infecting and replicating in human and chicken cells, a property of amphotropic 
viruses (Hiller et al.. 1985). 
To prevent PAM from being packaged, viral sequences shown to be necessary 
in cis for encapsidation were deleted. It has been shown (Shank and Linial. 
1980; Mann et al . , 1983) that the packaging signal for Mo-MuLV RNA lies between 
the Bal I and Pst I sites at the 5' end of the virus between viral nucleotide 
positions 214 and 565 (Shinnick et al., 1981). The Bal I site Is 6 bp 
downstream from the splice donor site and the Pst I site Is 55 bp upstream from 
ATG for Pr65 gag. pAM was digested with Bal I and Pst I, deleting the 350 base 
pair segment between these two sites (Miller et al., 1985) (Fig. 7). The Bal 1 
site was llnkered with Pst I polylinker and joined to the 5' Pst I site after 
partial Pst I digestion, making pPAM. 
PA-12 
pPAM was co-transfected into NIH 3T3 TK" cells (Shank and Linial, 1980) 
along with the herpes simplex thymidine kinase gene as a selectable marker at a 
1:20 ratio to pPAM (Graham and van der Eb. 1973; Wigler et al., 1979; Corsaro 
and Pearson. 1981: Miller et al.. 1985). The TK + clones were surveyed for 
their ability to package a wel 1 -characterized replication-defective virus pLPL 
encoding the hypoxanthine phosphorlbosyl transferase (HPRT) gene (Miller et al.. 
1983). One of the TK + transfected cell clones, which efficiently packaged pLPL 
as monitored by the conversion of HPRT’ cells to HPRT + after transient 
transfection with pLPL. was designated PA-12. PA-12 was the packaging cell 
line into which the SAX vector was transfected (see section on structure of 
cloned DNA In this document). 
Generation of helper virus contaminant 
At the time of construction. PA-12 did not produce helper virus when 
transfected with a variety of Mo-MuLV based DHFR vectors (Miller et al.. 
Recombinant DNA Research, Volume 12 
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