1985) (Table 1. Fig 8). When subsequently assayed, however. Miller, et al.. 
(1986) found that all but one neomycin phosphotransferase (Neo R ) producing N2 
virus infected PA-12 clones produced helper virus (Table 2). N2 is a Moloney 
based vector with a Mo-MuLV long terminal repeat (LTR) promoted Neo R gene 
replacing most of the gag , the pol_ and the erw genes, with an intact packaging 
sequence (Keller et al., 1985) (Fig 9). 
The level of helper virus was very low (about 200 focus forming units 
(FFU) ml compared with cells productively infected with amphotropic helper 
(106-10 7 FFU/ml), but was clearly detectable. The one clone which initially did 
not produce helper, did so after three weeks of culture. These results were 
reproduced with N2 producing clones that were generated with repeat N2 virus 
infection of PA-12 cells. It was postulated (Miller et al., 1986) that the 
frequent, if not unavoidable, co-production of helper virus upon infection of 
PA-12 cells with N2 virus is the result of a recombination event between 
identical 5'ends of Mo-MuLV and N2, comprising about 500 bp of N2 downstream of 
the packaging signal, including the gag sequences (Fig. 9). Evidence to 
D 
support this hypothesis was generated by the construction of a Neo vector pLNL 
(Miller et al., 1986) (Fig 9) lacking the gag region. PA-12 cells infected with 
this virus produced pLNL to high titers but did not produce helper (Table 2). 
Construction of PA - 317 
In light of these findings, several new designs for packaging cell lines 
were constructed (Fig 10). The one that was chosen, PA-317 contained several 
mutations in addition to the deletion of the packaging signal (Miller and 
Buttimore, 1986). Helper virus production was not detected, even with vectors 
found to transfer packaging function to PA-12. 
The 3' LTR of pPAM (Miller et al., 1985) (Fig 10) was replaced by the late 
SV40 polyadenyl ation signal, constructing pPAM2. In addition to the 
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