Biochemistry: Yu el al. 
A 
U3 
Proc. Natl. Acad. Sci. USA 83 (1986) 3195 
R [U5| // 1 U3 I R [ifi| ProvrtDNA 
LTR 
• R -- ^ » 13 Virol RNA 
t , 
B 
C 
Detrtion 1 299 bp I 
U5 ] M-MuLV ITS 
Fig. 1. Structure of the Mo-MuLV genome and the principle of 
SIN vectors. 04) Structure of the viral DNA, which is present in an 
integrated form in the chromosome of the infected cell (proviraJ 
DNA), and the structure of the viral RNA, which is incorporated into 
virions. (B) A deletion in the U3 region of the 3' LTR of the provira) 
DNA is transferred to the 5' LTR of the progeny proviral DNA (see 
text). (C) The deletion introduced into the 3' LTR of SIN vectors. 
The 299-bp deleted segment includes most of the two 72-bp repeats 
associated with the viral enhancer and the presumptive promoter 
region, which contains the canonical "CAAT" box (20, 21). E, 
enhancer; P, promoter; dLTR, LTR containing the deletion shown in 
C. 
of a provirus serves as a template for the formation of both 
U3 regions in the progeny provirus (12). Therefore, as shown 
in Fig. LB, a deletion in the U3 region of the 3' LTR 
(encompassing the enhancer and promoter sequences) will be 
transferred to both LTRs in the progeny provirus, provided 
that the deleted sequences do not encode other essential 
functions — i.e., functions involved in replication or 
polyadenylylation. As a consequence of the removal of the 
enhancer and promoter sequences from both the 5' and 3' 
LTR in the progeny provirus, the viral transcriptional unit is 
eliminated. The deletion that we have introduced in the U3 
region of the Moloney murine leukemia virus (Mo-MuLV) 3’ 
LTR, which extends from a Pvu II site to a Sac I site, is 
shown in Fig. 1C. This deletion of 299 base pairs (bp) removes 
most of the two 72-bp repeats, which contain enhancer 
activity as well as the promoter region (20, 21), but leaves 
behind the TATA box. 
The structure of a prototype SIN vector derived from 
Mo-MuLV carrying the deletion in the 3' LTR is shown in 
Fig. 2. This vector contains a selectable marker gene ex- 
pressed from an internal promoter as well as two cloning sites 
for insertion of genes, either 5’ to (BamHI) or 3’ to (Xho I) the 
selectable marker. The presence of a selectable gene within 
the vector facilitates the isolation and characterization of the 
transfected cells (when the recombinant DNA constructed in 
Escherichia coli is introduced into tissue-culture cells) as well 
as the infected cells (when the corresponding virus is used to 
infect the target cell). An internal promoter is essential for the 
continued expression of the selectable gene in the infected 
cells. If the selectable gene is expressed from the viral 5' 
LTR, h will not function in the infected cells because the 
Prototype 
LTR v t. 
o»! 
dLTR 
tpm 
MT-N 
SV-N 
Nh»I 
— 3400 1 3200 ) 
El. l 
xo 
Am 
► 3200 KT 
* 2200 WT 
>t‘* 
* X00MT 
► 2200 NT 
r* 
R 
Fig. 2. Structure of SIN vectors derived from Mo-MuLV and 
the structure of the corresponding proviruses in the infected cells. 
{Upper) A Mo-MuLV-derived prototype SIN vector consists of four 
parts; (0 An intact LTR (black box) followed by -300 bp (thin line), 
which contains the viral packaging signal (14). The 3' end of this DNA 
fragment was generated by BAL-31 digestion and maps *»60 bp 
upstream from a Pvu I site present in this region of the Mo-MuLV 
genome; (if) a DNA fragment containing a promoter driving the 
expression of (Hi) a selectable gene; and (iv) a second LTR containing 
a DNA fragment containing the deletion shown in Fig. 1C. The 5' end 
of this DNA fragment corresponds to the Mo-MuLV Cla I site 
present 140 nucleotides upstream from the LTR. MT-N and SV-N 
are two SIN vectors carrying the nee/ gene as a selectable marker, 
which is expressed from the mouse MT or early SV40 promoter. 
Digestion of MT-N and SV-N with Nhe I generates a characteristic 
3400- and 3200-bp DNA fragment, respectively (reflecting the dif- 
ference in size of the MT and SV40 DNA fragments). MT-N 
generates a 3200-nucleotide RNA transcript initiated in the 5' LTR 
and a 2200-nucleotide RNA transcript initiated in the MT-derived 
DNA fragment. Similarly, SV-N generates a 3000- and a 2200- 
nucleotide RNA transcript. (Lower) Genomic DNA was digested 
with Nhe I and analyzed by Southern blotting. Vector-specific DNA 
was detected by hybridization with a u P-labeled nee/ probe. SV-N 
and MT-N contain 2-5 pmol of vector DNA and 10 pg of N1H 3T3 
cell DNA. Lanes S-l to S-3 and M-l to M-3, cell lines infected with 
virus corresponding to SV-N and MT-N, respectively. Each cell line 
was derived by infection with virus that originated from an indepen- 
dently transfected +1 cell. Each lane contains 10 pg of DNA. 
enhancer and promoter sequences have been removed (Fig. 
1). Indeed, when virus derived from a SIN vector in which the 
nee /- gene expressed from the viral LTR was used to infect 
NIH 3T3 cells, the neo '- gene was not expressed and no 
G418-resistant cells could be isolated (data not shown). 
A number of different SIN vectors with internally promot- 
ed selectable genes were constructed. Two such vectors 
shown in Fig. 2 contain the bacterial nee /- gene as a selectable 
marker (22), driven by the mouse metallothionein I (MT), or 
by the early simian virus 40 (SV40) promoter. Similar vectors 
have been constructed that contain the hamster methotrex- 
ate-resistant dihydrofolate reductase cDNA as a selectable 
marker (23). The DNA constructs shown in Fig. 2 were 
introduced into t/»2 cells by the calcium phosphate-mediated 
Recombinant DNA Research, Volume 12 
[ 241 ] 
