Overview 
Figure 5. The deleted LTR trick. An initial vector is constructed in a plasmid such that the pro- 
moter/enhancer elements are deleted from the 3 ’ LTR (dLTR). Upon transfection into a packag- 
ing cell line, this structure is maintained. When the resulting vector is introduced into target cells, 
the 3 ' LTR is used as a template for both LTRs during the process of reverse transcription. Therefore 
the provirus in the target cell has both LTR promoter/enhancer sequences deleted, leaving the vec- 
tor genes reliant on internal promoters for expression. 
the regulation of a metallothionein (MT) or SV40 (SV) promoter. After infection, the 5 ’ LTR would 
be disabled as described in Figure 5. Therefore, only the shorter transcripts produced with the in- 
ternal promoter would be generated. 
resistance) gene. Interestingly, if one ex- 
amines the structure of the vector, it 
should not have worked at all. It was 
originally made as part of a study to 
determine the length of the packaging 
sequence (D. Armentano, Ph.D. Thesis, 
Princeton University, 1986). In contrast 
to other VIP vectors such as N4, in N2 
41 8 bp of the viral gag coding sequences 
(including the start code word) has been 
retained, but the Neo R gene is out of 
frame and therefore one might have 
assumed that it would not be expressed. 
However, it appears that it is this gag 
coding sequence which is responsible 
for the higher levels of virus generated 
from N2 vectors. It turns out that this 
DNA has unexpectedly introduced a 
cryptic 3 ' splice site just upstream from 
the Neo R gene which generates a 
spliced RNA form that serves as the 
mRNA for the Neo R gene (Armen- 
tano, et al., submitted). Evidence is 
accumulating from several laboratories 
that the N2 vector and its derivatives are 
exceptionally useful retroviral vectors, 
especially for the transfer of genes into 
suspension grown lymphoid cells (13) 
and bone marrow progenitors of several 
mammalian species (5, 9, 15, 16). (More 
on this topic below.) 
Self-Inactivating (SIN) Vectors 
SIN vectors are the latest addition to 
retroviral vectors (29). The LTRs at the 
two ends of the retroviral genome con- 
tain an element called an enhancer 
which not only can affect the expression 
of the vector’s gene but, when inte- 
grated into a cell’s chromosome, can 
also activate adjacent oncogenes (a 
process called insertional activation) (8). 
SIN vectors are designed to eliminate 
these two effects by “self-inactivating”. 
During the process of reverse transcrip- 
tion and integration, a portion of the 
virus DNA which includes the enhancer 
and promoter sequences becomes 
deleted. As a result, the proviral DNA 
in the infected cells becomes transcrip- 
tionally inactive, thus producing two 
consequences: The uninhibited expres- 
sion of the foreign gene and the reduc- 
tion of insertional activation. 
How this self-inactivation works is 
shown in Figure 5. SIN vectors contain 
a 299 bp deletion in the 3 ' LTR (dLTR). 
This deletion encompasses the pro- 
moter and enhancer sequences which 
control the accurate and efficient 
transcription of the viral genome. Only 
the sequences in the 5 ' LTR are 
necessary for the generation of viral 
RNA. Therefore, their removal from 
the 3 ' LTR, as shown in Fig. 5, does not 
affect viral functions. A consequence of 
the replication of retroviruses (25) is that 
a region of the 3 ' LTR (which encom- 
passes this deletion) is the template for 
the synthesis of that region in both the 
5 ' and 3 ' LTRs. Since the viral 
enhancer and promoter are absent from 
508 BioTechniqucs Vol. 4, No. 6 (1986)" 
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Recombinant DNA Research, Volume 12 
