Overview 
Figure 2. Generation or retroviral vectors. A native retrovirus (M-MuLV: Moloney-Murine Leukemia 
Virus) is genetically engineered such that its structural genes are replaced by an exogenous gene (5). 
This construct is cloned into E. coli and the DNA is then introduced by conventional transfection 
(step 1 -see text) into a packaging cell line. These cells provide the structural proteins in trans to per- 
mit both packaging of the vector and budding of vector-containing viral particles (infectious virus) 
into the medium. Target cells are infected (step 2) with these viral particles, where the natural cycle 
of reverse transcription and integration occurs. The stably integrated vector provirus then generates 
its gene product(s). 
fori is currently underway in several 
laboratories to improve this technology. 
Two parameters define an efficient 
retroviral gene transfer system. First is 
the capacity to infect a large proportion 
of the target cells, a property dependent 
at least in pan on the ability to generate 
a high concentration (or titer) of recom- 
binant virus. Second is the capacity to 
have the gene expressed properly. 
RETROVIRAL VECTORS 
One of the key components of this 
technology is the nature of the retroviral 
vector itself. It is the vector that will 
determine both the titer of recombinant 
virus obtained and the ability of the 
transduced gene to be expressed. The 
development of an all-purpose superef- 
ficient retroviral vector has proved 
elusive, arising from the fact that we do 
not understand many of the details of 
the structure and biology of this group 
of viruses. Ongoing research on the 
basic biology of these viruses is, 
therefore, vital for the dev elopment of 
improved vector systems. 
Three main strategies of retroviral 
vector construction are shown in 
Figures 3-5. A short description and 
discussion of their properties will 
follow. Note that these vectors cany not 
one but two genes. One gene is the gene 
of interest and the second one is a selec- 
table gene. This second gene is not ab- 
solutely essential to include in the vec- 
tor but its presence greatly facilitates 
their use. A selectable gene, as its name 
implies, enables the identification and 
isolation of cells harboring the retroviral 
vector as w-as discussed above. In some 
cases, the gene of interest is a selectable 
gene and therefore only one gene is in- 
corporated (4, 12, 20, 21). 
Double Expression (DE) Vectors 
The structure of a prototype double 
expression DE vector is shown in Fig. 
3. In retroviruses, most of the important 
cis functions are present at the ends of 
the viral genome and are maintained in 
the corresponding vectors. The termini 
of retroviruses are redundant and are 
called long terminal repeals, or, in 
short, LTRs. They are represented in 
Fig. 3 and henceforth by black boxes. 
As shown in Fg. 3, the MuLV viral 
506 QioTcchniqucs 
genes are expressed from two mRNA 
forms. The gag and pol genes are ex- 
pressed from an unspliced RNA species 
which is colinear with the viral genome. 
The env gene is expressed from a spliced 
RNA form generated from the un- 
spliced RNA species by the removal of 
the long intron. In the cytoplasm of the 
infected cells, similar amounts of both 
mRNA species are present and, there- 
fore, the retroviral genome must 
regulate the efficiency with which the in- 
tron is removed. DE vectors as shown 
in Fig. 3 contain two foreign genes, one 
replacing in effect the gag/pol genes 
(which is expressed from the unspliced 
RNA form) and a second gene replac- 
ing the viral env gene (which is expressed 
from the spliced RNA form). The dis- 
tinguishing feature of this type of vec- 
tor is that it provides not only the cis 
functions for the transmission of the 
foreign genes into the target cells but 
also provides the cis functions for their 
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Recombinant DNA Research, Volume 12 
