(wavy lines): Unipliced (jij/pol) and spliced (en») RNA. both under the regulation of the LTR* 
(black boxes). In a DE vector the gag pol and env genes are replaced with exogenous genes A and 
B. Then, the norma) splicing mechanisms are used to generate mRNAi for genes A and B. with ex- 
pression of bosh regulated by the 5 ' LTR. 
Flgere 4. VIP vectors. VIP vectors rely only on the very J ' portion of the retronra) genome for 
their function. A selectable gene product (e g.. Neo*) is generated from a transcript regulated by 
the retroviral J ' LTR. Asecond gene of interest is insened distaHy. under the regulation of its own 
promoter, generating a separate, unique mRNA. Titers (viral particles'' ml) are listed it the right, 
showing wild type titers with those of two VIP vectors. N4 and N2. 
expression, i.e., the enhancer and pro- 
moter present in the 5 ' LTR. the poly- 
adenylation signal in the 3 ' LTR, and 
the intron splice sites encoded internal- 
ly. Such a DE type vector was first 
reported by Cepko, et al. (2), and used 
successfully in several studies (3, 7, 17, 
24. 27, 28). 
There are two major drawbacks in 
using DE vectors. First, the expression 
of the two genes introduced into DE 
vectors is dependent on the efficient for- 
mation of the two viral RNA species as 
shown in Fig. 3, which, in turn, is de- 
pendent on a properly regulated splic- 
ing process. In the construction of the 
DE vectors described by Cepko. et al. 
(2). it was assumed that h is the viral 
splice junction sequences which regulate 
the splicing process. However, there is 
now mounting experimental evidence 
that this is not the case; rather, se- 
quences within the viral intron play an 
essential role (10. 11. 19) in modulating 
the levels of spliced and unspliced RNA 
forms present in the cytoplasm. The 
second major drawback of the DE type 
of vector is that the transduced gene is 
expressed from the retroviral promoter 
and. therefore, its usefulness will be 
limited to target cells where the viral 
promoter is active and to those ex- 
periments where this is the promoter 
that one wants to study. 
Vectors with I ntemaJ Promoters (VIP) 
VIP vectors were designed to circum- 
vent these limitations. As shown in Fig. 
4, in these vectors the selectable gene is 
directly linked to the left end of the viral 
DNA and is therefore expressed from 
the viral promoter. The gene of interest, 
however, is fused to another DNA frag- 
ment containing a promoter which is 
responsible for its expression. This pro- 
moter DNA fragment can be derived 
from any gene and, therefore, these vec- 
tors possess the flexibility to express the 
transduced gene in a manner most 
appropriate for a particular target cell 
(6. 23). 
Figure 4 also shows the structure of 
our primary VIP vector, N2. This vec- 
tor yields very high titers of virus (1-3 x 
10* cfu/ml) compared to other vectors 
(of the DE or VIP design) and efficient- 
ly expresses the selectable gene, in this 
case the bacterial Neo* (neomycin 
Vot 4. No. 6 (19t6) 
Recombinant DNA Research, Volume 12 
BioTerbniques JOt 
[229] 
