Galpin/DA/N2 IIIBenv 
individual producer cell clone, DA/N2 IIIBenv. 2C3, was selected using the above 
criteria. 
2.3.1 Characterization of DA/N2 IIIBenv.2C3 Producer Cell Line (PCL) 
( 1 ) Numbers of Transduced Copies Per Cell: 
Southern blot analyses of DA/N2 IIIBenv. 2C3 DNA probed with neo r (obtained 
from N2 IIIBenv) indicate the presence of a single full length proviral copy per 
cell. An additional band consistent with integration of a rev proviral copy was 
also observed. 
(2) Expression of HIV- 1 EIIBenv/rev: 
The N2 IIIBenv retroviral vector construct necessarily encodes the genes for both 
HIV-1 IIIBenv and rev as rev is required for env expression. Because HIV-1 
IIIBrev is encoded by two separate exons, a splicing event is required for the 
generation of rev mRNA. As shown in Figure 2, when N2 IIIBenv is introduced 
into a retroviral packaging cell, spliced and unspliced RNA species are produced. 
Because both spliced and unspliced RNA's contain the packaging signal, both 
species can be packaged within the PCL to produce either HIV-1 IIIBenv/rev 
virions or HIV-1 IIIBrev virions. The full length RNA will deliver and express 
both HIV-1 IIIBenv and rev proteins, whereas the spliced rev RNA has the 
capacity to express only HIV-1 IIIBrev. In order to assess the relative frequency 
with which the rev and env/rev vectors would be expected to be delivered in vivo , 
mouse BC10ME cells were transduced at multiplicity of infection (MOI) of 1 and 
10 in tissue culture. Southern blots of DNA from pooled transductants showed 
that the two provectors were present at a ratio of approximately 1:1. 
To examine HIV-1 env protein production in transduced cells, increasing amounts 
of HIV-IT (V) were used to transduce human HT-1080 cells. Forty-eight hours 
after transduction, cell lysates were prepared and analyzed by Western blotting 
using an anti-HIV- 1 env-specific monoclonal antibody (Figure 3). Both full 
Recombinant DNA Research, Volume 17 
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