Galpin/DA/N2 IIIBenv 
the production of full length and spliced RNAs (vector genomes), which are encapsidated 
into non-replicating vector particles. When transduced into target cells, the full length 
provector has the capacity to produce both HIV-1 IIIB env and rev proteins, whereas the 
spliced vector can produce only rev. 
Rather than directly transfecting the vector into the packaging cell line, the N2 
IIIBenv vector was transduced into the canine packaging cell line (DA). This procedure 
was favored, as transduction generates a defined proviral integration event and leads to 
higher titer producer cell lines than those generated by transfection 35 . 
Amphotropic PCLs are blocked for transduction by amphotropic vector as the cell 
receptor is occupied (a process known as "viral interference"). To overcome this 
transduction blockage, vectors containing alternative envelopes were first generated. One 
such alternative is the VSV G-protein, which binds to cell receptors other than the 
amphotropic receptor. The VSV G-protein is incorporated into the surface of a retroviral 
vector in a process termed "pseudotyping". VSV G-pseudotyped retroviral particles can 
efficiently transduce a wide array of cell lines 36 . 
A human embryonal kidney cell line 293 37 containing MoMLV gag/pol (termed 293 
2-3) was cotransfected with N2 IIIBenv provector and the plasmid, MLP-G, which 
expresses the VSV-G envelope protein (constructed at Viagene). The VSV-G 
pseudotyped N2 IIIBenv retroviral particles were used to transduce the canine packaging 
cell line (DA), and individual producer cell clones resistant to the antibiotic, geneticin 
(G418), were isolated. 
The clones were screened for maximal N2 MBenv vector production as determined 
both by their neo r titer and their ability to transduce and express HIV-1 IIIBenv in a 
human fibrosarcoma cell line, (HT-1080), as assessed by Western blot analysis. An 
35 Miller, et al., Somol. Cell Mol. Genet., 12: 175-183, (1986). 
36 Emi, et al.,7. Virol., 65:1202-1207 (1991). 
37 ATCC#CRL 1573. 
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