, and none of the clones 
secreted helper virus. This removal of the gag 
sequences from the Neo-virus N2 resulted in 
the elimination of helper virus production in 
infected PA 12 cells. 
DISCUSSION 
We have described an improved tech- 
nique for production of cell lines which secrete 
helper virus-free retroviral vectors. Starting 
with vector DNA, the method involves only 
one cloning step for isolation of the ceil lines 
and is thus relatively rapid. Clones can then be 
screened to insure that virus obtained is 
unrearranged and produced at high titer. 
Another factor which contributes to the utility 
of our method for producing helper-free virus 
is that viruses introduced by infection appear 
to be expressed more efficiently than viruses 
introduced by transfection (33). Thus, the 
titer of vector produced from infected lines is 
in general higher than that from transfected 
lines. In the case of HPRT-virus, we found an 
improvement of about 10-fold (3). 
It is not fair to assume that retrovirus 
vectors produced using packaging cell lines 
are always helper-free, thus screening the 
clones for the absence of helper virus is abso- 
lutely required. We have documented recur- 
rent helper virus production using the Neo- 
virus construct pN2, which could be explained 
by a recombination event involving homolo- 
gous sequences common to the N2 virus and 
the virus packaging system DNA. This region 
is not present in the Neo-virus LNL2, which is 
otherwise similar to N2. LNL2 does not gen- 
erate helper virus after infection of PA 12 
cells. The DHFR-virus SDHT contains the 
region of overlap, but does not generate helper 
virus after infection of PA 12 cells. However, 
this vector is based on spleen focus-forming 
virus, and the region of overlap can be esti- 
mated to be only 68% homologous with the 
packaging system DNA (34, 35), as compared 
with N2 where the homology is 100%. Thus 
recombination to yield helper virus should be 
much lower in the case of the DHFR-virus 
SDHT. 
Recombination to produce helper virus 
may occur at the DNA level during infection 
of the packaging cells with the vector or at the 
RNA level following secretion from the pack- 
aging cells. It has been shown that retroviral 
vectors without packaging signals can be 
incorporated into virions and transmitted at 
low frequency (19), and if the same were true 
of the packaging system RNA, then a virion- 
containing vector and packaging system RNA 
might allow recombination between the two at 
the RNA level during reverse transcription. 
We conclude that avoidance of overlap 
Recombinant DNA Research, Volume 12 
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