GaJpin/DA/N2 IIIBenv 
treated with IUdR and dexamethasone and tested negative for the presence of cryptic 
retroviruses. 
2.2.2 Construction of Packaging Cell Line 
The D-17 canine cell line was initially transfected with the plasmids pSCVIO 
and pFR400, which express MLV gag/pol and a methotrexate resistance marker, 
respectively, and an individual subclone selected which expressed high levels of MLV 
gag proteins. This clone was then transfected with pCMV env Jun Dra and pUT507 30 . 
The pCMV env am Dra plasmid encodes the MLV amphotrophic envelope gene 
(env am ) and the pUT507 plasmid provides a phleomycin dominant selectable 
resistance marker (phleo 1 )- 
After selection in phleomycin, individual colonies were isolated, expanded and 
tested for production of MLV gag and env am by Western blot analysis. A resulting 
packaging line clone, 4(15)SC1 #23, designated DA, was selected. 
The plasmids encoding the structural genes, pSCVIO and pCMV env am Dra, 
were constructed at Viagene, Inc. These plasmid constructs use the human 
cytomegalovirus (CMV) early transcriptional promoter 31 for structural gene 
expression. The MoMLV gag/pol was obtained from the MoMLV proviral plasmid, 
MLV-K 32 . The env am gene was obtained from the murine retrovirus MLV 4070A 
proviral clone 33 . The gag/pol and env lun constructs use the SV40 late transcriptional 
termination signal 34 . 
2.3 GENERATION OF THE PRODUCER CELL LINE (DA/N2 IIIBENV) USED 
TO GENERATE HIV-IT (V) 
The N2 IIIBenv retroviral vector, designated HIV-IT (V), is produced by introducing 
the N2 IIIBenv provector DNA into an amphotropic packaging cell line. This results in 
3 ^ Mulsant, et at, Somat. Cell. Mol. Genet., 14:243-252, (1988). 
31 Boshart, et al„ Cell, 41:521-530, (1985). 
32 Miller, et al„ Mol. Cell. Biol., 5:431-443, (1985). 
33 Chattopadhyay, et al., J. Virol., 39 .-777-791, (1981). 
34 Southem and Berg, J. Mol. App. Genetics, 2:327-341 (1982). 
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