so 
pSDHT 
pN2 
pPAM 
pLNL2 
SA 
— I— jdhlr j - foURj 
Ikb 
SD , s. 
SD 
j5 LTR 
/ / 
/ / 
000 
I 8*9 
po» 
SA 
•nv 
J {3'LTH( 
•\ 
I \ 
EsiyOEZl- 
Viral vectors. The DHFR-virus plasmid pSDHT was constructed as follows. A Hindi! I to Ncol fragment from 
the plasmid pFR400 (28) that contains the coding region for a methotrexate-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 XmalH and Clal sites. 
The mRNA encoding DHFR is presumably a spliced message, as is theSFFV envelope mRNA.TheNeo-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 (IS). 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 TnS, that contains the coding sequences for neomycin phosphotransferase, into the BamHI site of a 
Mo-MLV-based 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. 
Fig. 9. (Miller et al . , 1986) 
[180] 
Recombinant DNA Research, Volume 12 
